Blog

May 07, 2023

PROTAC: grandes oportunidades para la academia y la industria (una actualización de 2020 a 2021)

Transducción de señales y terapia dirigida

Signal Transduction and Targeted Therapy volume 7, Article number: 181 (2022) Cite this article

21k Accesses

33 Citations

38 Altmetric

Metrics details

PROteolysis TArgeting Chimeras (PROTACs) technology is a new protein-degradation strategy that has emerged in recent years. It uses bifunctional small molecules to induce the ubiquitination and degradation of target proteins through the ubiquitin–proteasome system. PROTACs can not only be used as potential clinical treatments for diseases such as cancer, immune disorders, viral infections, and neurodegenerative diseases, but also provide unique chemical knockdown tools for biological research in a catalytic, reversible, and rapid manner. In 2019, our group published a review article "PROTACs: great opportunities for academia and industry" in the journal, summarizing the representative compounds of PROTACs reported before the end of 2019. In the past 2 years, the entire field of protein degradation has experienced rapid development, including not only a large increase in the number of research papers on protein-degradation technology but also a rapid increase in the number of small-molecule degraders that have entered the clinical and will enter the clinical stage. In addition to PROTAC and molecular glue technology, other new degradation technologies are also developing rapidly. In this article, we mainly summarize and review the representative PROTACs of related targets published in 2020–2021 to present to researchers the exciting developments in the field of protein degradation. The problems that need to be solved in this field will also be briefly introduced.

In 2001, Crews group and Deshaies group reported the first example of PROTACs.1 As a novel chemical biology technology, PROTACs present a chemical knockdown strategy by hijacking the ubiquitin–proteasome system with bifunctional small molecules that can simultaneously bind target protein and E3 ubiquitin ligase and induce the target protein to be ubiquitylated and then be degraded by proteasome (Fig. 1). In the past 20 years, especially since dBET1 PROTAC based on pomalidomide as the E3 ligase ligand successfully degraded BET protein in 2015,2 the field of PROTACs has ushered in a period of rapid development (Fig. 2a). So far, a variety of PROTACs derived from different E3 ligase and protein ligands have been disclosed to achieve the degradation of various types of interesting proteins.

The mechanism of PROTAC-mediated protein degradation

The researches on PROTAC from 2001 to 2021. a The publications on PROTACs from 2001 to 2021. b The structure of ARV-110 and ARV-471. c The comparison of PROTAC targets on different diseases between 2001–2019 and 2001–2021. d Classification and percentage of degradable kinases

Due to its unique mode of action, PROTACs technology has received great attention in the industry and has been applied to the possible treatment of cancers, immune disorders, viral infections, neurodegenerative diseases, etc. Arivinas, a clinical-stage biopharmaceutical company, recently disclosed the structures of the androgen receptor (AR) degrader ARV-110 and the estrogen-receptor (ER) degrader ARV-471 (Fig. 2b).3,4,5 The previously announced results indicate that ARV-110 is safe and effective for patients with metastatic castration-resistant prostate cancer(mCRPC). This is PROTAC's first clinical trial data and presents a milestone in the transformation of PROTAC technology into a new treatment strategy. As the first targeted estrogen-receptor-degrading agent to enter clinical trials, ARV-471 is another potential best-in-class drug that may bring hope to breast cancer patients. Besides the AR and ER targeting PROTAC molecules, more and more PROTAC degraders have entered the clinical trial stage in the past 2 years. For instance, the new targets of these PROTAC molecules include BCL-xL, IRAK4, STAT3, BTK, BRD9, MDM2, etc., among which most are the first-in-class targets. In addition to being used as possible clinical treatments, PROTAC is an efficient protein knockdown tool that can directly control protein levels without gene editing operations. It can be used as a useful supplement to existing genetic research tools and provide possible answers to many fundamental biological questions. With the in-depth understanding of the mechanism of PROTAC and its great potential in biological research and disease treatment, more and more researchers have begun to pay attention to this field, and more targets have been proven to be degradable by PROTAC molecules. In 2019, we wrote a review of PROTACs in this journal,6 summarizing that there were about 40 proteins that could be degraded at that time. According to the latest statistics in December 2021, the reported PROTAC targets have reached more than 130 (Fig. 2c). The number of degradable targets reported in 2020–2021 (about 90) has completely exceeded the total amount of the previous 18 years, indicating that the era of protein degradation has arrived.

In the reported studies, researchers are more inclined to choose kinases as the targets of protein degradation. According to incomplete statistics, about 54 kinases can be degraded by different degraders based on the PROTAC technology, accounting for 45% of the total targets (Fig. 2d). The main reason is that most kinases have known and effective inhibitors or ligands, which can be easily modified to connect linkers and maintain sufficient binding affinity. In addition, the kinase has a deep binding pocket, which can promote the binding of PROTACs, thereby inducing the interaction between the kinase and the E3 ligase, and then ubiquitinating and finally degrading the kinase. Moreover, although the kinase protein has a high degree of homology, PROTAC can selectively degrade different subtypes of kinases and can even be developed from nonselective inhibitors. How is the selectivity achieved by PROTAC? Nonselective inhibitors will target the highly conserved ATP-binding pocket to exert their inhibitory effects. As is known, the residues in the ATP-binding pocket of different subtypes of kinases are highly similar, which usually results in poor selectivity for these inhibitors. When nonselective inhibitors serve as POI ligands for PROTACs, not only the protein ligands can recognize the corresponding kinases, but the degraders can induce specific protein–protein interaction between POI and E3 ligase to form a ternary complex. The two-step recognition mechanisms can lead to selectively degradation of targets.

There are 518 kinds of kinases that have been discovered so far, which are involved in a variety of physiological regulatory processes such as cell survival, proliferation, differentiation, apoptosis, and metabolism.7 These kinases have been divided into nine categories based on their structure and function, namely, tyrosine kinases (RTKs), TKL kinases (TKLs), STE kinases (STEs), CAMK kinases (CAMKs), and AGC kinases (AGCs)), CMGC kinase group (CMGCs), atypical protein kinase group (atypical), CK1 kinase group, and other groups (others). According to the classification of the human kinase profile, we have marked the kinases that can be degraded as degradable kinases (Fig. 3). In the "degradable" kinase table, according to the classification method of the kinase profile, it was noticed that only CK1 and CAMK have no reported PROTACs. The first and second targets are RTKs and CMGCs, with 19 (35.2%) and 14(25.9%) targets, respectively, accounting for more than half of the total kinases (Fig. 2d). Based on the kinases marked in the human kinase map, we have compiled the current "list of human ‘degradable’ kinases based on PROTAC". We believe that more and more kinases will be degraded by PROTAC technology in the future. In addition, we look forward to the joint efforts of scientists all over the world to achieve the goal of ‘each protein has a corresponding small-molecule degrader’ in the future.

The list of human "degradable" kinases based on PROTAC

In the past 2 years, the field of protein degradation has seen a rapid development and there has been many new literatures and technologies about not only PROTAC, but also molecular glue, LYTACs, AUTACs, ATTECs, RIBOTACs, PhosTACs, etc. A number of excellent reviews8,9,10,11,12,13 have been published to discuss these areas and progress. Due to space limitations, we will focus on the discussion about PROTAC degradation technology in this article over the period of 2020–2021. In the following pages, we will introduce the advances of PROTACs in disease fields, other PROTACs technologies, and the clinical progress of PROTACs. In different disease fields, cancers currently account for the largest part of the research on small-molecule degraders. We will generally introduce them in different sub-fields such as signaling pathways, transcriptional regulation, and cell cycle, etc. At the end of this paper, a brief summary and perspectives will be given.

Androgen receptor (AR) is a member of the nuclear hormone receptor superfamily, which plays a vital role in the maintenance of male secondary sexual characteristics and the development of the prostate. Androgen receptor disorder is the main cause of prostate cancer. Metastatic castration-resistant prostate cancer(mCRPC) remains incurable and lethal. A number of AR antagonists have been developed to treat advanced prostate cancer, such as Enzalutamide, Apalutamide, and Darolutamide.14 Unfortunately, patients with these AR antagonists ultimately developed drug resistance. Most tumors are resistant to AR antagonists due to AR signaling continue to function and drive tumor growth and progression.15 There is an urgent need to develop new treatment strategies to treat prostate cancer, especially metastatic castration refractory prostate cancer (mCRPC).

Wang group has been committed to the development of AR degraders. Since 2019, they have reported a number of high-efficiency AR degraders by mainly using AR antagonists as AR ligands and linking them with different E3 ligands such as VHL or CRBN to obtain different degraders. In the subsequent optimization process, they respectively modified the AR ligands, E3 ligands, linkers and achieved good results. In 2019, they first reported the degrader 1 (ARD-69, Fig. 4),16 a high-efficiency degrader obtained by connecting an AR ligand to VHL, could efficiently induce the degradation of AR protein in LNCaP cells, VCaP cells, and 22Rv1 AR+ cells. The DC50 were 0.86 nM, 0.76 nM, and 10.4 nM, respectively. It could also effectively reduce the level of AR protein in mouse xenograft tumor tissues and had a strong inhibitory activity in AR+ prostate cancer cell lines.

The representative PROTACs targeting AR

Subsequently, they optimized the VHL ligand and obtained the degraders 2 (ARD-61, Fig. 4)17 and 3 (ARD-266, Fig. 4)18 based on ARD-69. They found that the AR degradation activities of these two degraders were slight worse than that of ARD-69 in LNCaP cells and VCaP cells. But they could effectively induce the degradation of AR in other AR+ breast cancer cell lines. Also, they were more effective than Enzalutamide in inhibiting cell growth and inducing cell cycle arrest and apoptosis.

In 2021, they used CRBN ligand to replace the VHL ligand and obtained the degrader 4 (ARD-2128, Fig. 4),19 which achieved 67% oral bioavailability in mice. Oral administration of 4 (ARD-2128) could effectively induce degradation of AR protein and effectively inhibit the growth of tumors in mice. This study demonstrated the possibility of developing an orally active AR degrader for the treatment of prostate cancer.

In order to improve its degradation activity, they further optimized and reduced the molecule weight and obtained the degrader 5 (ARD-2585, Fig. 4).20 They found that the new degrader 5 (ARD-2585) had a significant increase in the degradation activity of AR in LNCaP cells and VCaP cells with the DC50 of 0.1 nM and 0.04 nM, respectively, also it could effectively inhibit cell growth in those cell lines with the IC50 values of 1.5 nM and 16.2 nM, respectively. Moreover, it achieved excellent pharmacokinetics and 51% oral bioavailability in mice.

In 2021, Cravatt group discovered that DCAF11 can be used as the E3 ligases for protein degradation, and reported the ligand 21-SLF that can efficiently bind to DCAF11.21 So they synthesized an AR-targeting degrader 6 (21-ARL, Fig. 4) based on 21-SLF and found it could induce dose-dependent degradation of AR in normal 22Rv1 cells, but it could not cause changes in AR protein in DCAF11-KO cells. The degrader 6 (21-ARL) could induce the degradation of 90% AR protein at 10 μM in LNCaP cells. These data indicated that the electrophilic PROTAC (PROTAC that operate by covalent adduction of E3 ligases) combined with DCAF11 can promote the degradation of AR protein in human cells.

Subsequently, Hwang group reported a new series of AR degraders for the treatment of metastatic castration-resistant prostate cancer. Primarily, they utilized TD-106 as an E3 ligase ligand, a novel CRBN ligand identified in their previous studies.22 Among all the AR degraders, the representative degrader 7 (TD-802, Fig. 4) could effectively induce the degradation of AR protein with DC50 of 12.5 nM and the Dmax of 93% in LNCaP prostate cancer cells.23 In addition, the degrader 7 (TD-802) showed good liver microsomal stability and pharmacokinetic properties in vivo.

In order to find PROTACs with lower toxicity and better binding affinity than before, Wang group designed and synthesized several series of AR PROTACs by using CRBN/VHL E3 ligands and AR antagonists in 2021.24 They tested the cell inhibition of these synthetic compounds in AR-positive VCaP cells at different concentrations, and the degrader 8 (A031, Fig. 4) could inhibit 69.56% of the cell viability under 1.0 µM.

In 2021, another series of AR PROTACs based on Bicalutamide analogs and thalidomide were designed, synthesized, and biologically evaluated by the Kim group.25 Several novel PROTACs had their abilities to induce the degradation of AR. In particular, the degrader 9 (Fig. 4) induced the degradation of AR in a dose and time-dependent manner with DC50 of 5.2 µM in LNCaP cells.

In addition to the above examples, Enzalutamide had also been used in the design of AR degraders. The degrader 10 (Fig. 4) was reported by Skidmore group based on Enzalutamide in 2020.26 It was a potent degrader, whose DC50 was 10 nM and Dmax was 33% in LNCaP cells. In addition, it showed an inhibitory effect on the proliferation of prostate tumor cells. The discovery of Enzalutamide-based PROTACs were expected to overcome the drug resistance that conventional drugs bring to patients.

The degrader 11 (PAP508, Fig. 4) was developed as a new type of PROTAC for AR protein by Lin group in 2020.27 The results showed that the degradation activities of 11 (PAP508) on AR protein depended on the action of proteasome, and the degradation effect was concentration and time dependent in LNCaP and VCaP cells. Also, it could inhibit the proliferation, migration, and invasion of prostate cancer cells.

AR-V7 is an AR variant with a truncated C-terminus. It has been confirmed that AR-V7 expression was induced by ADT and was associated with prostate cancer cell resistance. Recently, Kim group used the PROTAC technology to develop the degrader 12 (MTX-23, Fig. 4) to simultaneously target and induce the degradation of AR-V7 and AR full-length (AR-FL) proteins.28 The experimental results showed that the DC50 of degrader 12 (MTX-23) induced degradation of AR-V7 and AR-FL were 0.37 µM and 2 µM, respectively. The degrader 12 (MTX-23) inhibited the proliferation of prostate cancer cells, and only induced apoptosis in androgen-responsive prostate cancer cells.

Also in 2021, Ke group developed the AR degrader 13 (A16, Fig. 4) based on AR agonist RU59063 and Phthalimide.29 They found these degraders could reduce the level of AR protein with the ranging from 6% to 32% at 20 µM, and the degrader 13 (A16) had the best activity (32% degradation at 20 µM) on reducing the level of AR protein in LNCaP cells.

In 2021, Samajdar group designed AR-V7 protein degrader 14 (Fig. 4) based on AR-DBD binder VPC-14228.30 The DC50 was 0.32 µM for AR-V7 protein in 22Rv1 cells and 14 was found to have modest oral bioavailability.

Finally, we compared the reported AR degraders (Table 1). It was found that there were various types of AR warheads currently in AR PROTACs design, but more of them were Pfizer AR antagonist derivatives, followed by Enzalutamide derivatives and other binders were less used. In terms of the selection of E3 ligases, currently CRBN and VHL were both frequently selected, and other E3 ligases were used less frequently.

RAF kinase family regulates cell proliferation, growth, differentiation, and survival through the RAS–RAF–MEK–ERK signaling pathway. BRAFV600E expression has been reported in a wide variety of human cancers.31,32,33 Small-molecule drugs targeting BRAFV600E mutation include Dabrafenib, Vemurafenib, and Encorafenib.34 They have shown good effects in clinical application, but the generation of drug resistance limits their long-term use. These small-molecule inhibitors mainly play roles by binding to the catalytic pocket of RAF, but cannot inhibit dimerization, another key link of RAF activation, so they can not completely inhibit the activity of RAF.35,36 The limitations of current RAF inhibitors provide a rationale for the exploration of alternative therapeutic strategies by employing novel inhibitor mechanisms of action.

In 2019, Gou group designed a series of BRAF degraders based on RGS and pomalidomide.37 They found that degrader 15 (Fig. 5) could induce the degradation of BRAF protein in MCF-7 cells and it could effectively show antiproliferative activity on cancer cells by inducing apoptosis.

The representative PROTACs targeting BRAF and BRAFV600E

In 2020, Cullgen used Vemurafenib and BI882370 as ligands for BRAFV600E protein. By analyzing the binding mode of inhibitors and proteins, they designed a series of degraders and found that 16 (Fig. 5) based on Vemurafenib and 17 (Fig. 5) based on BI882370 had better degradation activity of BRAFV600E protein.38 They also found that degrader 16 could induce the degradation of BRAFV600E protein at 12 nM, while degrader 17 had degradation effect at 37 nM. At the same time, experiments had also shown that the two degraders had no degradation activity to wild-type BRAF protein. Finally, they performed anti-proliferation experiments with 16 and 17 on A375 cells and HT-29 cells, respectively. The experimental results showed that the inhibitory effect of Vemurafenib-based degrader 16 on A375 cells was worse than that of inhibitor Vemurafenib. The IC50 in HT-29 cells was 124 nM. And BI882370-based degrader 17 had the same inhibitory effect on A375 cells and HT-29 cells with IC50 of 46.5 nM and 51 nM, respectively.

Similarly, in 2020, Sicheri group developed a series of novel PROTACs targeting BRAF based on different BRAF inhibitors and E3 ligands (pomalidomide and VHL).39 The most effective degrader 18 (P4B, Fig. 5) induced the selective degradation of BRAFV600E but not the wild-type BRAF, although degrader 18 (P4B) had the same affinity for BRAFWT and BRAFV600E. Downregulation of BRAFV600E induced by degrader 18 (P4B) suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. In addition, the degrader 18 (P4B) displayed effectively BRAFV600D and BRAFG466V mutant cells. These findings highlighted a new approach to modulating the functions of oncogenic BRAF mutants and provided a framework to treat BRAF-dependent human cancers.

Recently, Crews group developed a VHL-based degrader 19 (SJF-0628, Fig. 5) and negative control degrader 20 (SJF-0661, Fig. 5) by coupling Vemurafenib to VHL through a rigid piperazine linker.40 They found the degrader 19 (SJF-0628) could induce the degradation of BRAFV600E protein in a variety of cell lines but did not induce the degradation of BRAFWT protein. In SK-MEL-28 cells, the DC50 to BRAFV600E was 6.8 nM and the Dmax exceeded 95%. It could not only induce the degradation of BRAFV600E, but also induce the degradation of a variety of BRAF mutants in a variety of cell lines, including BRAF-p61V600E, BRAFG469A, BRAFG466V, and so on. Subsequently, they tested the inhibitory effect of the degrader 19 (SJF-0628) on tumor cells. They found that in SK-MEL-28 cells (BRAFV600E) the EC50 of the degrader 19 (SJF-0628) was 37 nM. In SK-MEL-239 C4 cells(BRAF-p61V600E), the EC50 was 218 nM. In SK-MEL 246 cells (BRAFG469A), the EC50 was 45 nM.

eEF2K (eukaryotic elongation factor 2 kinase) is known as Calmodulin-dependent kinase III (CaM kinase III). eEF2K can phosphorylate its only intracellular substrate protein eEF2 and inhibit the peptide-chain extension stage during protein synthesis, and reduce the consumption of amino acids and energy so that the cell can survive under metabolic stress.41,42 The expression and activity of eEF2K in some tumor cells can affect the proliferation, migration, invasion, and other physiological processes43,44,45,46 But eEF2K shows different effects in different tumor cells. However, the eEF2K inhibitors have not achieved the expected effect in cancer treatment.

In 2021, Ouyang group analyzed the binding mode of eEF2K inhibitor A484954 and eEF2K protein, and obtained a series of degraders by linking it to different E3 ligase ligands.47 Western blotting experiments determined that the degrader 21 (Fig. 6) had the best eEF2K degradation activity in MDA-MB-231 cells, and the degradation activity Dr value was as high as 56.7%. It was also found that the degrader could induce apoptosis in MDA-MB-231 cells.

The representative PROTAC targeting eEF2K

EGFR, a member of the epidermal growth factor receptor (HER) family, is a glycoprotein belonging to tyrosine kinase-type receptor, which contains three regions: extracellular ligand-binding region, transmembrane region, and intracellular kinase region. As ligands, EGF and TGFα can activate EGFR to cause dimerization and guide the phosphorylation of downstream proteins, including MAPK, AKT and JNK pathways. Studies have shown that EGFR is overexpressed and abnormally expressed in many solid tumors and is associated with cell proliferation, angiogenesis, tumor invasion and tumor metastasis.48 Among them, non-small cell lung cancer(NSCLC), one of the most aggressive cancers, is closely related to aberrant EGFR signaling.49 Although three generations of small-molecule EGFR inhibitors have been approved by FDA for the treatment of NSCLC patients, drug resistance resulting from continuously heterogeneous mutations (EGFRC797S) remains a problem that inhibitors cannot overcome.50

In 2019, Jin group designed a new class of EGFR PTOACs based on Gefitinib and different E3 ligands.51 The degrader 22 (MS39, Fig. 7) and degrader 23 (MS154, Fig. 7) which were based on VHL and CRBN potently induced the selective degradation of EGFR mutants but not EGFRWT and inhibited lung cancer cells proliferation. In HCC-827 (EGFRe19d) cells and H3255 (EGFRL858R) cells, both the degrader 22 (MS39) and degrader 23 (MS154) could efficiently induce the degradation of mutant EGFR proteins, with DC50 of 5.0 nM, 3.3 nM, and 11 nM, 25 nM, respectively. While EGFR could not be degraded in OVCAR8(WT) cells and H1299 (WT) cells, indicating that the degrader 22 (MS39) and degrader 23 (MS154) had selective degradation activity against mutant EGFR. In addition, degrader 22 (MS39) had sufficient in vivo PK properties and was suitable for in vivo efficacy studies.

The representative PROTACs targeting EGFR

At the same time, a new class of EGFR PROTACs based on Osimertinib and lenalidomide was disclosed by Zhang group in 2020.52 The degrader 24 (Fig. 7) could effectively induce degradation EGFRDel19 with a DC50 of 161 nM and Dmax value of 68% in PC9 cells. The degrader 24 had good antiproliferative activity against a variety of EGFR mutant cells, such as PC9(EGFRDel19) cells, HCC-827(EGFRDel19) cells, and H1957(EGFRL858R/T790M) cells, with IC50 of 413 nM, 1.34 µM, and 657 nM, respectively. In addition, apoptosis and G0/G1 phase arrestation of PC9 cells was significantly induced by the degrader 24 unsurprisingly.

In 2020, Zhou group also reported other EGFR degrader based on Gefitinib and VHL.53 They found that the degrader 25 (Fig. 7) could not only induce the degradation of EGFRL858R protein, but also inhibit PD-L1 and IDO1 activities. The degrader 25 also significantly inhibited the growth of H3255 cells and enhanced the antitumor immune response of NSCLC.

In 2020, Zhang group published two works on EGFR PROTACs. In the first work, they developed EGFR degraders based on an inhibitor with pyrido[3,4-d] pyrimidine moiety, which was a fourth-generation EGFR-TKI that displayed potent inhibitory activity against EGFRL858R and EGFRL858R/T790M/C797S. The promising degrader 26 (Fig. 7) and degrader 27 (Fig. 7) induced degradation of EGFR in HCC-827(EGFRe19d) cells with the DC50 values of 45.2 nM and 34.8 nM, respectively.54 It was found that the apoptosis and the G1 phase arrestation of HCC-827 cells were significantly induced by the two degraders. In another work, they conjugated a purine-containing derivative which was discovered as a highly potent EGFR-TKI with lenalidomide and VHL ligand to obtained a PROTAC library.55 The most potent degrader 28 (P3, Fig. 7) effectively induced the degradation of mutant EGFR with a DC50 of 0.51 nM, Dmax of 80.4% and DC50 of 126 nM, Dmax of 90.3% in HCC-827 (EGFRe19d) cells and H1975 (EGFRL858R/T790M) cells, respectively. The degrader 28 (P3) also showed significant antiproliferative activity on HCC-827 (EGFRe19d) cells and H1975 (EGFRL858R/T790M) cells with IC50 of 0.76 nM and 203 nM. In addition, it also induced cell apoptosis and arrested cell cycle.

In 2020, Ding group also reported PROTACs targeting EGFRL858R/T790M based on a novel selective EGFRL858R/T790M inhibitor XTF-262, which was more than 100 folds selectivity over the wild-type EGFR and a panel of 465 kinases.56 Four E3 ligases (VHL, CRBN, MDM2, and cIAP1) were utilized for design of PROTACs. The promising degrader 29 (Fig. 7) with a VHL ligand effectively induced the degradation of EGFRL858R/T790M with DC50 value of 5.9 nM and showed significant antiproliferative activity on H1975 (EGFRL858R/T790M) cells with IC50 of 506 nM.

In 2021, Jiang group developed two highly selective and functional EGFR-targeting PROTAC 30 (SIAIS125, Fig. 7) and 31 (SIAIS126, Fig. 7) based on Canertinib and CRBN ligand.57 Interestingly, they induced sustaining and selective degradation of EGFRL858R/T790M in H1975 cells and EGFRe19d in PC9 cells rather than EGFREe19d/T790M in PC9Brca1 cells and EGFRWT in A549 cells, which led to the selective growth inhibition of EGFR mutant lung cancer cells instead of normal cells or A549 cells. Surprisingly, mechanistic studies showed that PROTAC-induced EGFR degradation acted through both ubiquitin–proteasome system and ubiquitin-autophagy-lysosome system. They also proved that elevated autophagy activities enhanced EGFR degradation and cell apoptosis induced by PROTACs.

Finally, we compared the reported EGFR degraders (Table 2). It was found that there were various types of EGFR warheads currently in EGFR PROTACs design, but more of them were already marketed drugs, followed by reported inhibitors. In the selection of E3 ligases, CRBN and VHL were currently used, and other E3 ligases were not used in EGFR degraders.

eIF4E (Eukaryotic translation initiation factor 4E) is a cap-binding protein that can specifically recognize the cap structure at the 5’-end of mRNA.58 It plays an important role in the initiation of eukaryotic translation.59 Studies have found that the overexpression of eIF4E is related to cancer and other diseases, and eIF4E levels are elevated in 30% of cancers, so eIF4E has become an attractive target for drug discovery.60

In 2021, Arthanari group developed a new type of eIF4E inhibitor i4EG-BiP based on the crystal structure and proved that the inhibitor could inhibit the proliferation of cancer cells.61 Then they designed and synthesized a series of eIF4E degraders based on the binding mode of i4EG-BiP and eIF4E.62 Through the degradation activity test, it was found that the degrader 32 (d4E-4, Fig. 8) and the degrader 33 (d4E-6, Fig. 8) showed certain eIF4E degradation activity. When the drug concentrations were 10 µM, the obvious downregulation of eIF4E could be observed after 0.5 h of drug treatment.

The representative PROTACs targeting eIF4E

Estrogen receptors include two types: type I receptors are the classical nuclear receptors, including ERα and ERβ, which are located in the nucleus and mediate the genotype effect of estrogen. Type II receptors are membranous receptors, including membranous components of classical nuclear receptors and GPER1 (GPR30), Gaq-ER, and ER-X, which belong to the G-protein-coupled receptor family. They mediate rapid non-genotypic effects and perform indirect transcriptional regulatory functions through the second messenger system. The distribution of these two types of receptors is tissue and cell-specific and they are involved in the regulation of various functions such as reproduction, learning, memory, cognition, and so on.

It is well-known that the overexpression of estrogen receptor α(ERα) may lead to ER-positive breast cancer, which accounts for 70% of breast cancer.63,64 Approved endocrine therapies include aromatase inhibitors(AIs) such as Letrozole, selective ER modulators (SERMs) such as Tamoxifen, and selective ER degrader (SERDs) such as Fulvestrant. But the long-term use of SERMs is prone to lead to drug resistance and Fulvestrant is limited by poor solubility and low oral bioavailability.65,66 The emergence of PROTACs provides a new method for the development of drugs targeting ERα.

Since July 2021, Pfizer and Arvinas have been collaborating to advance the study of oral PROTAC 34 (ARV-471, Fig. 9) targeting estrogen receptors (ER).5 The PROTAC 34 (ARV-471) was the first degrader developed by Arvinas in 2018 and entered clinical studies for ER+/HER2−, locally advanced or metastatic breast cancer. In December 2020, a phase I clinical study was completed and the results showed that the PROTAC 34 (ARV-471) induced degradation of ER potently with encouraging clinical efficacy and tolerability. Another two trials of 34 (ARV-471) were scheduled to start this year. With the advancement of 34 (ARV-471) research, PROTACs could not only provide tools for scientific research but also serve as an important approach for drug development.

The representative PROTACs targeting ER

PROTACs/SNIPERs, the peptide-based target protein-degradation inducers, tend to have low cell penetrability and poor intracellular stability as drawbacks. In 2019, Qian group attempted to improve stability and cell penetration by using a lactam cyclic peptide as ERα binding ligand.67 The optimized degrader 35 (I-6, Fig. 9) induced obvious ERα degradation and inhibited MCF-7 cell growth with an IC50 of 9.7 µM.

In 2019, Rae group reported a new ER degrader 36 (ERD-148, Fig. 9).68 They found that in wild-type MCF-7 and ERα LBD mutant cells, the degrader 36 (ERD-148) showed strong inhibitory activity, the IC50 values in MCF-7, cY537S, and cD538G cells were 0.8 nM, 10.5 nM, and 6.1 nM, respectively. At the same time, the degrader 36 (ERD-148) could not only induce the degradation of wild-type ERα but also showed strong degradation activity against Y537S and D538G mutant ERα.

In 2020, Tang group developed a two-stage strategy for PROTACs screening.69 In stage one, the ERα ligands with a hydrazide functional group reacted with E3 ligase ligands with a terminal aldehyde group in DMSO solution to form a more than 100 compounds library. Then the ELISA screening was conducted without further purification. Among them, the degrader A3 was screened out with DC50 of 10 nM and Dmax of 95%. Then they transformed the degrader A3 to a more stable degrader 37 (AM-A3, Fig. 30) with better degradation activity (DC50 = 1.1 nM, Dmax = 98%) and cell growth inhibition (IC50 = 13.2 nM, Imax = 69%) in MCF-7.

In 2021, X-Chem developed a novel class of PROTACs based on a new ERα binder which was based on DNA-encoded chemical library screening.70 They screened 120 billion DNA-encoded molecules and found the best warhead to ERα, then they conjugated the warhead to many kinds of E3 ligase ligands by click reaction to obtain some novel PROTACs. They found the degrader 38 (Fig. 9) could induce the degradation of ERα with DC50 of 37 nM and inhibit MCF-7 cells growth with GI50 of 165 nM, and effectively inhibited ER+ MCF-7 tumor growth in a mouse xenograft model of breast cancer.

AstraZeneca also announced its degraders that targeted the degradation of ERα.71 Their study found that the affinity of degrader 39 (Fig. 9) for ERα protein was as high as 0.8 nM. In MCF-7 cells, it had a strong degradation activity for ERα protein, and the DC50 was 0.3 nM.

Salem group reported the degrader 40 (UI-EP002, Fig. 9) could effectively induce the degradation of ERα, ERβ, and GPER.72 UI-EP002 would induce degradation of the plasma membrane and intracellular GPER and nuclear ERs, but could not affect others proteins lacking the estrogen targeting domain. The target specificity was further proved in human MCF-7 cells, which could effectively induce degradation of ERα, ERβ and GPER without affecting the PRs. The degrader 40 (UI-EP002) induced cytotoxicity and G2/M cell cycle arrest in MCF-7 breast cancer and human SKBR3 (ERα-ERβ-GPER+) breast cancer cells but did not induce proliferation inhibition of MDA-MB-231 breast cancer cells.

FGFR (fibroblast growth factor receptor) belongs to the tyrosine receptor kinase (TRK) family in the human genome, including FGFR1, FGFR2, FGFR3, and FGFR4 subtypes. The signaling pathways mediated by FGFR are required for normal cell growth and differentiation, and they are involved in physiological processes, such as neovascularization, cell proliferation, and migration, regulation of organ development, and wound healing.73,74,75 However, when FGFR is mutated or overexpressed, it will cause excessive activation of the FGFR signaling pathway and induce normal cell canceration. There are FGFR aberrations in almost all the detected malignant tumors, such as urothelial carcinoma, bile duct carcinoma, breast carcinoma, endometrial carcinoma, squamous carcinoma and so on.76,77,78 A number of companies are engaged in the research and development of FGFRs inhibitors. Although 2 FGFR inhibitors have been approved, they are all multi-target inhibitors.79,80 The FGFR subtype-selective inhibitors are both in the clinical development stage.81

In 2021, Gray group synthesized several glutarimide-based CRBN-targeting degraders with various linkers based on FGFR inhibitor BGJ398.82 Through binding affinity tests and protein-degradation experiments, it was found that the PROTACs based on BGJ398 could degrade TEL-FGFR2 in Ba/F3 cells, while the full-length FGFR2 was poorly degraded in Kato III cells. Therefore, they used VHL ligands to replace CRBN and found the degrader 41 (DGY-09–192, Fig. 10) based on BGJ398 and VHL ligands had nanomolar degradation activity for both wild-type and fusion-mutant FGFR2 proteins. Furthermore, the degrader 41 (DGY-09-192) showed highly selective degradation of FGFR1 and 2, as well as full-length FGFR2 or FGFR2 fusion proteins, despite retaining equivalent biochemical inhibition of all four FGFR isoform proteins. However, the degrader 41 (DGY-09-192) also had some limitations that need to be overcome. For example, the degrader 41 (DGY-09-192) showed no improvement in antiproliferative activity compared to the inhibitor BGJ398 and did not overcome BGJ398-induced point mutations in the FGFR protein.

The representative PROTAC targeting FGFR1/2

Insulin-like growth factor 1 receptor (IGF-1R) is a membrane receptor tyrosine kinase. Overexpression of IGF-1R plays a key role in the proliferation, transformation, and survival of various cancer cells, such as breast cancer, lung cancer, prostate cancer, and so on.83 The anti-apoptotic effect of IGF-1R can cause tumor cells to develop resistance to commonly used chemotherapy drugs or radiotherapy drugs. The protein encoded by the Src gene belongs to the Src family of kinases (SFKs), which consists of 9 members, namely Src, Lyn, Fyn, Lck, Hck, Fgr, Blk, Yrk, and Yes. Among them, Src is currently the most known member.84 It is a non-receptor tyrosine kinase, which is related to the survival and drug resistance of cancer cells. At present, studies have shown that the activation of Src is related to the drug resistance of IGF-1R inhibitors. Therefore, the dual inhibitory effect of IGF-1R and Src may be a feasible way to develop new antitumor drugs to overcome drug resistance.85

In 2020, Lee group designed a variety of degraders based on different types of IGF-1R inhibitors and CRBN.86 The ligands for the targeted protein included IGF-1R/Src dual-target inhibitors. During the screening process, it was found that the degraders 42 (CPR3, Fig. 11) and 43 (CPR4, Fig. 11) based on N2-phenyl-N4-(1H-pyrazol-3-yl) pyrimidine-2,4-diamine had IGF-1R/Src dual-target degradation activity. In MCF-7 and A549 cells, the obvious degradation of IGF-1R and Src could be observed at the concentration of 5 µM. Although the degradation effect was poor, it showed obvious antiproliferative activity on MCF-7 and A549 cells, and the IC50 were 3.3 µM, 2.7 µM and 4.2 µM, 7.6 µM, respectively.

The representative PROTACs targeting IGF-1R and Src

KRAS is a mouse sarcoma virus oncogene. There are three ras genes related to human cancer: HRAS, KRAS, and NRAS, which are located on chromosomes 11, 12, and 1, respectively. Among them, KRAS has the greatest impact on cancer because it serves as a molecular switch which cycles between an inactive(GDP-bound "off") state and an active (GTP-bound "on") state.87,88 It controls the pathways that regulate cell growth under normal conditions, when KRAS gene is mutated to be permanently activated, normal RAS protein can not be produced which leads to abnormal cell proliferation and cancerization. KRAS is one of the most frequently mutated oncogenes, high-frequency mutations(such as G12A, G12C, G12D, G12S, G12V, G13C, G13D) and some low-frequency mutations can activate KRAS.89 KRAS has mutations in a variety of cancers, among which pancreatic cancer has a mutation rate of 90%, colon cancer and lung cancer (mainly non-small cell lung cancer) have mutation rates of 30–50% and 19%, and cholangiocarcinoma accounts for about 26%. Since the KRAS protein does not have a suitable binding pocket for small inhibitors, the development of small inhibitors targeting KRAS has not made a breakthrough for a long time. However, people have been paying attention to the G12C mutation and have developed some covalent inhibitors. There have been many KRASG12C inhibitors were in clinical researches, but the results also showed that some patients have already developed drug resistance.90 PROTACs which had the advantages of overcoming drug resistance and targeting undruggable targets provide a complementary approach to cancer treatment.

In 2019, Arvinas announced its patent for using PROTAC technology to induce the degradation of KRASG12C. They designed a large number of KRASG12C degraders based on ARS-1620 derivatives and different E3 ligase ligands.91 They found that degraders 44 (Fig. 12) and 45 (Fig. 12) had good KRASG12C protein-degradation activity through degradation activity screening. In NCI-H2030 cells, the degradation of KRASG12C was less than 25% when the drug concentration was 300 nM, but the degradation activity was significantly enhanced as the concentration increased. The degradation activity was higher than 50% when the concentration was 1 µM. The results proved that the KRASG12C could also be induced degradation by PROTAC technology, which brought hope for the treatment of related diseases.

The representative PROTACs targeting KRASG12C

In 2020, Gray group also reported the PROTAC targeting KRASG12C. The CRBN ligands were tethered to covalent quinazoline-based ligand ARS-1620 at the 2-position on the quinazoline to construct a degrader library.92 Then KRASG12C degradation was screened by a fluorescent-activated cell sorting (FACS)-based assay in a high-throughput manner. But it was regrettable that these compounds only induced the degradation of an artificial GFP-KRASG12C fusion protein but not endogenous KARSG12C, which might be due to the inability of the lead degrader to effectively poly-ubiquitinate endogenous KRASG12C. However, the degrader 46 (XY-4-88, Fig. 12) showed good antiproliferative activity in different tumor cells, such as H358 cells (IC50 = 0.51 µM), MiaPaCa2 cells (IC50 = 4.14 µM) and MiaPaCa2(GILA) cells (IC50 = 0.51 µM). Nevertheless, they developed a series of critical assays for in vitro activity evaluation and laid the foundation for the emergence of subsequent KARS PROTACs.

In 2020, Crews group also reported the first-in-class endogenous KRASG12C degraders based on covalent KRAS inhibitor MRTX849 and VHL ligand.93 A variety of linkers were introduced at N-methyl moiety of the pyrrolidine to generate a PROTAC library. The degrader 47 (LC-2, Fig. 12) was identified as the most potent KRASG12C degrader through screening, which induced rapid and sustained degradation of endogenous KRASG12C with Dmax of 80% and DC50 of 0.59 µM in NCI-H2030 cells. In addition, they have also tested the degradation activity of the degrader 47 (LC-2) in a variety of cells and found that it could efficiently induce the degradation of KRASG12C protein in different cells. It also modulated downstream ERK signaling in homozygous and heterozygous KRAS-mutant cell lines.

Recently, Chen group designed and synthesized KRASG12C degraders based on KRas G12C-IN-3 and pomalidomide.94 They found the degrader 48 (KP-14, Fig. 12) showed the best KRASG12C degradation activity in NCI-H358 cells with a DC50 of 1.25 μM. Mechanism experiments have proved that degrader 48 (KP-14) selectively induced the degradation of KRASG12C through the protein–ubiquitin system, but could not induce the degradation of other KRAS mutants such as G13D. In addition, the degrader 48 (KP-14) exhibited effective antiproliferative activity and inhibited the formation of tumor colonies in NCI-H358 cells.

Also, based on the structure of LC-2, Lu group replaced its vinyl moiety to obtain a series of KRASG12C degraders.95 They found that degrader 49 (YF135, Fig. 12) had the best degradation activity in H358 and H23 cells. It could induce the degradation of KRASG12C protein with DC50 of 3.61 µM and 4.53 µM, respectively. At the same time, the degrader also showed good antiproliferative activity in these two tumor cells, and the IC50 were 153.9 and 243.9 nM, respectively. The degrader 49 (YF135) was the first reversible covalent PROTAC that was capable of recruiting VHL-mediated proteasomal to induce the degradation of KRASG12C.

MAP kinase kinase or mitogen-activated protein kinase (MEK) is an important signal molecule in Ras–RAF-ME- ERK pathway. MEK1 and MEK2 are two subtypes of the MEK family. MEK1 and MEK2 activate ERK in cell proliferation, apoptosis, cell differentiation and play an important role in tumorigenesis.

Jin group published two articles on MEK degraders in 2019 and 2020. In 2019, they synthesized the MEK degraders based on the structure of non-ATP competitive MEK inhibitor PD0325901 and VHL. They found that the degrader 50 (MS432, Fig. 13) showed the strongest degradation activity, but it was not selective for MEK1 and MEK2.96 In HT-29 cells, the degradation activity DC50 of MEK1 and MEK2 were 31 nM and 17 nM, and the inhibitory activity of GI50 was 130 nM. In SK-MEL-28 cells, the DC50 of MEK1 and MEK2 were 31 nM and 9.3 nM, and the inhibitory activity GI50 was 83 nM. It could also inhibit ERK phosphorylation in cells, and could inhibit the proliferation of colorectal cancer and melanoma cells more effectively than the negative control.

The representative PROTACs targeting MEK

In 2020, they also developed the other MEK degraders based on PD0325901 and different E3 ligases ligands. They obtained VHL-based degraders 51 (MS928, Fig. 13), 52 (MS934, Fig. 13), and CRBN-based degrader 53 (MS910, Fig. 13),97 and they found these degraders could effectively induce the degradation of MEK1/2 through the ubiquitin–proteasome system, but they were not selective for MEK1 and MEK2. These degraders also inhibited downstream signal transduction and cancer cell proliferation. The degradation activities and inhibitory activities of VHL ligand-based degraders 51 (MS928) and 52 (MS934) in HT-29 cells and SK-MEL-28 cells were equivalent to the degrader 50 (MS432), but the CRBN-based degrader 53 (MS910) was significantly weaker than the degrader 50 (MS432).

In 2020, Perry group reported the MEK1/2 degrader based on allosteric MEK inhibitor Refametinib derivative and VHL.98 They found the degrader 54 (Fig. 13) had the efficacy of inducing the degradation of MEK1/2 at 10 µM. The degrader 54 had a stronger effect on cell proliferation than inhibitor and showed a better efficacy of suppression of ERK1/2 phosphorylation and IL-6 secretion.

MYC is a broadly acting transcription factor that regulates cell differentiation and proliferation through multiple mechanisms.99 MYC gene is currently the most studied nuclear protein carcinoid gene, including C-MYC, N-MYC, L-MYC, and R-MYC. C-MYC is one of the most common activated proto-oncogenes.100 Cancers regulated by C-MYC account for about 20% of human cancers.101 However, because it is extremely difficult to develop drugs that directly target the MYC protein, MYC has become an "undruggable" target.102

Schneider group designed and synthesized a MYC degrader based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and thalidomide.103 They found the degrader 55 (MDEG-541, Fig. 14) could rapidly induce the degradation of MYC protein in HCT116 or PSN1 cells, and almost completely induced the degradation of MYC protein when the drug concentration was 20 µM. In addition, they also found that the degrader 55 (MDEG-541) could degrade GSPT1 and GSPT2 in a variety of cells.

The representative PROTAC targeting Myc

p38 mitogen-activated protein kinase (MAPK) family consists of p38α, p38β, p38γ, and p38δ.104 p38α is widely expressed in almost all cell types and attributed the main function in the p38 family. p38β expressed at a lower level and may have redundant functions with p38α. The function of p38α is highly dependent on cell type and environment. For example, during tumorigenesis, p38α usually plays a tumor-suppressive role in normal epithelial cells, while in malignant cells p38α tends to support tumor development.105 However, currently available p38α inhibitors have not shown the expected efficacy in clinical trials.

In 2020, Nebreda declared their design and synthesis of a series of novel p38α/β degraders based on an ATP competitive inhibitor of p38α/β PH-797804 and thalidomide analogs.106 The degraders 56 (NR-6a, Fig. 15) and 57 (NR-7h, Fig. 15) were the two representative degraders which could efficiently induce the degradation of p38α/β without other related kinases at nanomolar concentrations in several mammalian cell lines. The degraders 56 (NR-6a) and 57 (NR-7h) inhibited the p38α signaling pathway induced by stress and cytokines, which provided a useful tool to investigate function and influence of the p38 MAPK pathway in diseases.

The representative PROTACs targeting p38

KRAS is recognized as one of the targets of cancer treatment. However, no small-molecule drug targeting KRAS has been approved. PDE δ is a shuttling factor of RAS which can prevent KRAS binding to the endomembrane and promote its diffusion in the whole cell.107 Subsequently, KRAS is released from PDEδ by releasing factor Arl2 and transported to the plasma membrane. Several KRAS-PDEδ inhibitors with potent affinity have been reported, such as Deltarasin, Deltazinone, and Deltasonamide.108 However, Arl2 induces the fast release of high-affinity inhibitors from PDEδ and finally reduces their antitumor efficacy.109

In 2019, Waldmann group reported the development of degraders based on PDEδ inhibitor Deltasonamide and thalidomide.110 The degrader 58 (Fig. 16) could efficiently and selectively induce the degradation of PDEδ with DC50 of 48 nM and Dmax of 83.4% in Panc Tu-I cells. The application of the PDEδ degrader 58 increased sterol regulatory element binding protein (SREBP)-mediated gene expression of enzymes involved in lipid metabolism, resulting in elevated levels of cholesterol precursors. It demonstrated that PDEδ function played a role in the enzymatic regulation of the mevalonate pathway.

The representative PROTACs targeting PDEδ

In 2020, Sheng group developed a series of potent PDEδ degraders by connecting PDEδ inhibitor Deltazinone and pomalidomide.111 The most promising degrader 59 (Fig. 16) efficiently induced the degradation of PDEδ with the DC50 value of 3.6 μM, and exhibited significantly improved antiproliferative potency in KRAS-mutant SW480 cells. In addition, the degrader 59 also achieved significant tumor growth inhibition in the SW480 xenograft model. This approach offered an effective lead degrader for the treatment of KRAS-mutant cancer.

Src homology 2 domain-containing phosphatase 2 (SHP2) belongs to protein tyrosine phosphatase family.112 SHP2 mutations exist in most tumor cells. Moreover, this target has been confirmed to be related to a variety of signaling pathways. For example, in the RAS-ERK pathway, SHP2 acts as its upstream positive regulator, which promotes cancer cell proliferation by phosphorylating ERK. Therefore, the inhibition of SHP2 can inhibit the growth of cancer cells and induce apoptosis.113

In 2020, Wang group developed a SHP2 degrader 60 (SHP2-D26, Fig. 17) conjugated with the SHP2 inhibitor SHP099 and VHL ligand.114 The degrader 60 (SHP2-D26) could induce the rapid and efficient degradation of SHP2 protein in KYSE520 cells (DC50 = 6.0 nM) and MV4; 11 cells (DC50 = 2.6 nM), and was capable of reducing SHP2 protein levels by more than 95% in cancer cells. Compared to inhibitor SHP099, the degrader 60 (SHP2-D26) exhibited more than 30-folds of potent inhibition to cell growth in KYSE520 and MV4;11 cancer cell lines.

The representative PROTACs targeting SHP2

In 2020, Li group developed a novel SHP2 degrader 61 (SP4, Fig. 61) also conjugated with the SHP2 inhibitor SHP099 and CRBN ligand through a PEG linker.115 The degrader 61 (SP4) successfully induced the moderate degradation of SHP2 in HeLa cells with the Dmax about 40% at 500 nM after 24 h of treatment. At the same years, Zhou group developed another SHP2 degrader 62 (ZB-S-29, Fig. 17) conjugated with the potent and selective SHP2 inhibitor TNO155 and CRBN ligand.116 The degrader 62 (ZB-S-29) effectively induced the degradation of SHP2 protein in a time and dose-dependent manner with a DC50 of 6.02 nM in MV4;11 cells, and induced more than 90% SHP2 degradation at 500 nM after 24 h of treatment. Moreover, it exhibited significant cell proliferation inhibition in MV4;11 cells and induced apparent G1 phase arrest or apoptosis in a dose-dependent manner. Subsequently, another SHP2 degrader 63 (R1-5C, Fig. 17) developed by conjugating RMC-4550 with pomalidomide was reported.117 The degrader 63 (R1-5C) exhibited highly selective SHP2 degradation with low concentration, suppressed MAPK signaling, and inhibited cancer cell growth.

The serine/threonine kinase AKT is a central component of the phosphoinositide 3-kinase (PI3K) signaling cascade and a key regulator of critical cellular processes, including proliferation, survival, and metabolism. Hyperactivation of AKT, due to gain-of-function mutations or amplification of oncogenes (receptor tyrosine kinases and PI3K) or inactivation of tumor suppressor genes (PTEN, INPP4B, and PHLPP), is one of the most common molecular perturbations in cancer and promotes malignant phenotypes associated with tumor initiation and progression.118 Thus, AKT is an attractive therapeutic target.

In 2020, Toker group described a degrader 64 (INY-03-041, Fig. 18) by conjugating lenalidomide and the most advanced AKT inhibitor GDC-0068.119 The degrader 64 (INY-03-041) inhibited AKT1, AKT2, and AKT3 with the IC50 values of 2.0 nM, 6.8 nM, and 3.5 nM, respectively, while the IC50 values of GCD-0068 were 5.0 nM, 18.0 nM, and 8.0 nM, respectively. They found degrader 64 (INY-03-041) could induce the degradation of AKT1/2/3 in a dose-dependent manner and the maximal degradation activity was observed at 100 nM to 250 nM. Also they found degrader 64 (INY-03-041) had good antiproliferative activities in different cell lines, such as ZR-75-1 cells (GR50 = 16 nM). Besides, degrader 64 (INY-03-041) destabilized all the three AKT isoforms and reduced the downstream signaling effects even after degrader 64 (INY-03-041) was washed out. It suppressed cell proliferation more potently than GCD-0068, which indicated that it had potential therapeutic value for targeted degradation of AKT.

The representative PROTACs targeting AKT

In 2021, Jin group also designed the AKT degraders based on the structure of GDC-0068, which they coupled with different VHL and pomalidomide ligands to obtain VHL-based AKT degrader 65 (MS98, Fig. 18) and CRBN-based degrader 66 (MS170, Fig. 18).120 They found 65 (MS98) and 66 (MS170) showed AKT-degradation activities on BT474 cells with DC50 of 78 nM and 32 nM, respectively. They tested the inhibitory activity of the two degraders on different tumor cell lines, including BT474 cells, PC-3 cells and MDA-MB-468 cells. The results showed 65 (MS98) and 66 (MS170) both had inhibitory activity on these cell lines with GI50 were at the micromolar level. However, the degradation activities and growth inhibitory activities were both weaker than 64 (INY-03-041).

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase of the insulin receptor (IR) kinase subfamily. Fusion proteins of anaplastic lymphoma kinase (ALK) are emerging therapeutic targets for cancer and other human diseases, especially non-small cell lung cancer (NSCLC) and anaplastic large cell lymphoma(ALCL).121,122 So far, five ALK inhibitors, including Alectinib, Brigatinib, Ceritinib, Crizotinib, and Lorlatinib have been approved by the FDA for the treatment of ALK-positive NSCLC. Despite the initial response to these inhibitors, drug resistance was observed within 1–2 years in most patients partly due to acquired ALK-resistant mutations.123,124 Hence, novel therapeutic strategies are in demand to overcome drug resistance.

In 2019, the first multi-headed(several interconnected ligands for POI and E3 ubiquitin ligase) PROTAC was developed as a gold nanoparticle (GNP)-based drug delivery system for delivering PROTAC to target ALK.125 The degrader 67 (Cer/Pom-PEG@GNPs, Fig. 19) loaded with both Ceritinib and pomalidomide, and showed good stability in several media. 67 (Cer/Pom-PEG@GNPs) potently decreased the levels of ALK fusion proteins in a dose and time-dependent manner. And it specifically inhibited the proliferation of NCI-H2228 cells with IC50 of 4.8 µM. In comparison with small-molecule PROTACs, the new multi-headed PROTAC promoted the formation of coacervates of POIs/multi-headed PROTAC/E3 ubiquitin ligases, and POI and E3 ubiquitin ligase interacted through multidirectional ligands and a flexible linker, thereby avoiding the need for complicated structure optimization of PROTACs.

The representative PROTACs targeting ALK

In 2021, Li group also reported a new series of ALK degraders based on Ceritinib and pomalidomide.126 The degrader 68 (B3, Fig. 19) showed potent selective inhibitory activity to ALK (IC50 = 1.6 nM) and could decrease the level of ALK fusion protein in H3122 cells. Meanwhile, 68 (B3) showed better anticancer activity in vitro compared with Ceritinib in different cell lines and the antiproliferative activity to xenograft tumor model was acceptable. All the results demonstrated that the anticancer activities of 68 (B3) in vitro and in vivo were valuable for further investigation.

Apart from Ceritinib, Alectinib was also widely used in the design of ALK degraders. In 2020 and 2021, Jiang group reported several ALK degraders based on Alectinib and lenalidomide, such as 69 (SIAIS001, Fig. 19) and 70 (SIAIS091, Fig. 19).127 And they found these degraders had good ALK degradation activity in SR cells as the DC50 were 3.9 nM and 6.1 nM, and Dmax were 70.3% and 87.7%, respectively. At the same time, they also showed good anti-proliferation activity in SR cells, and the IC50 were 0.9 nM and 0.5 nM.

Subsequently, Xu group reported an ALK degrader 71 (Fig. 19) also based on Alectinib.128 The difference from Jiang group was that the E3 ligase ligand they used was pomalidomide, while Jiang group was lenalidomide. They found degrader 71 had the best ALK degradation activity, highest ALK binding affinity and best antiproliferative activity in such ALK-dependent cell lines as H3122 cells and Karpas 299 cells, whose DC50 were 27.4 nM, 116.5 nM and IC50 were 62 nM, 42 nM. The degrader 71 also had no obvious cytotoxicity in ALK fusion-negative cells. More importantly, it showed obvious antitumor proliferation activity in the xenograft mouse model.

Also in 2020, Jiang group reported the ALK degrader 72 (SIAIS117, Fig. 19),129 which was based on Brigatinib and VHL. It could not only induce the degradation of ALK protein, but also showed obvious antiproliferative activity on SR cells and H2228 cells, and the inhibitory activity was significantly better than that of the inhibitor Brigatinib. In addition, the degrader 72 (SIAIS117) could also induce the degradation of ALKG1202R mutant protein in vitro so it had potential anti-proliferation activity of small cell lung cancer. Then they replaced the VHL ligand with pomalidomide on the basis of 72 (SIAIS117) and obtained the degrader 73 (SIAIS164018, Fig. 19) with good degradation activity for ALK.130 It could not only induce the degradation of the wild-type ALK protein and ALKG1202R mutant protein, and even the EGFRL858R+T790M mutant protein. The degrader 73 (SIAIS164018) also had a strong inhibitory effect on the migration and invasion of a variety of tumor cells. Also, the kinase inhibition of 73 (SIAIS164018) was different from that of Brigatinib and it rearranged the kinase inhibition of Brigatinib.

In the BCL-2 family, overexpression of anti-apoptotic proteins including BCL-2, Bcl-xl, and myeloid cell 1 is a key sign of cancer partly evading apoptosis. B-cell lymphoma extra large (Bcl-xl) is a well-validated cancer target.131 Inhibition of these BCL-2 family proteins with small molecules has been widely studied as a cancer treatment strategy, resulting in the discovery of ABT-263 (BCL-2 and Bcl-xl dual inhibitor) and several selective Bcl-xl inhibitors as promising anticancer drug candidates.132 Although these inhibitors are useful for the treatment of certain hematological malignancies, such as CLL and AML, the on-target and dose-limiting thrombocytopenia induced by Bcl-xl inhibition has limited the clinical use of these inhibitors.133 Bcl-xl is mainly overexpressed in many solid tumor cells and leukemia cells, and its expression is highly correlated with resistance to cancer therapy. As one of the most important validated cancer targets without a safe and effective therapeutic, Bcl-xl needs more selective methods to inhibit its activity. E3 ligases are differentially expressed in tumor cells compared with normal tissues. Thus, a method that relies on PROTAC to induce protein degradation seems to perfectly overcome this problem.

Zhou and Zheng group has been devoted to the development of high-efficiency Bcl-xl degraders. They used different Bcl-xl inhibitors and E3 ligands to design and synthesize a large number of different Bcl-xl degraders, and obtained some Bcl-xl degraders with good degradation activity. In 2019, Zhou and Zheng group reported the first selective Bcl-xl degrader 74 (DT2216, Fig. 20) by coupling the toxicity ABT-263 with the VHL ligand to achieve efficient Bcl-xl degradation.134 The DC50 and Dmax were 63 nM and 90.8% in MOLT-4 T-cell acute lymphoblastic leukemia(T-ALL) cells. Since VHL was poorly expressed in platelets, degrader 74 (DT2216) was more potent against various Bcl-xl-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT-263 in vitro. In vivo, degrader 74 (DT2216), as a single drug or in combination with other chemotherapeutic drugs, can effectively inhibit the growth of several xenograft tumors without causing significant thrombocytopenia. These findings suggested that degrader 74 (DT2216) had greater clinical potential than ABT-263 or other Bcl-xl inhibitors.

The representative PROTACs targeting Bcl-xl

In addition to VHL, CRBN was also modestly expressed in platelets. Therefore, they replaced the VHL ligand with CRBN on the basis of DT2216 to obtain a series of degraders.135 In the degradation activity test, they found that the degrader 75 (XZ739, Fig. 20), a pomalidomide-dependent degrader to Bcl-xl, had the best degradation activity. It had a DC50 of 2.5 nM for the degradation activity of Bcl-xl in MOLT-4 T-ALL cells and had good antiproliferative activity against a variety of cells. In MOLT-4 T-ALL cells, its antiproliferative activity was 22-folds and the selectivity to human platelets was up to 120-folds than that of inhibitor ABT-263. Both the antiproliferative activity and the selectivity to platelets were significantly better than inhibitor ABT-263, thus showing great potential for application.

Furthermore, the group also introduced IAP ligands into the degraders in 2020.136 They found that the degrader 76 (Fig. 20) showed the best antiproliferative activity in MyLa 1929 cells with IC50 of 62 nM and it could efficiently induce the degradation of Bcl-xl protein in different cell lines, such as MyLa 1929 cells, A549 cells, MDA-MB-231 cells, SW620 cells, MeWo cells, SK-MEL-28 cells, and CHL-1 cells, which proved that it was feasible to replace CRBN and VHL with IAP ligands in the design of Bcl-xl degraders.

Subsequently, they still used ABT-263 and CRBN as ligands to design and synthesize the degrader 77 (PZ15527, Fig. 20).137 They found that the degrader could effectively induce the degradation of Bcl-xl protein in WI38 non-senescent cells (NCs) when the DC50 and Dmax were 46 nM and 96.2%, respectively. More importantly, it could effectively clear senescent cells and rejuvenate tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvements, Bcl-xl PROTACs have the potential to become a safer and more effective treatment than Bcl-xl inhibitors.

In 2021, on the basis of analyzing the binding mode of DT2216 and Bcl-xl protein, they connected the VHL ligand with ABT-263 from the methyl group of dimethylcyclohexene in ABT-263138 and finally found that the chiral compound 78 (PZ703b, Fig. 20) had the best degradation activity and cell growth inhibitory activity. In MOLT-4 and RS4;11 cells, the DC50 to Bcl-xl were 14.3 nM and 11.6 nM, and the IC50 were 15.9 nM and 11.3 nM, respectively. Its inhibitory activities were significantly better than the inhibitor ABT-263 and the degrader DT2216. It could not only induce the degradation of Bcl-xl, but also inhibit but not degrade BCL-2, which showed an unprecedented mixed dual-targeting mechanism in the PROTAC. They further found that PZ703b can form a stable {BCL-2:PROTAC:VCB} ternary complex in living cells, which may help PZ703b enhance its inhibitory effect on BCL-2.

In the same year, they also developed a pomalidomide-based degrader 79 (XZ424, Fig. 20)139 for Bcl-xl degradation by conjugating a potent and selective Bcl-xl inhibitor A-1155463. The degrader 79 (XZ424) selectively induced Bcl-xl protein degradation in a dose and time-dependent manner in MOLT-4 cells but not in platelets. The DC50 was 50 nM and the IC50 was 51 nM. Subsequently, they confirmed that the platelet-toxic Bcl-xl/2 dual inhibitor ABT-263 can be converted into platelet-sparing pomalidomide-based Bcl-xl specific PROTAC without reduction in activity or selectivity.

In addition to Zhou and Zheng group, Benowitz group also reported a degrader 80 (Fig. 20) based on A-1155463 and VHL ligand to target the degradation of Bcl-xl protein in 2020.140 They found that the degrader 80 can be used in THP-1 to induce the degradation of Bcl-xl protein efficiently. And the 1.9 Å heterotetramer structure composed of (ElonginB:ElonginC:VHL):PROTAC:Bcl-xl revealed the interaction between E3 ligase and target protein and PROTAC. The mode of action between the homologous part and the partner protein provided ideas for the subsequent design of protein–protein interaction inhibitors and degraders.

Then, Zhou and Zheng group obtained a series of degraders based on DT2216 and the binding mode of ABT-263 in Bcl-xl, which was linked the VHL to the methyl group of dimethylcyclohexene in ABT-263.141 They found 81 (753b, Fig. 20) was a better dual-target degrader targeting Bcl-xl and BCL-2, it could not only induce the degradation of Bcl-xl(DC50 = 6 nM), but also degrade BCL-2 (DC50 = 48 nM) in 293T cells. It was the first dual-targeted degrader for Bcl-xl/BCL-2. In addition, they also found 81 (753b) exhibited more potent antitumor activity than DT2216 in Kasumi-1 cells, which was also superior to that of ABT-263.

Through the comparative analysis of the degradation activity and inhibitory activity of these degraders, it was found that the degradation activities of the degraders were better, the inhibitory activities were better in most cells. But in some special cell lines, such as 293T and THP-1 cells, it was also reported that the degradation activities had no obvious influences with the inhibitory activities. The main reason may be that some cells were not sensitive to the changes of Bcl-xl protein.

Finally, we compared the reported Bcl-xl degraders (Table 3). It was found that there were two types of Bcl-xl warheads currently in Bcl-xl PROTACs design, ABT-263 and A-1155463. In the selection of E3 ligases, CRBN and VHL were currently used, and other E3 ligases were not used in Bcl-xl degrades. Therefore, more Bcl-xl degraders based on different Bcl-xl inhibitors need to be developed.

Chronic myelogenous leukemia(CML) is most often caused by the lack of autoinhibition of the c-ABL kinase domain in the oncogenic fusion protein BCR-ABL.142 When the ABL gene is translocated from chromosome 9 to the BCR gene on chromosome 22, BCR-ABL is generated. BCR-ABL activates downstream signaling pathways to cause CML cell proliferation disorder in patients.143 At present, three generations of BCR-ABL inhibitors have been approved for the clinical treatment of CML. As the first-generation ABL inhibitor, Imatinib becomes the paradigm for targeted cancer therapy. But the intolerance and drug resistance of Imatinib, especially for T315I, limits its clinical application. The second-generation (Nilotinib, Dasatinib, and Bosutinib) and third-generation (Ponatinib) ABL inhibitors provide multiple options for resistance patients. According to the difference between the inhibitors and the protein binding sites, ABL inhibitors also can be divided into five categories (type I–V), among which type I (such as Dasatinib), type II (Imatinib and Ponatinib), and type IV (Asciminib) have been paied more attention. However, these inhibitors could not inhibit all resistant mutants; severe side effects also limit their clinical application.144,145 Therefore, it seems that the degradation of BCR-ABL may overcome these problems.

In 2016, Crews group reported the first BCR-ABL PROTAC based on Dasatinib, but it only achieved the degradation of BCR-ABL at micromoles (>60% at 1 µM), but could not overcome the common drug-resistant mutants, especially for T315I mutant.146 In 2019, Crews group designed and synthesized a series of VHL-based BCR-ABL degraders by using the allosteric GNF family compounds of BCR-ABL.147 They optimized the linker portion of the degraders to improve both potency and cell permeability, obtaining the lead degrader 82 (GMB-475, Fig. 21). The degrader induced rapid proteasomal degradation and inhibition of downstream biomarkers in both human CML K562 cells and murine Ba/F3 cells. Besides, it inhibited the proliferation of BCL-ABL mutant Ba/F3 cells (T315I and G250E) more effectively than inhibitor Imatinib. Scaffold hopping is one of the common methods for structural modification of drugs in medicinal chemistry, which is a method to obtain novel core scaffold by changing the core structure of known active compounds. Subsequently, they employed a scaffold hopping approach to enhance the activity of GMB-475 to obtain the degrader 83 (GMB-805, Fig. 21).148 The new BCL-ABL degrader 83 (GMB-805) demonstrated more than ten-folds increase in ability to induce BCL-ABL degradation, and improved pharmacokinetic properties and in vivo activity.

The representative PROTACs targeting BCR-ABL

In 2021, Rao group developed some BCR-ABL degraders targeting all the three binding sites of BCR-ABL.149 These BCL-ABL degraders were designed and synthesized using four BCR-ABL inhibitors Imatinib, Dasatinib, Asciminib, and Ponatinib as target molecules. In this toolbox for inducing degradation of BCR-ABL from different binding pockets, the degraders designed on the other three BCR-ABL inhibitors all worked obviously well except the Imatinib-based degraders. Among them, three representative degraders, 84 (P22D, based on Dasatinib, DC50 = 10 nM, Fig. 10), 85 (P19As, based on Asciminib, DC50 = 200 nM, Fig. 10), and 86 (P19P, based on Ponatinib, DC50 = 20 nM, Fig. 10) exhibited effective degradation activity for wild-type BCL-ABL, and also showed good cytostatic activity. More importantly, 86 (P19P) could efficiently induce the degradation of wild-type and mutated BCR-ABL (Dasatinib-resistant T315I, Asciminib-resistant V468F mutants, E255K and H396R) in transfected HeLa cells without causing serious side effects. And it also exhibited better antiproliferative activity against T315I mutant BaF3 cell line, with EC50 of 28.5 nM. Through the above studies, it could found that PROTACs designed based on the type I inhibitor Dasatinib, the type II inhibitor Bonatinib and the type IV inhibitor Asciminib can successfully induce the degradation of the BCR-ABL protein. The degradation activities of PROTACs designed based on Dasatinib were significantly better than that of other PROTACs, while the PROTACs designed based on type II inhibitor Imatinib could not induce the degradation of BCR-ABL protein under the concentration of 30 µM. It indicated that the degraders based on type II inhibitors can be designed to induce the degradation of BCR-ABL protein, but it requires more careful consideration that the design of degraders based on Imatinib to induce the degradation of BCR-ABL. In summary, these PROTACs showed better selectivity and fewer adverse reactions than inhibitors, which indicated that PROTACs have great potential in overcoming the clinical resistance and safety issues of BCR-ABL.

Recently, Lu group described a new class of selective BCR-ABLT315I degraders based on a BCR-ABLT315I inhibitor GZD824.150 The degrader 87 (Fig. 21) exhibited the most potent degradation efficacy with DR of 69.89% and 94.23% at 100 and 300 nM, respectively. In addition, the degrader 87 had an IC50 value of 26.8 nM against Ba/F3T315I cells and also showed significant tumor regression in this mutation xenograft model in vivo.

In 2019, Jiang group reported the degrader 88 (SIAIS178, Fig. 21) by connecting Dasatinib and VHL by extensive optimization of linkers.151 The degrader 88 (SIAIS178) induced the effective degradation of wild-type BCR-ABL with the DC50 value of 8.1 nM in K562 cells, and several clinically relevant resistance-conferring mutations. Moreover, it achieved significant growth inhibition of the BCR-ABL+ leukemic cells in vitro, and induced substantial tumor regression in vivo against K562 xenograft tumors. Subsequently, Jiang group synthesized a series of CRBN-based degraders by conjugating Dasatinib to pomalidomide or lenalidomide.152 As an important example, a pomalidomide-based degrader 89 (SIAIS056, Fig. 21), possessing sulfur-substituted carbon chain linker, exhibited the potent degradation of wild-type and clinically relevant resistance-conferring mutations of BCR-ABL. Furthermore, the degrader 89 (SIAIS056), with favorable pharmacokinetics, induced significant tumor regression against K562 xenograft tumors in vivo. In addition, they reported a highly efficient protocol to construct a new IMiD-based azide library through click reaction. The degraders 90 (SIAIS629050, Fig. 21) and 91 (SIAIS629051, Fig. 21) showed good antiproliferative activity with IC50 of 1.5 nM and 2.9 nM in K562 cells, and exhibited potent degradation activity of wild-type BCR-ABL in a dose-dependent manner. This approach provided help for the rapid construction of degraders libraries.

Some examples reported recently have further enriched the tools for PROTAC-induced degradation of BCR-ABL. Based on the natural product Nimbolide, a covalent recruiter for the E3 ligase RNF114, Nomura group developed a novel PROTAC linking Nimbolide to Dasatinib to obtain the degrader 92 (BT1, Fig. 21), which could selectively induce the degradation of BCR-ABL over c-ABL in leukemia cancer cells.153 Compared with the previously reported cereblon or VHL-recruiting BCR-ABL degraders, it showed the unique degradation specificity profiles. These achievements pave a way to develop more drug-like PROTACs for degrading BCR-ABL in the future. In 2020, Jiang group also developed a novel photo-switchable azobenzene-PROTAC 93 (Azo-PROTAC-4C, Fig. 21).154 They could control the degradation of ABL and BCR-ABL proteins in live cells by changing the configuration of Azo-PROTAC with UV-C light.

Finally, we compared the reported BCR-ABL degraders (Table 4). It was found that there were various types of BCR-ABL warheads currently in BCR-ABL PROTACs design, but Dasatinib was still the most frequently used inhibitor. In the selection of E3 ligases, CRBN and VHL were currently used, and other E3 ligase such as RNF114 were also used in BCR-ABL degraders.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase, which is a member of the focal adhesion complex family.155,156 It mediates multiple signaling pathways, such as PI3K/AKT and RAS/MAPK, and also plays an important role in cell invasion and metastasis. FAK exerts kinase-dependent enzyme function and kinase-independent scaffold function which can't be investigated with kinase inhibitors.157 The emergence of PROTACs technology opens a new door for studying the non-enzymatic function of FAK.

In 2020, Gray group published the patent on FAK degraders.158 The patent showed that they designed a large number of FAK degraders based on FAK inhibitor VS-4718 and different E3 ligase ligands. In the subsequent activity screening, they found that most of the compounds have good FAK-degradation activity. Among them, the degrader 94 (Fig. 22) had relatively potent FAK-degradation activity, which could induce degradation of more than 85% FAK protein at 10 nM.

The representative PROTACs targeting FAK

Recently, GSK has also developed a new type of potent and selective degrader 95 (GSK-215, Fig. 22) based on VS-4718 and VHL ligand.159 Interestingly, it was confirmed that degrader 95 (GSK-215) which possessed a short and rigid linker generated a highly cooperative ternary complex by SPR and X-ray crystallography data. It induced the degradation of FAK with DC50 of 1.3 nM in A549 cells and induced cell proliferation inhibition in A549, MCF-7 cells rather than BT474 cells. The levels of other proteins would also be affected when the concentration was increased to 100 nM, such as CDK7, RPS6KA3, MET, and GAK. In addition, the degrader 95 (GSK-215) induced fast, effective, and durable FAK degradation in vivo in mouse liver.

p53 is an important tumor suppressor which can promote the apoptosis of cancer cells and prevent the development of tumors. Nearly 50% of human cancers are related to the abnormal activity of p53. The interaction between p53 and MDM2 is the main factor affecting the biological activity of p53.160 MDM2 is one of the key inhibitors of p53. It is highly expressed in a variety of tumors and plays an important role in the occurrence and development of tumors. Overexpression of MDM2 can downregulate the expression of p53, and inhibiting or degrading the MDM2 protein can block the MDM2–p53 interaction and upregulate the expression of p53, thereby exerting antitumor activity.161 Therefore, the development of antitumor drugs targeting MDM2–p53 has become one of the important methods to treat tumors. Although several MDM2–p53 inhibitors have entered clinical trials, no drugs have been approved for clinical use.

In 2018, Wang group reported the potent MDM2 degrader MD-224. Based on the structure of MD-224, they designed and synthesized other MD-224 analogs in 2019. They found that the degrader 96 (MG-277, Fig. 23) was not only a degrader for MDM2, but also a molecular glue for inducing degradation of GSPT1.162 The degrader 96 (MG-277) only induced moderate degradation of MDM2, but had very good degradation activity of GSPT1 with DC50 of 1.3 nM. It could not activate wild-type p53, but it had good antiproliferative activity in a variety of cells. For example, in RS4;11 and HL-60 cells, its IC50 were 3.5 nM and 8.3 nM, respectively. Subsequently, they also tested the antiproliferative activity of the degrader 96 (MG-277) in MDA-MB-231 siMDM2 and MDA-MB-468 siMDM2 cell lines, which IC50 were 19.3 nM and 19.8 nM, respectively, indicating that the degrader 96 (MG-277) exerted antiproliferative activity not only through the MDM2-p53 pathway, but also played a related physiological role through the degradation of GSPT1.

The representative PROTACs targeting MDM2

In 2021, Wang group designed and synthesized a series of degraders based on ursolic acid(UA) and pomalidomide.163 During the screening process, they found that the degrader 97 (1B, Fig. 23) had significant inhibitory activity in different tumor cells. The IC50 values in A549, Huh7, and HepG2 were 230, 390, and 380 nM, respectively. Then they conducted mechanism studies and the western blotting results showed that the degrader 97 (1B) induced the significant degradation of MDM2 and promoted the expression of P21 and PUMA proteins thereby inhibiting the proliferation of tumor cells and promoting the apoptosis of A549 cells. This was the first research on MDM2 degraders designed based on natural products, which provided ideas for the development of MDM2 degraders.

In 2021, Tang group reported a series of MDM2 ligands which were designed and synthesized based on the four-component Ugi reaction, and then synthesized MDM2 degraders based on these ligands.164 After extensive optimization based on antiproliferative activity and MDM2-degradation activity, the degrader 98 (WB214, Fig. 23) was determined to be the degrader with the best MDM2-degradation activity in leukemia cells. In RS4;11 cells, the MDM2-degradation activity DC50 was 4.1 nM. In addition, they also found that the degrader 98 (WB214) also induced the degradation of p53 with DC50 of 29 nM. Further studies have shown that it was a molecular glue that induced the degradation of MDM2. At the same time, it could effectively induce the degradation of GSPT1 and showed strong proliferation inhibitory activity on cells. In RS4;11 cells, its inhibitory activity IC50 was 1.2 nM.

Also in 2021, Sheng group reported the homo-PROTAC designed based on MDM2 inhibitor Nutlin-3 derivatives targeted degradation of MDM2.165 Since MDM2 was an E3 ligase, when MDM2 inhibitors were used as ligands, they could not only target MDM2, but also could combine with E3 ligase. The results showed that the degrader 99 (11a, Fig. 23) could effectively induce the dimerization of MDM2 with highly competitive binding activity and induce the degradation of MDM2 protein in A549 cells. The DC50 and Dmax were 1.01 µM and 95%, respectively. At the same time, it showed good antiproliferative activity on a variety of tumor cells. However, because the degrader 99 (11a) was a compound with chiral centers, they purified enantiomer 100 (11a-1, Fig. 23) and found that its antiproliferative activities in tumor cells were significantly better than that of degrader 99 (11a). It was found enantiomer 100 (11a-1) showed strong antitumor activity in vivo in the A549 xenograft nude mouse model.

FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that regulates hematopoiesis. It is expressed on the surface of many hematopoietic cells and is essential for the normal development of hematopoietic stem cells and hematopoietic cells. After FLT3 binds to ligands, it will dimerize or autophosphorylate and activate JAK-STAT, PI3K, and MAPK signaling pathways, which can promote tumor cell proliferation and differentiation or inhibit tumor cell apoptosis.166 FLT3 is expressed in most AML patients and exists 30% mutations. The mutations of FLT3 mainly include internal tandem duplication alteration(FLT3-ITD) and point mutations in the tyrosine kinase domain, accounting for 25% and 5%, respectively. Mutation or high expression of FLT3 may cause the continuous activation of the protein, leading to acute myeloid leukemia and acute lymphocytic leukemia.167 There are currently eight drugs that can act on FLT3 have been approved, but these drugs have poor selectivity and can cause gastrointestinal intolerance, long-term cytopenias, hand-foot syndrome and other side effects.168 In addition, the single drug has limited efficacy in AML patients with FLT3 mutations. So it is particularly important to use new technologies to develop treatment targeting FLT3 and mutations.

In 2021, Yang group obtained a series of PROTACs based on the binding model of Dovitinib and FLT3.169 After screening in vitro antiproliferative activity, it was found that the degrader 101 (Fig. 24) and 102 (Fig. 24) had significant antiproliferative effects on MOLM-13 and MV4-11 cells (FLT3-ITD-positive AML cells), which were better than the inhibitor Dovitinib. Subsequently, they tested the degradation activities of these two degraders on FLT3 in MOLM-13 and MV4-11 cells, and the results showed that both the two degraders could induce the efficient degradation of FLT3 protein. The DC50 of the degrader 101 to FLT3 in MOLM-13 and MV4-11 cells were 8.0 nM and 6.9 nM, respectively. While the DC50 of the degrader 102 in these two cells were 311 nM and 150 nM, respectively. In addition, they also proved that these two degraders completely block the downstream signaling pathways at low concentrations. And in the mouse model of vein transplantation, the degrader 101 and 102 could also show a significant inhibitory effect on the proliferation of MV4-11 cells in vivo.

The representative PROTACs targeting FLT3

Also in 2021, Chen group also reported the FLT3 degrader 103 (PF15, Fig. 24).170 They found 103 (PF15) could induce the degradation of FLT3 in BaF3-FLT3-ITD cells with DC50 of 76.7 nM.

Janus kinase (JAK) is a family of non-receptor tyrosine kinases, including JAK1, JAK2, JAK3, and Tyk2. Signal and activator of transcription (STAT), the substrate of JAK, dimerizes after being phosphorylated by JAK and then crosses the nuclear membrane into the nucleus to regulate the expression of related genes. JAK-STAT pathway is a major signal transduction mechanism of various cytokines and growth factors and has been implicated in a multitude of diseases from cancer to inflammatory diseases.171 Given the importance of the JAK-STAT pathway, blocking the function of JAK can silence the entire pathway, which has important implications for scientific research and disease treatment. Although several JAK kinase inhibitors have entered clinical research, they are usually difficult to achieve selectivity due to the high homology of JAK family proteins.172 Therefore, the rise of PROTACs technology provides a new strategy for the study of JAK proteins.

In 2020, GSK developed the first-in-class PROTACs targeting JAK proteins based on two pan-inhibitors which have pyrimidine and quinoxaline scaffold, respectively.173 They then tested the degradation activities of these degraders to endogenous JAK1 and JAK2 in THP-1 cells through automated western blotting. They found that the degraders 104–109 (J1-J6, Fig. 25) were able to induce the degradation of JAK1 and JAK2, which showed no selective degradation. And JAK3 protein was not detected in these cells. The results also proved that the PROTACs bearing an IAP E3 ligase ligand could induce significant degradation of JAK1 and JAK2 in THP-1 cells while VHL and CRBN-based PROTACs could not.

The representative PROTACs targeting JAK

In 2021, Mullighan group designed and synthesized a large number of PROTACs based on the binding modes of Ruxotinib and Barictinib with JAK protein.174 Through protein degradation and MTT experiment screening, it was found that most of the compounds had good JAK2 kinase degradation activity, but they also had certain GSPT1 degradation activity, especially the degrader 110 (Fig. 25) and 111 (Fig. 25). In order to minimize the molecular weight and polar surface area of PROTAC and improve its cell permeability, they also synthesized the N-propyl analog degrader 112 (Fig. 25). And in order to avoid GSPT1 degradation, degrader 113 (Fig. 39) was also designed and synthesized. The degradation activities of these degraders were tested on MHH-CALL-4 cells. They found that the degrader 110 and 111 could induce the degradation of JAK1/2/3, and also had good degradation activity on GSPT1/IKZF1. The degrader 112 could induce the degradation of JAK1/3 and also had good degradation activity for GSPT1/IKZF1, while the degrader 113 could only induce the degradation of JAK2.

Subsequently, they tested the antiproliferative activity of these degraders. They found that the activity of the degraders was better than that of the corresponding inhibitors. At the same time, the activity of the degrader 113 with better selectivity was significantly worse than that of degrader 110–112, which indicated that the antiproliferative activity was not only due to Janus kinase degradation. Finally, they tested the inhibitory activity of degraders in ALL tumor cells derived from different patients. These cells were in different genetic recombination backgrounds, including CRLF2r, EPORr, JAK2r, IL7R/SH2B3 mutations, and so on. Most tumors were sensitive to degraders but not sensitive to the corresponding inhibitors, and the inhibitory activities of the degrader 113 were relatively poor.

Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family of transcription factors that replicates and transmits signals from cell surface receptors to the nucleus.175 The continuous activation of STAT3 is often associated with the poor prognosis of human cancer because the activated STAT3 signal can not only promote the growth, survival, and metastasis of tumor cells but also inhibit the antitumor immune response.176 Therefore, STAT3 is an attractive target for the treatment of human cancer and other human diseases. Although scientists have been working tirelessly on this target for 20 years, targeting STAT3 is still very challenging.

In 2021, Wang group developed a new STAT3 degrader 114 (SD-91, Fig. 26),177 which was generated by the gem-difluoride of a previously reported STAT3 degrader SD-36178 convert into a ketone. The degrader SD-36 could convert into degrader 114 (SD-91) in the cell culture media rapidly, with nearly 50% of conversion observed at 2 h and 90% of conversion at 24 h. However, the degrader 114 (SD-91) exhibited excellent stability in the cell culture media and the in vivo dosing vehicle. The degrader 114 (SD-91) bound to STAT3 protein with a high affinity, and was more potent than SD-36 in inducing degradation of STAT3 in SU-DHL-1 and MOLM-16 lymphoma cells. A single administration of the degrader 114 (SD-91) could selectively and continuously reduce STAT3 in tumor tissues. Moreover, the degrader 114 (SD-91) achieved complete and long-lasting tumor regression in the MOLM-16 xenograft model in mice even with weekly administration. The degrader 114 (SD-91) represented a promising STAT3 degrader for the treatment of human cancers and other diseases related to STAT3 overactivation.

The representative PROTAC targeting STAT3

Among many signal pathways, the aberrant activation of Wnt/β-catenin signal is the most important for the occurrence and development of various cancer.179 More than 80% of cancer patients have inactivating mutations of APC or activating mutations of CTNNB1, leading to the messenger molecule β-catenin protein continuing to accumulate in cells, which eventually leads to the excessive activation of Wnt/β-catenin signal and the proliferation of cancer cells.180 As β-catenin is the central player of the canonical Wnt/β-catenin signaling and is frequently mutated in cancers, it is the most attractive target for cancer therapy.181 However, β-catenin has no enzymatic activity and small-molecule binding pockets, and has a large interaction interface with other proteins. Therefore, it is difficult for such small molecule inhibitors to completely inhibit the function of β-catenin.182 Thus, inducing the degradation of β-catenin is an ideal method for treating Wnt/β-catenin signal-related diseases.

Based on the Axin-derived peptide that binds to β-catenin, a stapled peptides xStAx was reported to impair Wnt/β-catenin signaling.183 Recently, β-catenin-targeting degrader 115 (xStAx-VHLL, Fig. 27) was developed by coupling xStAx with the VHL ligand to achieve efficient β-catenin degradation.184 Although they had clinical limitations as peptide-based PROTACs, they still displayed long-term degradation of β-catenin and strong inhibition of Wnt/β-catenin signaling in cancer cells and in APC/organoids. Furthermore, the degrader 115 (xStAx-VHLL) could effectively restrain tumor formation in BALB/C nude mice and potently inhibit the survival of colorectal cancer patient-derived organoids. In the future, it would be worthwhile to develop a β-catenin-targeting degrader with good pharmacodynamics for clinical therapies.

The representative PROTAC targeting β-catenin

The forkhead box superfamily is a class of transcription factors with a specific "winged helix structure" DNA binding domain, including several families such as FOXA, FOXC, FOXM, FOXO, and FOXP.185 FOXM1 (forkhead box protein M1) is one of the key genes controlling cell proliferation and its abnormal activation is closely related to the proliferation and division of cancer cells. It is generally upregulated in laryngeal cancer, gastric cancer, ovarian cancer, etc.185,186,187,188 And tumor gene expression profiling analysis also confirmed that FOXM1 is one of the most frequently upregulated genes in human malignant tumors,189 so it has become an important target for drug development.

Xiang research group synthesized a potent PROTAC degrader 116 (Fig. 28) based on the binding mode of FOXM1 inhibitor FDI-6.190 They proved that degrader 116 could induce the degradation of FOXM1 protein in MDA-MB-231 cells. The DC50 was 1.96 µM and GI50 for cell proliferation inhibition was 16.2 µM, which was slightly better than the corresponding inhibitor. This was the first time to use PROTAC technology to induce the degradation of FOXM1.

The representative PROTAC targeting FOXM1

Adrenergic receptors (AR) are divided into two categories: α subtype and β subtype. α adrenergic receptor is a type of G-protein-coupled receptor, mainly including α1 and α2, which mediate mostly excitatory function. Among them, α1-ARs play an important role in many physiological processes, including smooth muscle contraction, myocardial time-varying force, liver glycogen metabolism and so on.191,192 α1-ARs are divided into three subtypes: α1A, α1B, and α1D. α1A-AR is crucial in maintaining basic blood pressure,193 α1B-AR is of significance in the regulation of blood pressure by catecholamines,194 and α1D-AR is important in both physiological responses.195 Therefore, α1-ARs are important targets for the treatment of hypertension,196 benign prostatic hypertrophy,197 prostate cancer198 and other diseases. Especially, due to the fact that the α1A-AR has a close relationship to the proliferation of prostate cancer cells.199,200 Therefore, the development of α1A-AR selective degrader can provide a new method for the treatment of prostate cancer.

In 2020, Li group developed the α1A-AR degrader 117 (Fig. 29) based on 4-amino-6,7-dimethoxy-2-(piperazin-l-yl)-quinazoline core of prazosin derivatives,201 which endured antagonism to α1-ARs. This was the first PROTAC for GPCRs. The PROTAC was conjugated with prazosin and pomalidomide through different linkers. These PROTACs induced the slight degradation of α1A-AR in α1A-AR stably transfected HEK293 cells. And compound 117 showed the best degradation activity with the drug treatment for 12 h, and the DC50 value approximately was 2.86 µM in the treated HEK293 cells. For the HEK293 cells treated with 10 µM compound 117 for 12 h, the Dmax reached up to 94%. Then they test the antiproliferative activity of the compound 117 in androgen-independent PC-3 prostate cancer cells. They found that the antiproliferative activity of 117 (IC50 = 6.12 µM) was better than that of Prazosin (IC50 = 11.72 µM) in PC-3 cells. At last, they found compound 117 resulted in an inhibition in tumor growth when they conducted an in vivo study to examine its influence.

The representative PROTAC targeting α1A-AR

Lysine acetylation in histone tails has been associated with epigenetic. Bromodomian are highly conserved module which recognize acetylated lysine, it has been shown that 46 proteins contain 61 unique bromodomian so far.202 Most of them play important role in transcriptional regulation and chromatin remodeling and have been widely studied as a key target for the treatment of cancer.

The bromo and extral terminal domain family (BET) proteins, which include BRD2, BRD3, BRD4 and testis-specific BRDT, are the most widely studied class of bromodomain-containing proteins. In recent years, a large number of inhibitors targeting BET proteins have been reported,203,204,205,206,207,208 most of them showed good tumor suppression and partial compounds enter clinical studies successfully. Since the first degrader dBET1 was reported by Brander group in 2015,2 a number of PROTACs targeting BET proteins have been developed. However, due to the high homology of BET proteins, it is still difficult to achieve the selectivity of BET protein subtypes.

Since Polo-like kinase 1(PLK1) and bromodomain 4(BRD4) are both attractive therapeutic targets in acute myeloid leukemia (AML), Lu group developed BRD4 and PLK1 degrader 118 (HBL-4, Fig. 30) based on a dual-target inhibitor BI2536.209 The degrader 118 (HBL-4) induced efficient and fast degradation in human acute leukemia cells tested in vitro and vivo, such as MV4-11, MOLM-13, and KG1. It exhibited potent anti-proliferation in these cells and more effectively suppresses c-Myc levels than inhibitor BI2536. Meanwhile, the degrader 118 (HBL-4) induced dramatically improved efficacy in the MV4-11 tumor xenograft model compared with BI2536, which may be a potential therapy for the treatment of acute leukemia and other types of tumors.

The representative PROTACs targeting BRD

In 2019, Ciulli group introduced macrocyclization into PROTACs based on the structure of reported BET degrader MZ1. Macrocyclization could restrict the molecule in its bioactive conformation to reduce the energetic penalty, which has been widely used as a strategy to develop drug selectivity. They synthesized the degrader 119 (MacPROTAC-1, Fig. 30) which induced more obvious affinity discrimination between BD1 and BD2 to the BET degrader MZ1 by adding a cyclizing linker and solved the co-crystal structure of VHL-119 (MacPROTAC-1)-BRD4 BD2.210 Compared with MZ1, the degrader 119 (MacPROTAC-1) showed similar degradation activity and inhibition of cell proliferation in BET-sensitive human prostate carcinoma 22RV1 cells, although the binding affinity to BRD4 decreased.

Despite the pan-inhibitors or degraders targeting BET proteins have achieved great success in treating tumors, lack of subtybe selectivity limits their applications as biological tools and anticancer drugs which could lead unwanted side effects or toxicity. In 2020, Chen group reported degrader 120 (Fig. 30) which achieved potently and selectivity for BRD2 and BRD4 over BRD3 derived from a BD1 selective inhibitor and thalidomide.211 The degrader 120 completely induced the degradation of BRD4 at 1 μM with 8h treatment and effectively inhibited cell growth with low cytotoxic effect in both hematoma and solid tumor cells, especially for acute myeloid lymphoma cells MV4-11.

Recently, Ciulli group designed trivalent PROTACs consisting of a bivalent BET inhibitor and an E3 ligand tethered via a branched linker.212 Compared to bivalent PROTACs, the most active degrader 121 (SM1, Fig. 30), a VHL-based trivalent degrader, displayed more sustained and higher degradation efficacy for BET family proteins which led to more potent anticancer activity. Mechanistically, the degrader 121 (SM1) intramolecularly engaged BD2 and BD1 to form a 1:1:1 ternary complex with VHL and BRD4 with prolonged residence time. Although the molecular weight of the trivalent degrader had increased, the degrader 121 (SM1) exhibited better cell permeability and the remarkably favorable PK profile.

Besides several common structures such as JQ1, more and more BET inhibitors have been used in the design and synthesis of PROTACs. In 2019, Zhang group developed a new class of degrader based on a potent dihydroquinazolinone-based BRD4 inhibitor.213 Among them, the best degrader 122 (Fig. 30) completely induced the degradation of BRD4 at 1 μM with 3h treatment and inhibited cell growth with an IC50 of 0.81 μM in THP-1 cells, which was four-folds more potent than inhibitor in the antiproliferative assay.

Subsequently, ABBV-075 derivative was used to design PROTACs by Zhang group214 and Yu group215 successively. In 2020, Zhang group introduced linkers at the position of pyrrole ring to obtain the degrader 123 (Fig. 30) which induced the degradation of BRD4, cell cycle arrest and apoptosis effectively in human pancreatic cancer cell line BxPC-3. Antiproliferative activity of the degrader 123 against BxPC-3 cell line (IC50 = 0.165 μM) was improved about seven-folds compared with ABBV-075. In 2021, the sulfonyl group of ABBV-075 was removed to connect with E3 by Yu group and led to the discovery of the degrader 124 (Fig. 30) which induced the degradation of BRD4 with DC50 of 0.25 nM and 3.15 nM in MV4-11 cells and RS4-11 cells. It also induced the proliferation inhibition in MV4-11 and RS4-11 cells with IC50 of 0.5 nM and 4.8 nM, respectively.

Given the important role of BET protein in cells, PROTACs targeting BET will inevitably cause toxic effects on normal cells, which limit the clinical application of BET degraders. So there are some strategies were adopted to make PROTACs regulated in space and time.

The introduction of a photocaged group into PROTACs is a common method. In 2019, Pan group introduced the bulky 4,5-dimethoxy-2-nitrobenzyl (DMNB) group into dBET1 through the amide nitrogen of the linker to get the degrader 125 (pc-PROTAC1, Fig. 30) which induced the degradation of BRD4 in live cells and the expected changes of phenotypic in zebrafish only after light irradiation.216 As known that methylation of the imide nitrogen abolished degradation activity of a CRBN-based BRD4 degrader, Li group also installed DMNB groups on the glutarimide nitrogen of dBET1 to obtain the degrader 126 (N2, Fig. 30), which was proved to induce the degradation of BRD4 in HEK293T cells and inhibit tongue squamous cell carcinoma(TSCC) HN-6 cells growth in a zebrafish xenograft model.217 Subsequently, Deiters group successfully installed a 6-nitropiperonloxymethyl (NPOM) group into the glutarimide nitrogen to generate the BRD4 degrader 127 (Fig. 30), which induced the potent degradation of BRD4 by treatment with 365 nm light in HEK293T cells.218 And the DMNB group was attached to the hydroxyl group of VHL E3 ligase-recruiting ligand by Tate group in 2020.219 The degrader 128 (Fig. 30) could induce the degradation of BRD4 after a short irradiation time under the live-cell video microscopy.

It is well-known that more than 600 E3 ubiquitin ligases are encoded by the human genome, only a handful of E3 ligases(VHL, CRBN, IAPs, and MDM2) could be used by PROTACs technology. BRD4 is a commonly used target for screening new E3 ligases for PROTACs. In 2019, Cravatt group has validated that the E3 ligase subunit DCAF16 supported targeted protein degradation by synthesizing degrader 129 (KB02-JQ1, Fig. 30) which induced the degradation of BRD4 with the engagement of DCAF16.220 However, KB02-JQ1 could only induce the degradation of BRD4 at a concentration of 40 μM. In 2020, Chen group also developed a BRD4 degrader 130 (DP1, Fig. 30) based on JQ1 and DCAF15 ligand E7820.221 The results showed that 130 (DP1) was the most potent degrader with DC50 of 10.84 μM and Dmax of 98% in SU-DHL-4 cells. Recently, Jin group synthesized the degrader 131 (MS83, Fig. 30) by connecting a highly potent, selective, and noncovalent KEAP1 ligand KI696 to the BET bromodomain pan-inhibitor (+)-JQ1.222 Interestingly, the degrader effectively induced the degradation of BRD4 and BRD3 protein levels, but did not have an influence on BRD2 protein and selectively induced the degradation of BRD4 short isoform over long isoform in MDA-MB-231 cells. The CRBN-based PROTACs are known to be inherently unstable, readily undergoing hydrolysis in body fluids, which significantly affects their cell efficacy. Recently, Rankovic group developed novel CRBN binders, phenyl glutarimide (PG) analogs, which retained high affinity for CRBN and displayed improved chemical stability.223 Based on this novel ligand, their group synthesized a novel BRD4 degrader 132 (SJ995973, Fig. 30) which inhibited the MV4-11 cells proliferation with an IC50 value of 3 pM. Moreover, the degrader 132 (SJ995973) exhibited the most potent degradation efficacy with DC50 of 0.87 nM in MV4-11 cells.

In 2021, Fischer group developed a series of novel CRBN-based BRD4BD1L94V PROTAC based on a "bump-and-hole" approach.224 By using cells stably expressing the BRD4BD1L94V degron fused to EGFP systems, the corresponding degrader 133 (XY-06-007, Fig. 30) bound to BRD4BD1L94V with increased selectivity over the wild-type bromodomain, and exhibited potent and selectively degradation of BRD4BD1L94V with no degradation of wild-type and other BET family of bromodomains. In addition, the degrader 133 (XY-06-007) displayed acceptable pharmacokinetics profiles, which made it suitable candidates for future in vivo studies. In recently, Ciulli group also developed a novel VHL-based degrader 134 (AGB1, Fig. 30) based on the "bump-and-hole" approach.225 In an inducible BromoTag degron system, the degrader 134 (AGB1) not only formed a strong, cooperative ternary complex between VHL and the BromoTag-BRD2 but also completely induced the degradation of BromoTagged target proteins with low nanomolar potency. The degrader 134 (AGB1) exhibited exquisite selectivity over the native wild-type BET, which led it not cytotoxic in several cancer relevant cell lines. In summary, these two distinct methods provided a useful tool to study the effect and implications of rapid and highly selective degradation of a target protein.

Also in 2021, Jiang group reported the efficient construction of an IMiD-based azide compound library and applied this method to the development of BET protein degraders. The BET protein degraders 135 (SIAIS629048, Fig. 30) and 136 (SIAIS629049, Fig. 30) were obtained.152 These two degraders showed strong BET protein-degradation activity when the concentration was 50 nM. At the same, it could show strong anti-proliferation inhibitory activity on MV4-11 cells. Recently, Wang group reported the selective BRD4 degrader 137 (WWL0245, Fig. 30) with potent antiproliferative effects in AR-positive prostate cancer based on a dual BET/PLK1 inhibitor WNY0824226 The degrader 137 (WWL0245) could effectively induce the degradation of BRD4 and the Dmax was more than 99% in AR-positive prostate cancer cells. It had no degradation activity against other BRD subtype proteins. The degrader 137 (WWL0245) also showed good antiproliferative activity in BETi-sensitive cancer cells (including AR-positive prostate cancer cells) with an IC50 of 3 nM in MV4-11 cells.

Finally, we compared the reported BRD degraders (Table 5). It was found that although there were various types of BRD warheads currently used, JQ1 was used more frequently and others were used less. In the selection of E3 ligases, most of the currently used were CRBN and VHL, and some new E3 ligases have been mentioned in reports, such as DCAF15, DCAF16, etc. These cases were all in the development of new E3 ligases reportes. Therefore, more BRD degraders based on different BRD inhibitors need to be developed.

The paralogous chromatin regulators CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through chromatin lysine acetylation and transcription regulation, as well as the scaffold function mediated by multiple protein–protein interaction domains. Several potency, selectivity, and drug-like properties of small-molecule inhibitors targeting these domains have been developed, which provided unique tools for exploring various p300/CBP functions of enhancers.227 However, inhibiting a single domain alone cannot completely eliminate the p300/CBP activity in the cell. Thus, it is necessary to inhibit multiple functional domains at the same time, or even completely deplete the protein to ablate the p300/CBP-mediated enhancer activity.228

In 2021, Ott group described the first CRBN-based p300/CBP degrader 138 (dCBP-1, Fig. 31).229 The degrader 138 (dCBP-1) induced the highly potent, selective, and rapid dual degradation of CBP and p300, which highlighted the capability of applying PROTAC technology to exceptionally large proteins(>300 kDa). The degrader 138 (dCBP-1) had potent antiproliferative activity in multiple myeloma cells. Compared with the treatment of bromodomain and KAT domain inhibitors alone or in combination, it had enhanced effects on MYC gene expression programs, anti-proliferation, and chromatin structure in multiple myeloma. As an effective degrader of this unique acetyltransferase, degrader 138 (dCBP-1) was a useful tool to analyze the mechanisms of these factors coordinate enhancer activity in normal and cancer cells.

The representative PROTAC targeting CBP and p300

The YEATS domain is a new type of histone acetylation "reader". The human genome encodes four YEATS domain-containing proteins, namely ENL, AF9, YEATS2, and GAS41.230 Among them, ENL protein is essential for the development of acute myeloid leukemia(AML).231 When ENL is knocked out, it would induce anti-leukemia effect and inhibit the growth of leukemia in vivo and in vitro.

In 2021, Erb group discovered the highly effective ENL YEATS domain inhibitor SR-0813 based on high-throughput screening technology. On the basis of SR-0813, they designed and synthesized a degrader 139 (SR-1114, Fig. 32) that targeted ENL.232 It was found that the degrader 139 (SR-1114) could induce degradation of ENL protein in a variety of cell lines, and it had the best activity in the MV4-11 cells with DC50 of 150 nM.

The representative PROTAC targeting ENL

Histone deacetylases (HDACs) which serve as "epigenetic erasers" play an important role in chromosome structural modification and gene expression regulation by catalyzing deacetylation of substrate proteins. They make histones bind tightly to negatively charged DNA by deacetylating them, which densify chromatin and inhibit gene transcription. Until now, 18 human HDACs have been identified and classified into four classes: Class I (HDAC1, 2, 3, and 8), class II (HDAC 4, 5, 6, 7, 9, and 10), class III (SIRT1, 2, 3, 4, 5, 6, and 7) and class IV (HDAC11).233,234,235 Class I HDAC is mainly located in the nucleus, and HDAC3 is also present in the cytoplasm. Class II can respond to different cell signal responses and shuttle between the nucleus and the cytoplasm. Class III is a completely different type as the atypical histone deacetylase family of other HDACs.236,237 Type IV is mainly located in the nucleus.

HDAC6, the only protein in the HDACs family that has two tandem domains, is mainly distributed in the cytoplasm. HADC6 deacetylates α-tubulin, HSP90, cortactin and interacts with many proteins such as dynein, ubiquitin, which make it participate in cancer progression, neurodegenerative diseases, and inflammatory disorders. Rao group has been working on developing PROTACs targeting HDAC6 since 2019,238 they linked pomalidomide to the benzene ring of NexA unlike NP8, which led to the discovery of HDAC6 degrader 140 (NH2, Fig. 33) in 2019.239 When it was compared with NP8, the degrader 140 (NH2) exhibited comparably excellent degradation activity in MM.1S cells with the DC50 of 3.2 nM. The two degraders extended from different directions of the inhibitor exerted the same degradation activity revealed the extremely large flexibility of the ternary complex.

The representative PROTACs targeting HDAC

In 2020, on the basis of previous work, Tang group replaced pomalidomide in the structure of HDAC6 degrader with VHL and obtained the first selective HDAC6 degrader.240 Among them, the degrader 141 (Fig. 33) showed the best degradation activity in MM.1S with DC50 of 7.1 nM and the Dmax of 90%. In addition, they also tested its HDAC6 degradation activity in mouse cells. In the mouse 4935 cell line, the DC50 was 4.3 nM and the Dmax was 57%. Subsequently, they developed a competition assay to evaluate binding affinity of different E3 ligands in cells and screened a library of thalidomide analogs, including those with partial linkers. After screening, the most active E3 ligase ligands were conjugated to pan-inhibitor SAHA led to the discovery of a selective HDAC6 degrader 142 (YZ167, Fig. 33).241 The DC50 of the degrader 142 (YZ167) was 1.94 nM in MM.1S cells. At the same time, it was found that the degrader 143 (YZ268, Fig. 33) designed based on HDAC6-selective inhibitor Next-A also had selective degradation activity for HDAC6 during the screening process, without influence on neo-substrate IKZFs and GSPT1.

In 2021, He group also reported another HDAC6 degrader based on CDK/HDAC6 inhibitor derived from Indirubin.242 They evaluated the degradation activities of HDAC6 in K562 cells. Interestingly, the PROTAC 144 (Fig. 33) demonstrated the most superior HDAC6 degradation with DC50 of 108.9 nM and Dmax of 88%. Also, they found the degrader 144 (Fig. 33) upregulated acetylation of α-tubulin without any obvious degradation of HDAC1 and CDK2 in K562 and HeLa cells.

In addition to these degraders that selectively targeting HDAC6, other research groups have also reported degraders targeting other subtypes of HDAC in 2020. For example, Hodgkinson group reported a degrader 145 (Fig. 33) targeting class I HDAC (HDAC1/2/3) designed based on VHL and HDAC inhibitor CI-994.243 They found that the degrader 145 could induce the degradation of HDAC1/2/3 in HCT116 cells. Zhang group attempted to design PROTACs targeting HDAC6 based on Bestatin and SAHA by recruiting cellular inhibitor of apoptosis protein 1(cIAP1) E3 ubiquitin ligase.244 However, effective degradation of HDAC1/6/8 could be observed in RPMI-8226 cells. What interesting was that the degradation effect of the degrader was not obvious when the cells were treated for 6 hours. However, the degradation effect on the three subtypes of HDAC was significantly enhanced when the treatment time was extended to 24 hours. The degrader 146 (Fig. 33) exhibited more potent aminopeptidase N (APN, CD13) inhibitory activities and anti-angiogenic activities than the approved APN inhibitor Bestatin, which made it work as a potent APN and HDAC dual inhibitor and HDAC1/6/8 degrader instead of just a degrader. The degrader 147 (Fig. 33) was designed by Hansen group based on pomalidomide and SAHA.245 Although it could show strong inhibitory activity against a variety of subtypes of HDAC, in subsequent experiments they found that the degrader 147 could only induce the degradation of HDAC1 and HDAC6, which proved the difference between the selectivity and affinity activity of degrader. It provided a reference for the subsequent design of selective degraders.

Epigenetic plays an important role in various biological functions. They are not only closely related to the normal functions of the human body, but also related to the occurrence and development of many diseases. Therefore, it an effective means that controlling epigenetic changes to treat diseases. Histone methylation is a reversible genetic modification. Histone methylation and demethylation are in a dynamic equilibrium process, which participates in the regulation of gene transcription and other biological events.246 The imbalance between histone methylation and demethylation may play an important role in the occurrence and development of tumors. Many histone demethylase inhibitors have been reported, but no drugs have been approved so far.247 Therefore, traditional small-molecule histone demethylase inhibitors have insufficient stamina in the treatment of diseases.248 So developing degraders targeting histone demethylases based on PROTAC technology has become a feasible solution.

In 2021, Suzuki group reported histone demethylase degrader 148 (Fig. 34) designed based on KDM5C inhibitor.249 They found that the degrader 148 had good degradation activity in prostate cancer PC-3 cells. When the concentration was 5 µM, obvious protein degradation could be observed. With the increase of drug concentration, the degradation activity was obviously enhanced. It was also proved that the antiproliferative activity of degrader 148 on tumor cells was significantly better than inhibitor. Although the degradation activity was not excellent, as the first case of histone demethylase degrader, it laid the foundation for the subsequent development of related target degraders.

The representative PROTAC targeting KDM5C

Nicotinamide phosphoribosyltransferase (NAMPT) is a cytokine that promotes B-cell maturation and inhibits neutrophil apoptosis.250 It is the rate-limiting enzyme that catalyzes the synthesis of nicotinamide adenine dinucleotide (NAD+). Since tumor cells have a high demand for large amounts of NAD+, inhibiting NAMPT expression can significantly inhibit tumor cell proliferation. It has been confirmed that NAMPT is highly expressed in various malignant tumors such as breast cancer, prostate cancer, gastric cancer, thyroid cancer, colon cancer and hematological tumors.251 Therefore, NAMPT is considered as a drug target for antitumor therapy.252

Based on the binding mode of inhibitor MS7 and NAAMPT, a series of PROTAC molecules were obtained by connecting MS7 with VHL ligands.253 Then it was found that 149 (A7, Fig. 35) was an effective degrader of NAMPT protein. Intracellular NAMPT (iNAMPT) could be degraded by 149 (A7) through the ubiquitin–proteasome system, thereby in turn decreases the secretion of extracellular NAMPT (eNAMPT). At the same time, they also found that the inhibitor MS7 did not have the function of inhibiting tumor-infiltrating MDSCs, and the degrader could boost antitumor efficacy through this effect, so the degrader 149 (A7) exhibited stronger antiproliferative activity than the inhibitor MS7.

The representative PROTAC targeting NAMPT

Histone lysine methyltransferase catalyzes the transfer of methyl groups to specific lysine side chains at the ends of histones H3 and H4 to form histone methylation marks, thereby affecting gene transcription, DNA replication, and DNA repair. It plays an important role in maintaining chromatin stability and gene expression regulation. Representative lysine methylases include PRDM6, G9a, EZH2, DOT1L, NSD, and SUV420H1.254 NSD is a nuclear receptor-binding SET domain protein (NSD) family, including three members of NSD1, NSD2 (also known as MMSET or WHSC1) and NSD3 (also known as WHSC1L1). NSD3 is a Lysine methyltransferase at position 36 of histone H3(H3K36), catalyzes the dimethylation of H3K36.255 NSD methyltransferases are mutated, amplified, and overexpressed in a variety of human cancers, including multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, breast cancer, prostate cancer, and lung cancer.256 Therefore, NSD is considered as a potential target for the development of novel anticancer drugs.

BI-9321 is a reported NSD3 PWWP1 antagonist. By analyzing its binding mode with NSD3 protein, Wang group obtained a series of PROTAC molecules by connecting BI-9321 with different E3 ligase ligands and then screened the degradation activities of NSD3.257 They found that 150 (MS9715, Fig. 36) had the best NSD3 degradation activity with DC50 of 4.9 µM and Dmax of greater than 80% in MOLM-13 cells. After quantitative proteomic analysis, it was found that 150 (MS9715) was highly selective and had no degradation activity on other proteins. In addition, 150 (MS9715) could effectively inhibit the expression of NSD3 and cMyc-related genes, while the corresponding inhibitor BI-9321 did not have this function.

The representative PROTAC targeting NSD3

The polycomb repressive complex 2 (PRC2) is an epigenetic modulator of transcription, which is mainly constituted of four subunits: EZH1/2 (enhancer of the zeste homolog 1/2), EED (embryonic ectoderm development), SUZ12(suppressor of the zeste 12 protein homolog), and RbAp46/RbAp48 (retinoblastoma (Rb)-associated proteins 46/48).258 It catalyzes methylation of H3K27. The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive epigenetic mark that regulates gene expression, and hyper-trimethylation of H3K27 can be observed in several types of tumors. EZH2 is the catalyst subunit of PRC2 and mutation will be happened in several cancers, such as DLBCL.259 EPZ6438 is a selective EZH2 inhibitor approved for epithelioid sarcoma.260 In fact, EZH2 also mediates transcriptional activation independent of EZH2/PRC2 catalytic activity in some cancers.

In 2019, Bloeche group reported an EED degrader 151 (Fig. 37) by conjugating a potent EED inhibitor MAK683 with VHL ligand.261 The degrader 192 not only induced rapid degradation of EED but also EZH2 and SUZ12 within the PRC2 complex. It selectively inhibited the proliferation of PRC2-dependent cancer cells. At the same time, James group reported that degrader 152 (UNC6852, Fig. 37) also reduced the protein level of EED (DC50 = 0.79 µM) and EZH2 (DC50 = 0.3 µM) on HeLa cells.262 In summary, these data demonstrated that the degradation of the subunit of the PRC2 complex could cause the complex to lose its function, which was expected to be a method for PRC2-mediated cancer treatment.

The representative PROTACs targeting PRC2 (EZH2, EED)

In 2020, Jin group utilized a hydrophobic tag to link an EZH2 inhibitor to generate the degrader targeting EZH2.263 The degrader 153 (MS1943, Fig. 37) could reduce EZH2 protein levels in MDA-MB-468 cells at 5 µM and suppress the H3K27me3 mark. Compared with inhibitor, the degrader 153 (MS1943) showed significant inhibition on cell growth and induced cell death in TNBC cells in vitro and in vivo.

Based on a clinical EZH2 inhibitor EPZ6438 and pomalidomide, Yu group developed the degrader 154 (E7, Fig. 37) which induced degradation efficacies of all PRC subunits(EZH2 72%, SUZ12 81%, EED 75%, RbAp48 74%) at 1 µM in WSU-DLCL-2 cells.264 The degrader 154 (E7) also displayed stronger anticancer abilities than EPZ6438 in WSU-DLCL-2 cells and SWI/SNF-mutant cancer cells.

In 2021, Wen group reported a series of degraders with VHL ligands. The degrader 155 (YM181, Fig. 37) induced the degradation of 50% EZH2 protein level at 1 μM, and the degradation could be detected at 2 h.265 It showed superior antiproliferative effects against lymphoma cell lines.

The PRMT family is a group of enzymes that can modify the nitrogen atom of the arginine guanidine group with monomethyl and dimethyl groups. Nine members have been found in mammals. According to the different methylation products, PRMTs can be divided into type I and type II. Both type I and type II enzymes can catalyze the production of monomethylated modification. The difference is that type I can further catalyze the production of asymmetric dimethylarginine (aDMA), while type II catalyzes the formation of symmetric dimethylarginine (sDMA). PRMT5 is the first type II methyltransferase to be isolated as a JAK2 binding protein.266 Overexpression of PRMT5 leads to infectious diseases, heart diseases, and cancers(such as breast, lung, and liver cancer).267,268,269

In 2020, Jin group developed a degrader 156 (MS4322, Fig. 38) conjugated with the PRMT5 inhibitor EPZ015666 and VHL ligand through a PEG linker.270 As the first-in-class PRMT5 degrader, 156 (MS4322) induced the efficient degradation of PRMT5 in MCF-7 cells with the DC50 and Dmax value of 1.1 µM and 74%, respectively. The antiproliferative activity of the degrader 156 (MS4322) (IC50 = 18 nM) was better than EPZ015666 (IC50 = 30 nM) in MV4-11 cells. Moreover, it was highly selective for PRMT5 by proteomic study and exhibited good plasma exposure in mice. So the degrader 156 (MS4322) was a valuable chemical tool for exploring the PRMT5 functions in disease.

The representative PROTAC targeting PRMT5

Sirtuins protein is a nicotinamide adenine dinucleotide (NAD+) dependent histone deacetylase.271 There are seven recognized members in the human sirtuin family: SIRT1-SIRT7, all of which have highly conserved NAD binding domains and catalytic functional domains. The sirtuin protein family can regulate the acetylation modification and ADP ribosyl modification of a variety of proteins. SIRT2 is mainly distributed in the cytoplasm and is particularly important for the growth and metabolism of tumor cells, which makes SIRT2 an attractive target for cancer treatment.272

In 2020, Lin group developed a SIRT2 degrader 157 (TM-P4-Thal, Fig. 39) conjugated with the thiomyristoyl lysine-based SIRT2 selective inhibitor TM and CRBN ligand through a PEG linker.273 The degrader 157 (TM-P4-Thal) successfully induced the degradation of SHP2 at 0.5 µM in MCF-7 cells, which led to simultaneous inhibition of its deacetylase and defatty-acylase activities in living cells.

The representative PROTAC targeting SIRT2

The chromatin-associated WD40 repeat domain protein 5(WDR5) acts as a functional subunit of the mixed lineage leukemia (MLL) histone methyltransferase complexes. WDR5 is critical for the methylation of histone H3 lysine 4 (H3K4) on chromatin catalyzed by the MLL1 complex and MLL1 complex-mediated regulations of gene transcription.274 Numerous studies have identified WDR5 as a promising potential therapeutic target. Efforts on targeting the WIN or WBM binding site of WDR5 have led to discovery of several inhibitors that potently and selectively block PPIs between WDR5 and its binding partners.275 However, these WDR5 PPI inhibitors rely on receptor occupancy pharmacology and target only some but not all WDR5 oncogenic functions, exerting poor antiproliferative activity on tumor cells.276

In 2021, Knapp group reported different WDR5 degraders based on two diverse WIN site binding scaffolds (OICR-9429 modified scaffold and pyrroloimidazole modified scaffold) and different E3 ligase ligands.277 In MV4-11 cells, OICR-9429 modified scaffold derived degrader 158 (Fig. 40) induced the degradation of WDR5 of 58% with a DC50 value of 53 nM, and pyrroloimidazole modified scaffold derived degrader 159 (Fig. 40) induced the degradation of WDR5 of 53% with a DC50 value of 1.24 µM.

The representative PROTACs targeting WDR5

In the same year, Jin group also reported the WDR5 degraders based on OICR-9429 and VHL ligase ligands.278 Firstly, they designed WDR5 degrader 160 (MS33, Fig. 40) and found it could induce the degradation of WDR5 with DC50 of 260 nM. Subsequently, they used the degrader 160 (MS33) successfully resolve the structure of the VHL-MS33-WDR5 ternary complex. Based on the crystal structure, they modified the OICR-9429 ligand and shortened the linker to obtain a new WDR5 degrader 161 (MS67, Fig. 40). They found that the degrader 161 (MS67) could quickly induce the degradation of WDR5 with DC50 of 3.7 nM, which was about 70-folds higher than the degrader 160 (MS33). At the same time, they also resolved the structure of the VHL-MS67-WDR5 ternary complex. Subsequently, the antiproliferative activity of the degrader 161 (MS67) was tested on a variety of AML cells, which showed that it had good anti-proliferation activity. In addition, it inhibited the malignant growth of MLL-r AML cells in vitro and in vivo.

Aurora kinases are serine/threonine protein kinases that regulate centrosome and microtubule functions during mitosis. They are mainly divided into three subtypes: Aurora A, Aurora B, and Aurora C. Aurora A and Aurora B are essential for the cell cycle progression of most cell types and Aurora C mainly distributes in testis.279 Aurora A kinase is related to centrosome replication and mitotic exit. When Aurora A kinase is inhibited, it can effectively cause cell mitosis to arrest in the G2/M phase and quickly induce apoptosis.280 Aurora B kinase plays a role in coordinating the function of chromosomes and cytoskeleton in the cell cycle. When Aurora B kinase is inhibited, the cytoplasm of the cell cannot be divided and cell growth is arrested.281 Since both Aurora A and Aurora B kinases are related to tumorigenesis, Aurora kinase inhibitors have been extensively carried out in clinical researches on various diseases, such as lung cancer, breast cancer, esophageal cancer, colorectal cancer, acute myeloid leukemia and so on, but currently no drugs have been approved for clinical use.

The Wolf group reported the first selective Aurora A degrader 162 (JB170, Fig. 41) in 2020 based on the inhibitor alisertib (MLN8237),282 which had a known binding mode and high binding affinity with Aurora A kinase and easy derivation. They found that degrader 162 (JB170) had strong binding ability and degradation activity to Aurora A kinase, its degradation activity was significantly better than that of other designed molecules, and it had a Dmax of 300 nM and a DC50 of 28 nM for Aurora A kinase. After MV4-11 cells were treated with degrader 162 (JB170) or alisertib, degrader 162 (JB170) reduced the level of Aurora A by 73%, which was 57% lower than alisertib. And among the 4259 proteins detectable in this experiment, no other proteins were downregulated, including Aurora B. Subsequently, they found that degrader 162 (JB170) only induced cell accumulation in S phase, which was different with alisertib that induced the G2/M phase arrest of cells. They speculated this may be due to the fact that degrader 162 (JB170) had regulated the non-enzymatic activity of Aurora A. In order to study the effect of degrader 162 (JB170) on the proliferation activity of cancer cells, they used degrader 162 (JB170) to treat MV4-11 cells. The MV4-11 cells were inhibited by 32% after 72 h of treatment with 1 µM of JB170. Similar results were observed in the colony formation assay using IMR5 cells.

The representative PROTACs targeting Aurora A

Fischer group reported the second case of selective Aurora A degrader 163 (dAURK-4, Fig. 41) in 2021,283 which was also based on alisertib as the target protein ligand. They verified the degradation activity by western blot in the MM.1S multiple myeloma cell line, and also proved that the antiproliferative activity of degrader 163 (dAURK-4) in the MM.1S multiple myeloma cell line was significantly better than that of the inhibitor alisertib.

Lindon group reported the third Aurora A degrader 164 (PROTAC-D, Fig. 41) in 2021.284 They verified its degradation activity on Aurora A in the U2OS cell lines and also proved that it did not induce Aurora B kinase degradation. At the subcellular level, they found that the distribution of Aurora A on the spindle was different from that of the centrosome. Therefore, the therapeutic phenotypic result of degrader 164 (PROTAC-D) was different from the result mediated by alisertib. At the same time, they also confirmed that in intermitotic cells, degrader 164 (PROTAC-D) mediated the degradation of Aurora A in the non-centrosome, but had no effect on the level of Aurora A in the centrosome.

The cell-division cycle protein 20 (Cdc20) is the substrate receptor of anaphase synthesis complex or cyclosome (APC/C). It coordinates late initiation and exit of mitosis through safety and time-dependent degradation.285 Cdc20 is a key mitotic factor, which controls the beginning of anaphase and the exit of mitosis. Moreover, Cdc20-APC/C plays a key role in cancer progression and drug resistance. Furthermore, Cdc20-APC/C cell processes beyond the cell cycle, including apoptosis, neurogenesis, stem cell expansion.286 Meanwhile, it has a strong connection between the aberrant upregulation of Cdc20 and various types of cancers. Cdc20 is a potential biomarker and an ideal target for cancer treatment.287

In 2019, Wan group described the first Cdc20 degrader 165 (CP5V, Fig. 42).288 The degrader 165 (CP5V) induced the degradation of Cdc20 resulted in significant inhibition of breast cancer cell proliferation and resensitization of paclitaxel-resistant cell lines. The degrader 165 (CP5V) played an important role in inhibiting the progression of breast tumors. In cultured cells and preclinical breast cancer models, it induced the degradation of Cdc20 to induce mitosis inhibition, thereby inhibiting cancer cell proliferation. As a result, it played an important role in inhibiting breast cancer tumor progression in vivo. CP5V-induced degradation of Cdc20 may be an effective treatment strategy for breast cancer anti-mitotic therapy.

The representative PROTAC targeting Cdc20

Inactivation of cyclin-dependent kinase 2(CDK2), which overcomes the differentiation arrest of acute myeloid leukemia(AML) cells, maybe a promising approach for the treatment of AML.289,290,291 However, there are no available selective CDK2 inhibitors.292,293

In 2020, Chen group used AT-7519 as the CDK2 targeting ligand connected to CRBN ligand through different linkers to obtain a series of degraders.294 They found the degrader 166 (9A, Fig. 43) had a good selective degradation of CDK2 through activity screening. And it had no effect on CDK5 and CDK9. Subsequently, the degrader 166 (9A) showed good inhibitory activity against cell proliferation with IC50 of 0.84 µM in PC-3 cells.

The representative PROTACs targeting CDK2

In 2021, Rao group developed a first-in-class CDK2 degrader 167 (CPS2, Fig. 43) by conjugating a CRBN ligand and a nonselective CDK2 ligand JNJ-7706621.295 The degrader 167 (CPS2) induced the rapid and potent degradation of CDK2 in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.

Also in the same year, Zuo group reported the another CDK2 degrader 168 (PROTAC-8, Fig. 43) based on AZD5438 and VH032.296 They found the degrader 168 (PROTAC-8) could selectively induce the degradation of CDK2 protein. The DC50 was about 100 nM to CDK2 while no degradation activity for other subtypes of CDK protein. They also found that it could be used to prevent and treat cisplatin ototoxicity and excitotoxicity of kainic acid in zebrafish. It the first report that CDK2 degrader could be used to prevent and treat acquired hearing loss.

In 2020, Gray group modified the structure of a pan-CDK inhibitor TMX-2039 to obtain a compound TMX-3010 that increased the selectivity for CDK1/2/5, but still had a certain constant activity on CDK4/6 (IC50 ≈ 100 nM).297 TMX-3010 improved the selectivity to CDK1/2/5, possibly because the linker moiety significantly impaired the inhibitory activity on CDK4 and CDK6. Based on TMX-3010, they developed a selective degrader 169 (TMX-2172, Fig. 44) for CDK2 and CDK5 over other CDKs. In OVCAR8 cells, the degrader 169 (TMX-2172) selectively induced the degradation of CDK2 and CDK5 in a time and dose-dependent manner, while having no influence on other CDKs. Although CDK5 deletion did not cause growth defects in OVCAR8 cells, a more selective CDK2 degrader was in urgent need of development.

The representative PROTAC targeting CDK2/5

CDK4 and CDK6 are a family of serine-threonine kinases that interact with cyclins to phosphorylate Rb, thereby regulating the G1/S transition of the cell cycle. Phosphorylation of Rb leads to the release of the transcription factor E2F to activate many transcription genes that are responsible for cell cycle progression.298 Among CDKs, CDK4/6 play an important role in the cell cycle and are often overexpressed or overactivated in tumor samples.299 However, the current clinical use of CDK4/6 inhibitors have limited its application due to its drug resistance and off-target effects.300 So far, many selective and nonselective degraders of CDK4/6 have been reported.301,302,303,304,305,306 Here, we make a supplement to the CDK4/6 degraders reported in recent 2 years.

In 2021, Dominici group reported their work on the development of selective CDK6 degraders.307 The results showed that the degradation of CDK6 was more effective than inhibition with the dual CDK4/6 inhibitor Palbociclib in suppressing Ph-positive ALL in mice, suggesting that the growth-promoting effects were CDK6 kinase-independent in Ph-positive ALL. The most effective degrader 170 (YX-2-107, Fig. 45) induced the rapid and selective degradation of CDK6 over CDK4 in Ph-positive ALL cells, and significantly inhibited S-phase cells and inhibited the expression of phosphor-RB and FoxM1 regulated by CDK6. The degrader 170 (YX-2-107) showed good antitumor effects in Ph-positive ALL xenografts and it had a good safety window. However, it was fast metabolized in mice with a t1/2 of 1 h, suggesting that further improvement in PK parameters was warranted.

The representative PROTACs targeting CDK2/4/6

In addition to CRBN and MDM2 ligands, the VHL and cIPA ligands have also successfully been utilized in PROTAC for the degradation of CDK4/6. In 2020, Kronke group designed and generated series of VHL and cIPA-based CDK4/6 degraders.308 The VHL-based degraders were either specific degradation for CDK6 or exhibited dual degradation for CDK4 and CDK6. The most representative degrader 171 (Fig. 45) was a selective CDK6 degrader with the DC50 value of 5.1 nM and Dmax more than 95% in MM.1S cells. However, cIAP-based degraders induced a combined degradation of CDK4/6 and IAPs resulted in synergistic effects on cancer cell growth. In conclusion, the results showed that VHL and cIAP-based degraders were an attractive approach for targeted degradation of CDK4/6 in cancer.

A growing number of reports indicated that inhibitors targeting CDK2/4/6 could act as a more feasible chemotherapy strategy. In 2021, Wei group developed a degrader 172 (Fig. 45) based on Ribociclib derivative. The degrader 172 could selectively induce the degradation of CDK2/4/6 over other CDKs.309 It also showed reduced protein levels of CDK2/4/6 in a dose and time-dependent manner as well as inhibition of the retinoblastoma (Rb) protein phosphorylation in malignant melanoma cells. Moreover, it also remarkably induced cell cycle arrest and apoptosis of melanoma cells. However, the degrader 172 showed poor oral bioavailability. By adding a labile group to the degrader 172 they got the new degrader 173 (Fig. 45), whose oral bioavailability significantly increased up to 68%. In B16F10 xenograft model, oral administration (200 mg/kg) of the degrader 173 showed a significant reduction in tumor growth. This may also provide a universal solution for oral CRBN-based degraders.

Cyclin-dependent kinases (CDKs) are a class of Ser/Thr protein kinases that have key roles in cell cycle regulation or cell transcription.310 CDK9 is a member of the family of CDKs and is crucial in transcription regulation that regulates most of the cancer suppressors and oncogenes.311 It overexpressed in a variety of tumors such as leukemia and malignant melanoma. CDK9 inhibitors can inhibit the kinase activity and phosphorylation of CDK9, thereby preventing P-TEFb-mediated activation of RNA Pol II, and inhibiting gene transcription of many anti-apoptotic proteins.312 Although a large amount of data indicates that CDK9 is a promising target for cancer treatment, the development of highly selective CDK9 inhibitors is still a huge challenge. Indiscriminate inhibition of CDK family kinase activity can cause adverse side effects and undesirable toxicity.313 In recent years, some examples of selective CDK9 degrader have been reported.

In 2017, Rana group reported the first CDK9 degrader by conjugating a CDK9 inhibitor Nopyrazole and thalidomide. The degrader could potently induce 56% CDK9 to degrade at 10 μM, which was not enough for tumor treatment.314 Recently, Natarajan group reported a similar degrader 174 (Fig. 46) with better degradation activity by changing the length of linker. The degrader 174 could selectively induce degradation of CDK9 with DC50 of 158 nM while sparing other CDK family members.315 These results indicated that the length of the linker was important for degradation activity and selectivity of CDK9.

The representative PROTACs targeting CDK9

In 2021, Bian group reported a selective degrader 175 (B03, Fig. 46) for CDK9 by conjugating a selective CDK9 inhibitor BAY-1143572 and pomalidomide.316 The degrader 175 (B03) induced the degradation of CDK9 in acute myeloid leukemia cells with DC50 of 7.6 nM, which was superior to the reported CDK9 degraders. In addition, the degrader 175 (B03) strongly induced apoptosis and inhibited cell proliferation of MV4-11 cells (IC50 = 25 nM), which was better than the inhibitor BAY-1143572 (IC50 = 560 nM). Moreover, the degrader 175 (B03) could induce the degradation of CDK9 in vivo. It was the lead degrader of further development and CDK9 degradation was a potentially valuable treatment strategy for acute myeloid leukemia.

Based on the previous results and through the analysis of the CDK9 ligands binding modes, Chen group also designed and developed a series of new CDK9 protein degraders in the same year.294 They found the degrader 176 (PROTAC 45, Fig. 46) had better activity in inhibiting the growth of TNBC cells, the DC50 reached up to 4 nM in BT-549 cells. And the degrader 176 (PROTAC 45) could effectively and selectively induce degradation of CDK9 in a short period of time. In addition, mechanistic studies have shown that the degrader 176 (PROTAC 45) could effectively downregulate the downstream targets of CDK9 (such as MCL1 and MYC) at the transcriptional level. Pharmacodynamic studies have shown that the degrader 176 (PROTAC 45) could effectively induce tumor regression in a TNBC xenograft model. This was the first reported in vivo evaluation of the therapeutic activity of CDK9 degrader in the treatment of TNBC, which proved that CDK9 degradation therapy was a new method for the treatment of TNBC.

In 2020, Chen group first developed a series of novel degraders for CDK2/9 degradation by connecting CDK9 inhibitor FN-1501 and CRBN ligand.317 The representative degrader 177 (Fig. 46) potently induced degradation of both CDK2 (DC50 = 62 nM) and CDK9 (DC50 = 33 nM) in PC-3 cells, and inhibited cell proliferation by effectively blocking the cell cycle in S and G2/M phases. The degradation of CDK2/9 by dual PROTAC may be a potentially effective therapeutic approach.

CDK12 can phosphorylate RNA polymerase II to regulate transcription elongation, and it can also play a key role in RNA splicing, DNA-damage response (DDR) and the maintenance of genome stability.318,319 It is overexpressed in breast cancer, ovarian cancer, prostate cancer and other cancers, and is a valuable target for cancer valuable.318 Loss of CDK12 function by silencing and using selective covalent CDK12/13 inhibitor THZ531 leads to a decrease in DDR-associated genes transcription and increases sensitivity to PARP inhibitors and platinum chemotherapy. Thus, CDK12 inhibitors can be used in combination with DNA damaging agents for HR-deficient cancers.320,321 However, the development of CDK12 inhibitors is particularly challenging due to high sequence similarity with its close homolog CDK13.322

In 2021, Gray group developed a selective CDK12 degrader 178 (BSJ-4-116, Fig. 47) by linking piperidine moiety of optimized segment to thalidomide.323 As expected, the degrader 178 (BSJ-4-116) demonstrated strong inhibitory activity on CDK12 enzyme activity and effectively induced the degradation of CDK12 in a dose and time-dependent manner in Jurkat cells, while CDK13 protein levels were minimally affected. Moreover, it exhibited potent antiproliferative effects when used alone or in combination with the PARP Olaparib as well as used alone against cell lines that resistant to covalent CDK12 inhibitors. Interestingly, chronic exposure of degrader 178 (BSJ-4-116) to MOLT-4 cells and Jurkat cells resulted in acquired resistance phenotypes via mutations of Ile733Val and Gly739Ser on CDK12 which resulted in the weaken of degrader binding affinity.

The representative PROTACs targeting CDK12

Then Zhu group synthesized a potent PROTAC 179 (PP-C8, Fig. 47) based on CDK12/13 noncovalent dual inhibitor SR-4835.324 They demonstrated that the degrader 179 (PP-C8) could selectively induce the degradation of CDK12, while there is no degradation activity on CDK13 in different cell lines. And they also found that the degrader could lead to the degradation of cyclin K, and the degradation activity DC50 for CDK12 and cyclin K were 416 and 412 nM, respectively. They also proved the degrader 179 (PP-C8) was highly selective to CDK12-cyclin K complex by quantitative proteomics. They have also shown that 179 (PP-C8) and PARP inhibitors exhibited good synergy in triple-negative breast cancer (TNBC).

Wee1 is a tyrosine kinase that regulates the G2/M cell cycle checkpoint by phosphorylating and inactivating CDK1 in response to extrinsic DNA damage and errors in DNA synthesis, thereby preventing mitotic entry.325 Cancer cells often have a deficient G1/S checkpoint which frequently via mutation of p53 to leave them reliant on the G2/M checkpoint to avoid mitotic catastrophe. Therefore, abrogation of the G2/M checkpoint by inhibiting Wee1 can sensitize tumors to DNA damaging therapies. In previous studies, Wee1 inhibitor AZD1775 has been discovered.326 However, it has obvious off-target and side effects, which limits its clinical application.

In 2019, in an attempt to overcome these limitations, Gray group developed Wee1 degraders by conjugating AZD1775 to pomalidomide.327 The degrader 180 (ZNL-02-096, Fig. 48) displayed potent Wee1 degradation in MOLT-4 cells while having no influence on PLK1. In addition, it induced G2/M phase arrest at 10 folds lower doses than AZD1775 and synergized with Olaparib in ovarian cancer cells.

The representative PROTAC targeting Wee1

The cereblon (CRBN) is a subunit of the E3 ubiquitin ligase complex cullin-RING ligase 4 (CRL4CRBN) and is the substrate receptor for the damaged DNA binding protein 1 (DDB1) and Cul4A.328,329 CRBN is also the direct target protein of thalidomide and immunomodulatory imide drugs (IMiDs) and is crucial for thalidomide teratogenicity.330 CRL4CRBN is a unique E3 ubiquitin ligase with substrate selectivity altered by ligands such as IMiDs.331 It can be efficiently recruited to a protein of interest (POI) when binding with IMiD-based proteolysis-targeting chimeras(PROTACs), leading to the protein ubiquitination and proteasomal degradation.332 As the primary IMiD target, CRBN is a protein of huge potential practice.333 CRBN degraders are considered to be useful tools to figure out the molecular mechanism of thalidomide analogs334.

In 2018, Gütschow group designed a series of PROTACs that targeted CRBN for the first time, proving the feasibility of a degrader-induced degradation of CRBN.330 They synthesized so-called homo-ROTACs by linking the two pomalidomide molecules. They found the degrader 181 (Fig. 49) could selectively induce the degradation of CRBN with minimal effects to other related proteins such as IKZF1, IKZF3 and CRL4. The maximum degradation activity of CRBN was achieved at 6h after treatment with 100 nM degrader 181. The homo-PROTACs could overcome the low selectivity of traditional chemical probes and also provide a new tool for exploring the mechanism of CRBN and IMiDs. Subsequently, they also reported several hetero-PROTACs by assembling CRBN and VHL in 2019. One of the particularly potent CRBN degrader 182 (CRBN-6-5-5-VHL, Fig. 49) had the best degradation activity of CRBN with DC50 of 1.5 nM in MM1S cells.334 Compared with the degrader 181, hetero-PROTACs such as CRBN-6-5-5-VHL, CRBN-6-6-6-VHL and CRBN-4-4-4-6-VHL all could achieve more efficient degradation activities of CRBN while inducing almost negligible degradation of the neo-substrates IKZF1 and IKZF3.

The representative PROTACs targeting CRBN

In 2019, Ciulli group reported their most active VHL-CRBN hetero-dimerizing degrader 183 (Fig. 49) which induced the rapid and efficient degradation of CRBN with a DC50 of 200 nM in HeLa cells (within 1 h of treatment). Their work provided proof-of-concept for hijacking different E3 ligase to induce degradation of a particular one.335

Subsequently, Kim group developed a series of VHL-CRBN hetero-dimerizing degraders which effectively induced degradation of CRBN but not VHL.336 To develop VHL-CRBN hetero-dimerizing PROTACs they designed different linkers to connect pomalidomide with VH032. The linkers were designed to connect the amino group of pomalidomide and the terminal acetyl group of VH032. The most potent and selective degrader 184 (TD-165, Fig. 49) could induce the degradation of CRBN with DC50 of 20.4 nM in HEK293T cells.

Recently, Gray group developed six homo-PROTACs and six hetero-PROTACs adopting a similar strategy as the previous ones. By combining protein immunology and genomics analysis, they successfully screened out two effective hetero-PROTACs which could selectively induce the degradation of CRBN, which provids a reference for follow-up research. The degradation efficiency of degrader 185 (ZXH-4-130, Fig. 49) and degrader 186 (ZXH-4-137, Fig. 49) were better than degrader 183 and degrader 184 (TD-165).337

The 26S proteasome consists of a 20S core and a 19S regulatory subunit. hRpn13 is a component of the 19S subunit, which contains two functional domains, an N-terminal pleckstrin-like receptor for ubiquitin(Pru) domain and a C-terminal deubiquitinase adaptor (DEUBAD) domain.338,339,340

In 2021, Walters group screened an inhibitor XL5 with better binding ability based on the reported structure of hRpn13 inhibitor RA190.341 On the basis of XL15, they used different E3 ligase ligands to connect with it to obtain a series of PROTAC molecules. They found that 187 (XL5-VHL-2, Fig. 50) was able to induce the degradation of DEUBAD-lacking hRpn13Pru species in RPMI-8226 wild-type cells, but not hRpn13. The degradation activity of DEUBAD-lacking hRpn13Pru species was DC50 of 39 µM and Dmax of 81%. When treated with 187 (XL5-VHL-2), it only resulted in ubiquitination of hRpn13 protein without degradation.

The representative PROTAC targeting hRpn13Pru

E3 ubiquitin ligases are emerging as attractive targets for small-molecule modulation and drug discovery and gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs.342 However, E3 ligases do not comprise deep and "druggable" active sites for binding to small molecules. E3 ligase inhibition may be ineffective or fail to recapitulate genetic knockout or knockdown.343 New chemical modalities to target E3 ligases are therefore demanded.

In 2017, Ciulli group described proof-of-concept of homo-PROTACs using diverse molecules composed of two instances of a ligand for the VHL E3 ligase.344 The most active degrader 188 (CM11, Fig. 51) dimerized VHL with high avidity in vitro and induced potent, rapid, and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. As a novel chemical probe for selective VHL degradation, the degrader 188 (CM11) would find wide use in investigating and dissecting the biological functions of pVHL.

The representative PROTAC targeting VHL

ZFP91 is a newly discovered ubiquitin E3 ligase that is upregulated in prostate cancer, acute myeloid leukemia, and colon cancer tumors. It maintains the stability of NIK protein through K63-ubiquitinated NIK, thereby activating the NF-κB signaling pathway and promoting the occurrence and development of tumors.345

In 2021, Neamati group designed a ZFP91 degrader 189 (XD2-149, Fig. 52) based on Napabucasin, which was undergoing multiple clinical trials and was reported to inhibit STAT3, and the degrader 189 (XD2-149) resulted in inhibition of STAT3 signaling in pancreatic cancer cell lines without inducing proteasome-dependent degradation of STAT3.346 Proteomics analysis showed that it could induce the degradation of the E3 ubiquitinprotein ligase ZFP91 with DC50 values of 80 nM in BxPC-3 cells. At the same time, it also showed a moderate antiproliferative activity, and the IC50 was about 0.9 µM in MIA PaCa-2 cells and BxPC-3 cells.

The representative PROTAC targeting ZFP91

As a key regulator in the B-cell receptor(BCR) signaling pathway, BTK is a non-receptor cytoplasmic tyrosine kinase and plays a key role in B-cell lymphomas.347 In 2013, although the covalent inhibitor ibrutinib was approved for treating MCL and activated B-cell-like (ABC)-DLBCL by FDA, the patients developed resistance due to the missense BTK mutation of C481S.348 With the advent of PROTACs technology, there have been many noncovalent degraders which can degrade both the wild-type and ibrutinib-resistant C481S BTK effectively.349,350,351,352,353 The next stage may be aimed at improving druggability for a clinical study.

In 2019, Lin group developed a new BTK degrader 190 (SPB5208, Fig. 53) by conjugating Ibrutinib and thalidomide.354 The degrader 190 (SPB5208) could inhibit the growth of the JeKo-1 cells and induce the degradation of BTK protein both in vitro and in vivo.

The representative PROTACs targeting BTK

In 2020, Crews group illustrated a new series of BTK degraders based on MT802 which was reported previously with potent degradation activity of BTK and poor pharmacokinetic properties. To develop an appropriate degrader for in vivo study, they modified the linker and E3 ligands while keeping their length constant. Fortunately, the degrader 191 (SJF620, Fig. 53) exhibited both potent degradation activity and encouraging pharmacokinetic profile.355

In 2021, Wang group designed a novel series of PROTACs targeting BTK based on ARQ531, a reversible noncovalent BTK inhibitor that inhibits both wild-type (WT) and mutated BTK. In addition to having moderate membrane permeability and good plasma stability, the most potent degrader 192 (Fig. 53) also could induce the degradation of BTKWT/BTKC481S and inhibit BTKWT/BTKC481S TMD8 cells growth effectively.356

In 2021, Rao group developed a new type degrader of targeted protein degradation by merging PROTAC and molecular glue for inducing the degradation of BTK and GSPT1 proteins concurrently. They designed a BTK PROTACs library with short linkers to keep the balance of the efficiency of PROTACs and the features of molecular glue.357 The representative degrader 193 (GBD-9, Fig. 53) induced the fast and effective degradation of BTK and GSPT1 in DOHH2 cells and exhibited more potent antiproliferative activity than Ibrutinib on a variety of DLBCL and AML cell lines. This work not only provided a new example for the design of double-mechanism degraders with the characteristics of molecular glue and PROTACs, but also expanded the indications of PROTACs targeting BTK, which may have broader clinical application prospects.

In 2021, Pfizer solved the unique BTK-degrader-cIAP1 ternary complex crystal structure by using the degrader 195 (BC5P, Fig. 53) which was derived from aminopyrazole series in previous work and it was shown that BTK was flexibly tethered to cIAP1 with a multitude of sampled conformations. To restrict this flexibility, a shorter and more rigid linker was induced to obtain the degrader 194 (BCPyr, Fig. 53) which enhanced protein–protein interactions, restricted conformational dynamics but weakened BTK- degradation activities. This indicated that the cooperativity of ternary complex did not necessarily correlate with degradation activity.358

Although Ibrutinib is an irreversible covalent inhibitor, most PROTACs targeting BTK are based on noncovalent binding. Certainly, there have been many attempts to design covalent PROTACs in recent years. In 2020, Pan group illustrated a new class of BTK PROTACs based on an irreversible covalent binder. Although the degrader 196 (Fig. 53) could induce the degradation of BTK protein with a DC50 of 136 nM and a Dmax of 88%, its potency was reduced due to the loss of substoichiometric activity.359 In 2020, Nir group360 and Wang group361 successively reported reversible covalent BTK PROTACs which enhanced the binding affinity of inhibitor without compromising the substoichiometric activity of PROTACs. Coincidentally, they both developed reversible covalent PROTACs based on cyanoacrylamide. Interestingly, in Wang work, the reversible covalent degrader 197 (RC-1, Fig. 53) had high target occupancy and worked as both an inhibitor and a degrader which exhibited more potent degradation activity of BTKWT and BTKC481S than the equivalent irreversible covalent and reversible noncovalent degrader. Conversely, the degrader 198 (RC-3, Fig. 53) was the best reversible covalent BTK PROTACs in Nir work, even less potent than the equivalent irreversible covalent and reversible noncovalent degrader. But Nir work mainly illustrated the molecular mechanism that part of the degradation by irreversible covalent PROTACs was driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drave degradation primarily by covalent engagement.

Finally, the authors compared the reported BTK degraders (Table 6). It was found that although there were various types of BTK warheads currently used, Ibrutinib was used more frequently and others were used less. In the selection of E3 ligases, most of the currently used was CRBN, and VHL and cIAP have been mentioned in some reports. Therefore, more BTK degraders based on different BTK inhibitors and E3 ligases need to be developed.

CCR9 is a chemokine receptor expressed on memory/effector CD4+ T cells and selectively binds the chemokine CCL25.362 The expression of CCL25 is significantly increased when the intestine is inflamed and drives lymphocyte migration to the intestinal tissue through interaction with CCR9.

It a treatment for Crohn disease to prevent the migration of lymphocytes to the intestinal tissue by inhibiting the interaction of CCR9 and CCL25. Vercirnon is a small-molecule CCR9-selective antagonist that binds to the CCR9 receptor on the intracellular side of the GPCR, but not on the extracellular side of the cell where GPCR ligands normally bind.363 By binding to this allosteric site on the CCR9 receptor, Vercirnon prevents CCR9 from interacting with intracellular signaling molecules, thereby achieving an antagonistic effect.

Based on the binding mode of Vercirnon and CCR9, Schiedel group obtained a more active antagonist.364 Based on the structure of the new antagonist, they designed a CCR9-targeted degrader 199 (CCR9-PROTAC, Fig. 54) and then they tested the degradation activity of the degrader on CCR9 in HEK293T cells. They found the degrader 199 (CCR9-PROTAC) could induce the degradation of CCR9 at 1 nM, but the "hook effect" also appeared as the concentration increased. This was the first PROTAC targeted the intracellular allosteric binding site of GPCRs and provided an unprecedented approach to modulate GPCR activity.

The representative PROTAC targeting CCR9

CD147 is a transmembrane glycoprotein, a member of the immunoglobulin superfamily. As a tumor-associated antigen, it is highly expressed in various tumors, including malignant melanoma (MM). As a pivotal role in tumor progression, including affecting tumor cell apoptosis, invasion and metastasis. Therefore, CD147 may provide a potential drug target for tumor diagnosis and treatment.365

To date, the anti-CD147 monoclonal antibody drug Licartin has been used in clinical treatment of liver cancer, but the radioactive I130 contained in Licartin may have inconvenience and risk of radiation leakage in clinical application.366 Pseudolaric acid B (PAB), a recently reported natural product, was an antagonist of CD147, which could avoid the above-mentioned problems.367 So in 2020, Chen group reported the first CRBN-based CD147 degrader 200 (Fig. 55) derived from PAB.368 The degrader 200 could effectively induce the degradation of CD147 with DC50 of 6.72 µM in Sk-Mel-28 cells and inhibited melanoma cells in vitro and in vivo. In summary, the degrader 200 as the novel type of anticancer agent provided a new method for CD147-mediated cancer treatment.

The representative PROTAC targeting CD147

Cytoplasmic retinoic acid-binding proteins, CRABP-I and CRABP-II, are found in all vertebrates and are conserved across species. CRABP-I is thought to be related to metabolism of retinoic acid(RA) and resistance to RA in cancer cells, while CRABP-II is suggested to be associated with nuclear transportation of RA.369 CRABP-II is expressed in tissues such as the choroid plexus, gut endoderm, and interdigital mesenchyme, which do not express CRABP-I.370 CRABP-I/II are related to Alzheimer disease and various cancer.371,372 Therefore, CRABPs could be target proteins for treatment of these diseases. However, it is challenging to directly inhibit the functions of CRABPs by small molecules.

In 2010, Hashimoto group developed the first cIAP1-based PROTAC by hijacking cIAP1-E3 ligase (bestatinmethyl ester, MeBS) and all-trans retinoic acid (ATRA) to induce the degradation of CRABP-I/II.373 Based on this work Naito group developed new class CRABPs degrader 201 (β-NF-ATRA, Fig. 56) that used β-naphthoflavone (β-NF) as a ligand to replace the MeBS to recruit aryl hydrocarbon receptor(AhR) E3 ligase complexes in 2019.374 The degrader 201 (β-NF-ATRA) could effectively induce the degradation of CRABP-I/II in a dose and time-dependent manner in MCF-7 and IMR-32 cells, and induced AhR self-ubiquitylation and was degraded by the proteasome, which may cause the potential for synergistic anti-tumorigenic activity.

The representative PROTAC targeting CRABP

Heat shock protein 90 (HSP90) is a ubiquitous, highly conserved and highly active protein in cells, which is more abundant in tumor cells than in normal cells. As a molecular chaperone, HSP90 assists different kinds of oncoproteins to fold, stabilize and mature.375 The service proteins of HSP90 contain a large number of signal transduction molecules such as kinases and transcription factors, which play an important role in tumor formation and growth.376 HSP90 inhibitors are a class of compounds that can bind to HSP90-regulatory sites, cause conformational changes of HSP90 and induce degradation of substrate proteins, thereby exerting an inhibitory effect.377 HSP90 inhibitors also can cause multiple signal transduction pathways of tumor cells to be inhibited. Many kinds of HSP90 inhibitors have entered the clinical phase.

Wu group designed and synthesized a series of PROTACs based on the binding mode of HSP90 inhibitor BIIB021 to the protein.378 Then they tested their degradation activity on HSP90 protein and proliferation inhibitory activity in MCF-7 and MDA-MB-231 cells, they found the degrader 202 (BP3, Fig. 57) could induce the degradation of HSP90 protein with a DC50 of 0.99 µM and an IC50 of 0.63 µM for cell proliferation inhibitory activity in MCF-7 cells. When used as a single drug, degrader 202 (BP3) could effectively inhibit the tumor proliferation in mice.

The representative PROTAC targeting HSP90

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme enzyme that catalyzes the first and rate-limiting steps of the decomposition of tryptophan into N-formyl-aminoylinosine. It acts on a variety of tryptophan substrates, including D-tryptophan, L-tryptophan, 5-hydroxytryptophan, tryptophan, serotonin, and so on.379 IDO1 plays a role in various pathophysiological processes, such as antibacterial, antitumor defense, neuropathology, immunomodulation, and antioxidant activity.380 Recently, a large number of reports have confirmed that IDO1 was overexpressed in a variety of cancers and plays an important role in cancer immune escape. Therefore, IDO1 has become one of the important targets in the field of tumor therapy. In fact, several highly effective and selective IDO1 inhibitors have entered the clinical development stage for the treatment of human cancer.381

In 2020, Xie group reported the application of PROTAC technology in the targeted degradation of IDO1 enzymes. They designed a series of targeted IDO1 degraders based on pomalidomide and IDO1 inhibitor Epacadostat, and evaluated their degradation activity and anti-proliferation in HeLa cells.382 They found that the degrader 203 (Fig. 58) had the best degradation activity of IDO1, its DC50 was 2.84 µM, Dmax was 93%. And its antiproliferative activity on HeLa cells was 37.43 µM, which was significantly better than the inhibitor Epacadostat.

The representative PROTAC targeting IDO1

Liver X receptors (LXRs) are members of the orphan nuclear receptor transcription factor superfamily and ligand-dependent transcription factors, including two subtypes of LXR-α (NR1H3) and LXR-β (NR1H2).383 LXR-α is mainly expressed in the liver, spleen, kidney, lung, intestine, adipose tissue, and macrophages, and LXR-β is widely and lowly expressed throughout the body.384 When LXRs are activated by ligands or synthetic agonists, they can participate in the regulation of sugar, lipid, cholesterol metabolism, immune response, and inflammation to regulate the transcription and expression of target genes.385

In 2021, Demizu group synthesized some LXR-β degraders based on the LXR-α/LXR-β agonist GW3965 and different E3 ligase ligands through different connection methods and then found that the VH032-based degrader 204 (GW3965-PEG5-VH032, Fig. 59) had the best degradation activity of LXR-β, the obvious degradation of LXR-β could be observed when the drug concentration was 3 µM.386

The representative PROTAC targeting LXR-β

Macrophage migration inhibitory factor (MIF) is the first cytokine with the function of multiple inflammatory mediators. It is also an important endocrine hormone.387 As an inflammatory chemokine, MIF plays a very important role in many diseases: metabolic diseases (such as atherosclerosis), autoimmune diseases, cancers, infectious diseases(such as sepsis) and wound healing.388 Therefore, MIF can be used as a biomarker and target of these diseases.

In 2021, Dekker group designed and synthesized a variety of degraders based on the MIF tautomerase inhibitors and pomalidomide.389 Among them, the degrader 205 (MD13, Fig. 60) could induce the degradation of MIF with a DC50 of 100 nM in A549 cells. It induced cell cycle arrest at the G2/M phase and also inhibited ERK phosphorylation.

The representative PROTAC targeting MIF

The nuclear protein PARP1 has a well-established role in the signaling and repair of DNA, and is a validated therapeutic target for cancers and other human diseases.390 Cancer cells bearing mutations in BRCA1 or BRCA 2 are exquisitely sensitive to PARP1 inhibitors, which is called synthetic lethality. To date, several small-molecule PARP1 inhibitors, such as Olaparib, Rucarparib, Niraparib, and Talazoparib, have been approved for the treatment of BRCA-mutation ovarian and breast cancers.391 However, there are still challenges in terms of PARP1 inhibitors usage that limit their therapeutic utility, including the acquisition of drug resistance, the low proportion of BRCA1 or BRCA 2 mutations in cancer cells, and cytotoxicity caused by PARP1 trapping.392,393

In 2019, Yu group reported their works on the development of PARP1 degraders.394 The degrader 206 (iRucaparib-AP6, Fig. 61) induced robust degradation of PARP1 at concentrations as low as 50 nM in primary rat neonatal cardiomyocytes. Knocking down the protein level of PARP1, the degrader 206 (iRucaparib-AP6) protected muscle cells and primary cardiomyocytes from DNA-damage-induced energy crisis and cell death. In summary, the degrader 206 (iRucaparib-AP6), by blocking both the catalytic and scaffolding functions of PARP1 without causing PARP1 trapping, provided an ideal approach for the treatment of cancers and other diseases caused by PARP1 hyperactivation.

The representative PROTACs targeting PARP1

In 2020, Shen group developed several PARP1 degraders based on the combination of PARP1 inhibitor Olaparib and lenalidomide.395 The representative degrader 207 (Fig. 61) effectively induced the degradation of PARP1 at 10 µM in SW620 cells. At the same time, Chen group reported the design and synthesis of PARP1 degraders based on Olaparib and thalidomide/lenalidomide.396 The representative degrader 208 (SK-575, Fig. 61) potently inhibited the growth of cancer cells bearing BRCA1/2 mutations, and induced potent and specific degradation of PARP1 in various human PARP1-positive cancer cells with the Dmax more than 95%. It was worth noting that the PK and PD data showed that a single dose of degrader 208 (SK-575) was fully exposed to plasma for more than 24 h and effectively induced PARP1 degradation in SW620 xenograft tumor tissues. Moreover, it exhibited durable tumor growth inhibition in mice when used as a single agent or in combination with cytotoxic agents.

PARP14 is an interferon-stimulated gene that is overexpressed in a variety of tumors. By regulating IFN-γ and IL-4 signals, affects the polarization of pro-tumor macrophages and inhibits the antitumor inflammatory response. Catalytic inhibitors of PARP14, such as RBM012042, can reverse IL-4 driven pro-tumor gene expression in macrophages. However, it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. However, the role of non-enzymatic functions of PARP14 in PARP14-driven immunity and inflammation is still unclear.397

Kuntz group developed the first-in-class degrader 209 (RBN012811, Fig. 62) based on a PARP14 inhibitor RBN012042.398 The degrader 209 (RBN012811) could selectively induce the degradation of endogenous PARP14 with DC50 about 5 nM in KYSE-270 cells, and have no effect on the total protein levels of other PARP enzymes. It also could induce a dose-dependent reduction of IL-10 levels in primary human macrophages.

The representative PROTAC targeting PARP14

Programmed death receptor (PD-1), also known as CD279, is an important immunosuppressive molecule. It regulates the immune system and promotes self-tolerance by downregulating the response of the immune system to human cells and by inhibiting the inflammatory activity of T cells.399 PD-L1 is a ligand of PD-1. The combination of PD-1 and PD-L1 initiates the programmed death of T cells and enables tumor cells to obtain immune escape. PD-L1 is upregulated in a variety of tumor cells and it binds to PD-1 on T cells, inhibits T-cell proliferation and activation, inactivates T cells, and finally induces immune escape.400

In 2020, Chen group first reported a series of novel PD-L1 degraders.401 Most of the compounds exhibited excellent inhibitory activities against PD-1/PD-L1 interaction, especially the degrader 210 (P22, Fig. 63) with an IC50 value of 39.2 nM. The degrader 210 (P22) could moderately induce the degradation of PD-L1 in a lysosome-dependent manner, which may contribute to its immune effects. In 2021, Yang group reported another PD-L1 degrader 211 (Fig. 63) based on BMS-37.402 The degrader 211 could effectively induce the degradation of PD-L1 protein at micromolar in various malignant cells in a proteasome-dependent manner, such as MC-38, MCF-7, and Kasumi-1 cells. Moreover, it could significantly reduce PD-L1 protein levels of MC-38 cancer cells in vivo, and achieve significant tumor growth inhibition in the MC-38 xenograft model. This approach showed that PROTAC could be used as a new alternative strategy for tumor immunotherapy.

The representative PROTACs targeting PD-L1

Polo-Like Kinase 1 (PKL1) belongs to the polo-like kinase family, which is a type of serine/threonine kinase that is widely present in eukaryotic cells. The structure of PKL1 is highly conserved, with a kinase domain (KD) at the N-terminus and two conserved polo-box domains (PBD) at the C-terminus.403 PBD is related to the subcellular localization and function of PKL1. Normally, the PBD of PLK1 binds to the KD domain to inhibit the phosphorylation of T210 in the KD domain, thereby inhibiting its kinase activity. Once the PBD of PLK1 binds to its ligand, the PBD is immediately separated from the T loop of the kinase domain and PLK1 is activated. PLK1 kinase can interact with a variety of substrates through its kinase activity to regulate cell mitosis, cytokinesis, DNA-damage response, development, and other processes.404,405 There is also a destruction box (D box) between the KD and PBD domains, which is closely related to the degradation of PLK1.

In 2020, Lu group developed a dual degrader 118 (HBL-4, Fig. 64) conjugated with the PLK1 inhibitor BI2536 and CRBN ligand through a PEG linker.209 This was the first degrader for PLK1. The degrader 118 (HBL-4) induced the efficient degradation of BRD4 and PLK1 in MV4-11 cells with a DC50 of about 10–20 nM and 5 nM, respectively. Then they found the antiproliferative activity of the degrader 118 (HBL-4) (IC50 = 4.48 nM) was better than BI2536 (IC50 = 88.5 nM) in MV4-11 cells. At the same time, it induced dramatically improved efficacy in the MV4-11 tumor xenograft model compared with BI2536.

The representative PROTAC targeting PLK1

Retinoic acid receptors (RAR) belong to the nuclear receptor superfamily, including three subtypes of α, β, and γ. RAR-β is divided into β1, β2, β3, β4, and so on. RAR regulates the transcription of target genes by binding to their ligands, thereby exerting various biological effects. It plays an important role in mediating cell growth and apoptosis.406

In 2011, Hashimoto group developed an RAR degrader 212 (Fig. 65) conjugated with the RAR inhibitor Ch55 and cIAP1 ligand BE04 through a PEG linker.407 The degrader 212 could induce the degradation of RAR protein in HT1080 cells for the first time, while had no influence on the amount of CRABP-II.

The representative PROTAC targeting RAR

Nucleic acids with a four-stranded helix structure are called G-quadruplexes, which are a class of nucleic acid secondary structures with important regulatory functions and are believed to play an important role in gene expression and telomere maintenance.408 G-quadruplex binding proteins play an important role in the regulation of life in which G-quadruplexes are involved. G-quadruplex-binding proteins include a variety of proteins, such as BRCA1, PARP1, RHAU, TRF2, and so on.409 RNA helicase associated with AU-rich element (RHAU) proteins has been identified as a major source of quadruplex-resolving activity in cell lysates.410

Phan group reported the novel degrader 199 (G4-PROTAC, Fig. 66) generated from a combination of G4 warhead and different E3 ligase ligands.411 They found that a significant downregulation of RHAU protein could be observed at 199 (G4-PROTAC) concentration of 1 nM, and its degradation activity increased along with increasing degrader concentration. At the same time, the degrader also had obvious "hook effect". They also proved that the degradation of RHAU protein could be rapidly induced after using the degrader for 6 h, and the degradation effect gradually increased with the prolongation of the incubation time.

The representative PROTAC targeting RHAU

Receptor serine/threonine kinases are single-pass transmembrane protein receptors with serine/threonine protein kinase activity in the intracellular region. It mainly phosphorylates the serine or threonine in downstream signal proteins, transmits the extracellular signal into the cell, and then achieves a variety of biological functions by affecting gene transcription. Pattern recognition receptors NOD1 and NOD2 are located upstream of RIPK2 and regulate its activity.412 The transcriptional activation of a variety of inflammatory cytokine genes and a variety of immune diseases are related to the activation of this pathway.413

In 2021, Harling group developed a highly potent and selective RIPK2 degrader 214 (Fig. 67) based on a novel IAP ligase ligand.414 The degrader 214 successfully induced the efficient degradation of RIPK2 in the absence of cIAP1 autoubiquitination over a range of concentrations in human PBMCs. The DC50 and Dmax were about 1 nM and 94.3%, respectively. The antiproliferative activity of the degrader 214 (IC50 = 10 nM) was better than other RIPK2 degraders. It exhibited low total systemic clearance in both rat and dog which afforded prolonged in vivo degradation of RIPK2 in vivo. In addition, it reduced endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound.

The representative PROTAC targeting RIPK2

The spliceosome is a large protein-RNA complex that consists of five small nuclear RNAs (snRNAs) and a variety of associated proteins, such as splicing factor 3B (SF3B).415 SF3B subunit 1 (SF3B1) is one of the fundamental components of the multiprotein complex SF3B and is involved in 30 splice site recognition at intron–exon junctions during RNA splicing. The SF3B1 gene is frequently mutated in tumors, such as myelodysplastic syndrome (20%) and chronic lymphocytic leukemia (15%). Because the functions of individual components of the spliceosome are largely unknown, SF3B1 inhibitors (such as pladienolide B, spliceostatin A, and herboxidiene) are widely used to repress spliceosome-mediated alternative splicing and induce apoptosis in various tumor cells.416

In 2021, Cheng group reported the SF3B1 degrader 215 (PROTAC-O4I2, Fig. 68) by fusing thalidomide to O4I.417 They found the degrader 215 (PROTAC-O4I2) selectively induced the degradation of SF3B1 and induced cellular apoptosis in a CRBN-dependent manner. In a Drosophila intestinal tumor model, the degrader 215 (PROTAC-O4I2) increased survival by interference with the maintenance and proliferation of stem cells. Thus, their results demonstrated that SF3B1 could be degraded by utilizing noninhibitory chemicals, which expanded the PROTAC target proteins.

The representative PROTAC targeting SF3B1

Among the membrane proteins that constitute about 27% of all proteins in the human genome, the solute carrier(SLC) family is the second largest membrane protein family in humans, and consists of more than 400 types of proteins divided into 52 families. In recent years, phylogenetic analysis has shown that 15 SLC families can be divided into four phylogenetic clusters, namely α, β, γ, and δ. The sequence analysis has shown that 24 SLC families belong to three PFAM clans, MFS, APC, and CPA/AT.418 SLCs are responsible for the absorption and transportation of several substances on cell membranes, including amino acids, nucleotides, sugars, inorganic ions, and drugs. Nearly 100 human SLCs that transport amino acids have been proposed, 60% of them have been confirmed to transport amino acids, and the rest are closely related to amino acid transporters known phylogenetically.419

In 2020, Furga group developed a SLC degrader 216 (d9A-2, Fig. 69) conjugated with a SLC9A1 inhibitor and pomalidomide with an optimized PEG linker.420 The degrader 216 (d9A-2) was a first-class SLC PROTAC, which could induce the high-efficiency degradation of its homologous targeted SLC9A1, as well as the degradation of other SLC9 members, such as the transporters SLC9A2 and SLC9A4. The proliferation inhibitory activity of the degrader 216 (d9A-2) on KBM7 cells was consistent with the evidence of degradation in the cytotoxicity test. In addition, the degrader 216 (d9A-2) treatment resulted in a variety of cancer cell intracellular pH (pHi) restoration obstacles and toxicity.

The representative PROTAC targeting SLC

The ATP-dependent chromatin remodeling complex BAF/PBAF changes the position and composition of nucleosomes through slipping and elimination to achieve dynamic regulation of chromatin structure. About one-fifth of tumors occur are closely related to the complex somatic mutations. The studies have found that two ATPases in the complex SMARCA2/4 are potential cancer treatment targets.421 SMARCA4 plays a tumor suppressor effect in solid tumors, but in AML it is necessary to maintain the oncogenic transcription program and drive proliferation.422 It has been reported that the double allosteric inhibitor of SMARCA2/4 ATP enzyme activity effectively inhibited the proliferation of the SMARCA4 mutant xenograft model.423

In 2019, Ciulli group developed a series of SMARCA2/4 degraders conjugated with a bromodomain ligand and VHL ligand.424 The degrader 217 (Fig. 70) successfully induced the moderate efficient degradation of SMARCA2/4 in MV4-11 cells with the Dmax about 65–70%. Guided by high-resolution ternary complex crystal structures of SMARCA2BD-217-VCB, they also designed and developed the degrader 218 (ACBI1, Fig. 70), a potent and cooperative degrader of SMARCA2, SMARCA4, and PBRM1. The degrader 218 (ACBI1) could effectively induce the degradation of SMARCA2, SMARCA4, and PBRM1 in MV4-11 cells with the DC50 value of 6, 11, and 32 nM, respectively.

The representative PROTACs targeting SMARCA2/4

In 2021, Chinnaiyan group also linked the same bromodomain ligand with VHL and developed a degrader 219 (AU-15330, Fig. 70) for SMARCA2 and SMARCA4.425 The degrader 219 (AU-15330) could induce the degradation of SMARCA2 and SMARCA4 in HEK293 and HeLa cells, but it also showed a certain degradation activity to PBRM1.

Steroid receptor coactivator (SRC) is a type of transcription coactivator, which usually contains three subtypes, namely SRC-1, SRC-2, and SRC-3.426 SRC-1 is the first identified transcription coactivator that can promote the activity of various transcription factors (TF), such as estrogen receptor α, progesterone receptor and so on. As a coactivator, SRC-1 can not only promote the effect of transcription factor, but also promote the formation of protein complexes, so SRC-1 plays an important role in the organism.427 Studies have shown that the expression level of SRC-1 is low in normal physiological body, and it is abnormally activated and highly expressed in tumor cells. Therefore, SRC-1 is considered to be an oncogenic protein. Although corresponding inhibitors have been developed for SRC, most of them target SRC-1 and SRC-3. The inhibitors are lack selectivity to SRC-1 and are prone to cause side effects.428 Therefore, using PROTAC technology to induce degradation of SRC-1 protein has become a promising method.

In 2020, Lim group designed and synthesized the degrader 220 (CL1-LY2, Fig. 71) that targeted SRC-1 based on specific SRC-1 ligand stapled peptide YL2 and pomalidomide.429 Although the SRC-1 binder was stapled peptide, it showed good binding affinity to SRC-1. In MDA-MB-231 cells, the degrader 220 (CL1-LY2) could induce degradation of SRC-1 protein at 10 μM, while it could not induce degradation of SRC-1 protein in Colo205 cells with low CRBN expression. In addition, it did not induce degradation of SRC-3, indicating that it had good selectivity.

The representative PROTAC targeting SRC-1

Tyrosinase is the main rate-limiting enzyme of melanin synthesis, and it plays an important role in skin protection and pigmentation. So far, almost all tyrosinase inhibitors are based on cheap and easily available mushroom tyrosinase (mTYR) obtained through in vitro screening, such as hydroquinone, arbutin, l-ascorbic acid, ellagic acid, and tranexamic acid.430 However, most tyrosinase inhibitors have certain side effects. For example, hydroquinone is toxic to human cells and can cause skin irritation and bone marrow toxicity, l-ascorbic acid is easily sensitive to heat, ellagic acid is insoluble and has poor bioavailability.431 In addition, in order to produce an inhibitory effect, tyrosinase inhibitors need to constantly occupy the active site of the tyrosinase, but high doses can cause undesirable off-target effects and cause damage to the skin.432

In 2021, Tang group designed and synthesized a tyrosinase degrader based on L-Dopa and pomalidomide.433 The degrader 221 (TD9, Fig. 72) had the best effect in inducing degradation of human tyrosinase, and it also showed obvious dose and time-dependent manner. It also proved that the degradation mechanism was achieved through the protein–ubiquitin system. In addition, they also used the low-toxicity degrader 221 (TD9) on zebrafish to reduce the synthesis of zebrafish melanin, highlighting the potential for the treatment of tyrosinase-related diseases.

The representative PROTAC targeting tyrosinase

Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, and antipyretic properties. Its pharmacological effects are not fully understood. It is not only related to the activities of cyclooxygenase 1 and 2, but also inhibit the biosynthesis of phospholipase A2 (PLA-2) and microsomal prostaglandin E synthase 2 (mPGES-2).434 Studies have shown that it had good activity against coronavirus infections, so it had great development potential in the field of anti-inflammatory and antiviral.435

In 2021, Goracci group designed a series of PROTACs based on Indomethacin and VHL,436 it was found that the degrader 222 (Fig. 73 and 223 (Fig. 73) had good inhibitory activities against a variety of coronaviruses through activity screening. Molecular simulations showed that PGES-2 may be the potential target of INM-based antiviral PROTAC.

The representative PROTACs targeting pan-coronavirus antiviral

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 240 million individuals worldwide, and the death toll has exceeded 4.9 million and is still rising. Although some vaccines have been widely used to prevent COVID-19, due to the high mortality and the occurrence of mutations, the development of curable drugs has become more and more important. Although some drugs have entered the clinical trial stage, the therapeutic effect is still to be observed. So using new technologies to develop new treatments has become a strategy.

Telaprevir-based PROTAC has been reported to be used for the degradation of HCV NS3/4A protease. The Mpro protein plays an important role in SARS-CoV-2, so PROTAC technology can be used to induce degradation of the Mpro protein so as to achieve the purpose of curing SARS-CoV-2. The applicability of PROTAC technology on SARS-CoV-2 has been discussed recently by different groups.

In order to apply PROTAC technology to the treatment of SARS-CoV-2, Sobhia group firstly assessed the similarity of NS3/4A and Mpro residues. The sequence alignment results showed that Telaprevir could be used in the design of Mpro degraders. Subsequently, the degraders 224–226 (Fig. 74) which were most likely to induce degradation of Mpro protein was generated by constructing a ternary complex model and simulating the protein–protein interaction.437

The representative PROTACs targeting SARS-CoV-2

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Currently, eighteen HDAC isoenzymes, which can be divided into four classes, have been discovered. Among them, class I HDACs, containing HDAC1, 2, 3, and 8, are well-known for their importance in cell motility, immunoregulation, and proliferation.438 More and more studies have shown that HDAC3 is closely related to the occurrence and development of tumors.439 However, because HDAC3 has a highly conserved catalytic domain, it is quite challenging to develop an isoenzyme-specific HDAC3.

In 2020, Dekker group reported the development of a novel PROTAC targeting HDAC3 by tethering the CRBN ligand pomalidomide and o-aminoanilide-based class I HDAC inhibitors.440 The degrader 227 (HD-TAC7, Fig. 75) induced the degradation of HDAC3 with a DC50 value of 0.32 μM in RAW 264.7 macrophages, whereas without influence on HDAC1 and 2. In the same years, Liao group developed a novel VHL-based degrader 228 (XZ9002, Fig. 75) that could selectively and potently induce the degradation of HDAC3 in a dose and time-dependent manner.441 Furthermore, the degrader 228 (XZ9002) had potent antiproliferative activity against cancer cells.

The representative PROTACs targeting HDAC3

Hematopoietic prostaglandin D synthase (HPGDS) is an attractive target for the treatment of a variety of diseases, including allergic diseases and Duchenne muscular dystrophy.442 To date, several types of H-PGDS inhibitors have been developed as therapies for allergic and inflammatory responses. However, no HPGDS inhibitors have yet been approved into clinical studies.443 Therefore, the development of novel agents having other modes of action to modulate the activity of H-PGDS is required.

In 2021, Demizu group reported the first H-PGDS degrader 229 (PROTAC(HPGDS)-1, Fig. 76) by coupling the HPGDS inhibitor TCF-007 with CRBN ligand pomalidomide.444 The degrader 229 (PROTAC(HPGDS)-1) effectively induced the selective degradation of H-PGDS protein and suppression of prostaglandin D2(PGD2) production. It was worth noting that the degrader 229 (PROTAC(HPGDS)-1) continued to inhibit the production of PGD2 after the removal of the drug, whereas the production of PGD2 was restored after the removal of TFC-007. In the same year, by using a docking simulation of the ternary complex of H-PGDS-PROTAC (H-PGDS)-1-cereblon, this group also successfully developed the degrader 230 (PROTAC(H-PGDS)-7, Fig. 76) without any linker.445 The degrader 230 (PROTAC(H-PGDS)-7) exhibited potent and selective degradation of H-PGDS with DC50 value of 17.3 pM after 6 h treatment, and potent suppression of prostaglandin D2 production in KU812 cells. In addition, the degrader 230 (PROTAC(H-PGDS)-7) had better inhibition of inflammatory cytokines than TFC-007 in a Duchenne muscular dystrophy model using mdx mice with cardiac hypertrophy. Therefore, the degrader 230 (PROTAC(H-PGDS)-7) was expected to play a role in biological research and clinical treatment.

The representative PROTACs targeting H-PGDS

IRAK1, a class of serine-threonine protein kinases associated with IL-1R and TLR signaling, plays a key role in initiating the innate immune response against foreign pathogens. Upon activation, TLR or IL/1Rs dimerizes and allows its intracellular structural domain to recruit the myeloid differentiation primary response 88 (MyD88) adaptor protein. MyD88 molecules oligomerize through the N-terminal death domain (DD) and recruit IRAK containing the DD structural domain to form a macromolecular signaling complex called myddosome, which leads to the autophosphorylation and activation of IRAK4. Activated IRAK4 phosphorylates IRAK1 and/or IRAK2, followed by the complementation of TNFR-associated factor 6 (TRAF6) into the myddosome. Activated TRAF6 is then released into the cytoplasm, where it triggers activation of the IκCB kinase (IKK)-nuclear factor-κB (NF-KB) cascade and MAP kinase (MAPK) signaling pathways.446,447,448,449 Despite some advances in the study of IRAK1, IRAK1 remains a challenging target in the field of traditional small-molecule inhibitors, partly due to the lack of understanding of the structural domains primarily responsible for its scaffolding function.

In 2021, Dai group first reported a series of IRAK1-targeting PROTAC based on IRAK1 inhibitor JH-I-25 and VHL ligands.450 The most potent degrader 231 (JNJ-1013, Fig. 77) showed better kinase selectivity and potent IRAK1 degradation activity with DC50 of 3 nM and Dmax of 96% in HBL-1 cells. In ABC DLBCL cells with the MyD88L265P mutation, the degrader 231 (JNJ-1013) effectively blocked downstream signaling pathway of IRAK1, induced apoptosis, and displayed stronger antiproliferative effects compared to IRAK1 inhibitors, indicating that the scaffold function of IRAK1 played a key role in ABC DLBCL cell survival.

The representative PROTAC targeting IRAK1

IRAK3 (interleukin-1 receptor-associated kinase 3, also known as IRAK-m) is a class 1 pseudokinase member of the IRAK family(along with IRAK1, IRAK2, and IRAK4).451 IRAK3 is believed to perform biological functions through a non-kinase-catalyzed scaffolding action.452 The expression of IRAK3 is mainly restricted to leukocytes, and it has been reported to inhibit pro-inflammatory signaling in innate leukocytes(monocytes, macrophages and neutrophils).453 Knockdown of IRAK3 in mice leads to reprogram of myeloid cells toward immune activation and promotes proliferation of effector T cells, which in turn helps to overcome immune suppression and enhance the host response to checkpoint inhibition.454,455 Since IRAK3 is a pseudokinase, it is unclear whether the molecule bound to IRAK3 has a functional role, and it is essential to employ some techniques to address this issue.

In 2020, Edmondsonet group first reported potent and selective IRAK3 degraders by conjugating a byproduct of IRAK4 inhibitors with CRBN and VHL ligands.456 the degrader 232 (Fig. 78) induced the degradation of IRAK3 with DC50 of 2 nM and Dmax of 98% in THP-1 cells and primary macrophages. Although not yet fully optimized, the degrader 232 was an excellent tool to study the biology of IRAK3 degradation in vitro.

The representative PROTAC targeting IRAK3

Playing a key role in both toll-like receptors (TLRs) and interleukin-1 receptors (1L-1R) signaling pathways, IRAK4 has been identified as a potential drug target involved in the innate immune process and has attracted widespread interest as a novel modality for the treatment of inflammatory diseases and oncology.457 Upon activation of TLRs or 1L-1R receptors, MYD88 will be recruited to the TIR domain of the receptor, followed by the IRAK family to form a myddosome complex. IRAK4 bound to MYD88 will lead to phosphorylation of IRAK1 and IRAK2 in the complexed and subsequent activation of the IkB kinase (IKK)-nuclear factor-kB (NF-kB) and the mitogen-activated protein kinase (MAPK) signaling pathways. Several reports have revealed an important role of IRAK4 in myddosome assembly in addition to its kinase catalytic function and its kinase backbone function, suggesting that overall inhibition or elimination of IRAK4 has a great potential to completely eliminate IL-1R/TLR assembly or the resulting signaling.458 Several investigators have begun to explore to use PROTACs to induce the degradation of IRAK4.

In 2019, Anderson group reported a series of PROTACs by incorporating IRAK4 ligand PF-06650833 to different E3 ligases.459 The VHL-based degrader 233 (Degrader-3, Fig. 79) induced the most potent degradation of IRAK4 with a DC50 of 151 nM in PBMCs and 36 nM in dermal fibroblast cells. However, the degrader 233 (Degrader-3) did not show additional potential benefit upon TLR7/8 stimulation in PBMCs compared to PF-06650833. And the inhibition of IL-6 and TNF-α was also not observed in IL-1β stimulated human dermal fibroblasts. Nevertheless, the possibility of targeting IRAK4 kinase allowed new therapeutic chances to treat autoimmune and oncological diseases.

The representative PROTACs targeting IRAK4

In 2020, Dai group reported a series of potential PROTACs based on CRBN ligands and a highly selective IRAK4 inhibitor for the degradation of IRAK4.460 These PROTACs showed moderate affinities to CRBN-DBB1 with binding affinity values ranging from 490 to 1080 nM. Among all of the PROTACs, the degrader 234 (Fig. 79) only induced the degradation of IRAK4 protein, displayed a remarkable selectivity of IRAK4 degradation in OCI-LY3 ABC DLBCL cells in a proteome-wide analysis. The degrader 234 induced the rapid degradation of IRAK4 with a DC50 value of 405 nM in HEK293T cells after 24 h of treatment. The results showed that neither IRAK4 kinase inhibition nor degradation resulted in cell death or growth inhibition, suggesting that the role of IRAK4 in the survival of ABC DLBCL cells was redundant. IRAK4 PROTACs characterized in this study provided useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable through pharmacological perturbation.

In 2021, Duan group disclosed a set of PROTACs by conjugating an IRAK4 inhibitor to pomalidomide via flexible linkers, including hydrophilic polyethylene glycol (PEG) and hydrophobic all-carbon chains.461 The most potent degrader 235 (Fig. 79) induced the degradation of IRAK4 in OCI-LY10 and TMD8 cells in a dose and time-dependent manner. In addition, the degrader 235 efficiently blocked the IRAK4-NF-κB signaling pathway and displayed a substantial advantage in inhibiting the growth of cells expressing the MYD88L265P mutation compared with the parent IRAK4 inhibitor.

Glycogen synthase kinase 3 (GSK-3) belongs to a multifunctional serine/threonine protein kinase under the phosphotransferase family. There are two isomers of mammalian GSK-3, namely GSK-3α and GSK-3β.458 GSK-3β controls the synthesis of glycogen to regulate the metabolism of glycogen, and affects the permeability of mitochondria and the releases of cytochrome C to regulate apoptosis. GSK-3β also acts on activation of the transcription factors and functions in many biological processes including embryonic development, cell differentiation, and insulin response.459 GSK-3β is abundant in brain and is associated with many neurodegenerative diseases and neurological disorders. Recently, GSK-3β has been described as a tumor suppressor due to its ability to phosphorylate the pro-oncogenic moleculars that affect the cell cycle and DNA repair.460

The first GSK-3β degrader was reported by Sun group in 2021.461 The degrader 236 (PG21, Fig. 80) was a powerful GSK-3β degrader, it could effectively induce the degradation of GSK-3β in a dose-dependent manner, which could induce 44.2% protein degradation at 2.8 μM. Mechanistic investigation determined that the degrader 236 (PG21) needed to rely on the intracellular UPS to induce GSK-3β degradation. In addition, it protected against glutamate-induced cell death in HT-22 cells.

The representative PROTACs targeting neurodegenerative diseases

PARK8 is one of the genes associated with Parkinson's disease (PD), which encodes leucine-rich repeat kinase 2 (LRRK2).462 To date, a large number of disease-associated LRRK2 mutations have been identified and five mutations (R1441C, R1441G, Y1699C, G2019S, and I2020T) are associated with PD pathogenesis. LRRK2 mutations, particularly the most common G2019S mutation, are observed in patients with autosomal dominant PD and apparently disseminated PD. Pathogenic mutations in the LRRK2 gene increase LRRK2 kinase activity, and LRRK2 kinase inhibitors are neuroprotective in preclinical models of Parkinson's disease. These findings make LRRK2 to be one of the important targets for the treatment of PD and targeting LRRK2 has become an effective treatment for PD.463

In 2020, Dömling group firstly reported the PROTACs targeting LRRK2 degradation.464 Series of potential LRRK2 PROTACs were constructed based on the LRRK2 inhibitors PF-06447475 or GNE-7915 and CRBN ligand pomalidomide. For example, the degrader 237 (Fig. 80) was a representative degrader, although the PROTACs were effective in inhibiting kinase activity and performed good cell permeability, the western blotting did not show any significant changes in LRRK2 protein level after the PROTACs treatment, which suggested that these PROTACs were unable to induce degradation of LRRK2.

α-Synuclein is mainly expressed in human brain cells and central nervous system cells. It is a presynaptic 140 amino acid that has no secondary structure in many tissues. The misfolding and aggregation of these proteins is a pathological feature of Parkinson's disease (PD) and other neurodegenerative diseases.465

In 2020, Crew group published the patent on α-Synuclein degraders.466 They designed and synthesized different degraders and tested their degradation activity in HEK293 TREX α-synuclein A53T cells. The experimental results showed that the degrader 238 (Fig. 80) and 239 (Fig. 80) induced degradation of α-Synuclein, and the degradation activity were 30–65% at 1 µM, which proved neuronal diseases related to α-Synuclein accumulation and aggregation (AD, PD, Dementia, etc.) could be treated by targeted degradation of α-Synuclein.

Tau is a cytoskeletal protein that regulates the formation and stability of microtubules by binding to neuronal microtubules, and maintains the structure and function of cytoskeletal neurons. AD and other neurological diseases can occur when the tau protein is defective and cannot sufficiently stabilize microtubules.467 Hyperphosphorylation and aggregation of tau protein destroy the microtubule structure, leading to neurofibrillary tangles in AD neurons. More and more evidence showed that the hyperphosphorylation of tau protein and the increase of p-tau protein aggregation are closely related to the occurrence and development of AD.468

In 2021, Wang group developed a novel tau protein degrader 240 (C004019, Fig. 80) by recruiting VHL E3 ligase.469 It showed selective and potent tau protein degradation both in vitro cellular models (HEK293 and SH-5Y5Y) and in vivo mice models (hTau-transgenic and 3xTg-AD). Most importantly, both single and multiple doses (once per 6 days for a total of five times) of subcutaneous degrader 240 (C004019) significantly reduced tau levels in the brains of wild-type, hTau-transgenic, and 3xTg-AD mice with improved synaptic and cognitive function. Therefore, the degrader 240 (C004019) could be considered as a promising drug candidate in related diseases.

TRKs are receptor tyrosine kinases encoded by the NTRK1/2/3 genes and contain mainly three members(TRKA/B/C), which are associated with the development and function of neuronal tissues.470 When the extracellular structural domains of TRKs bind to their corresponding ligands, they will induce dimerization and activation of the intracellular kinase domains of TRKs thereby providing signals to downstream PI3K/AKT, RAF/MEK/ ERK, and phospholipase C gamma(PLCγ).471 This ultimately leads to constitutive activation of the TRK pathway and promotes cell proliferation, survival, and malignant transformation.

In 2020, Chen group reported a series of selective and potent TRKA degraders by conjugating a pan-TRK inhibitor GNF-8625 to CRBN ligand thalidomide.472 The degrader 241 (CG416, Fig. 80) and 242 (CG428, Fig. 80) were the most promising degraders, whose DC50 were 0.48 nM and 0.36 nM in KM12 cells. The degrader 241 (CG416) and 242 (CG428) also induced degradation of wild-type TRKA with DC50 values of 1.26 nM and 2.23 nM in HEL cells. These two degraders exhibited more potent inhibition of cell growth than the TRK inhibitor GNF-8625. Further, they also exhibited good plasma exposure in mice. These results suggested that they were valuable chemical tools for investigating TRKA function.

TRKC, a member of the TRK family, is closely related to the development and function of neuronal tissues. The decrease in TRKC has been found in a variety of neurodegenerative diseases including AD, PD, and HD.473 In addition, overexpression and aberrant activation of TRKC has been observed in a variety of human tumors. Aberrant activation of TRKC and TRKC fusion proteins significantly induces growth rate, epithelial–mesenchymal transition(EMT), and oncogenic capacity through constitutive activation of the PI3K-AKT, RAS-MAP kinase (MAPK), and JAK2-STAT3 pathways.474

In 2019, Zhao group reported the first TRKC PROTACs based on the TRKC binder IY-IY (Kd = 112 nM) and different E3 ligase ligands.475 pomalidomide-based degrader 243 (Fig. 80) showed better degradation activity of TRKC with an estimated DC50 of 0.1–1.0 μM. TRKC degraders may provide opportunities for alternative treatment strategies for TRKC-related diseases.

Cas protein (CRISPR-associated protein) is a type of nuclease in the CRISPR (Clustered regularly interspaced short palindromic repeats) system, which plays an important role in the adaptive immune function of CRISPR system.476,477 It assists CRISPR-Cas systems in targeting and ultimately degrading foreign nucleic acids under the guidance of CRISPR RNA (crRNA).478,479 Cas protein can be divided into two classes (Classes 1 and 2) and six types (types I–VI) based on their specific mechanisms of guiding RNA biogenesis and targeting interference.480,481 There are several most widely used examples such as Cas9 (type II), Cas12 (type V), and Cas13 (type VI).482,483,484 Although CRISPR-Cas system has become a powerful tool to manipulate the human genome for gene therapy, it may still cause some side effects owing to off-target edits.485

In 2021, Cheng group developed engineered CasFCPF proteins (Cas9, dCas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe(FCPF) amino acid sequence (known as the π-clamp system) into Cas proteins, which could be labeled by perfluoroaromatics carrying thefluorescein. Then they designed a perfluoroaromatics-based degrader 244 (PROTAC-FCPF, Fig. 81) by using lenalidomide as E3 ligases ligand. The degrader 247 (PROTAC-FCPF) could induce the degradation of Cas9FCPF with IC50 of 167.2 nM in HEK293T cells and 143.4 nM in HeLa cells.486

The representative PROTACs targeting Cas protein, HMGCR and VEGFR2

HMG-CoA reductase (HMGCR) is an eight-channel transmembrane protein located in the endoplasmic reticulum (ER). It is the rate-limiting enzyme in the cholesterol biosynthetic pathway and a classical drug target of statins which can lower the cholesterol and preventing cardiovascular disease (CVD).487,488 Statins competitively bind to the active site of the enzyme through its HMG-like part to prevent the production of mevalonate and downstream derivatives including cholesterol. However, statins may cause a compensatory increase in protein expression489 which limits the maximal effectiveness of the drug and provoking some side effects including skeletal muscle damage.490,491 With the development of PROTACs, a promising therapeutic approach to induce degradation of proteins of interests, it might be able to solve this problem by chemical knockdown instead of small-molecule inhibition.492

The first degrader of HMGCR was reported by Rao group in 2020.493 They synthesized a class of PROTACs by linking Atorvastatin with CRBN ligands. The most potent and selective degrader 245 (P22A, Fig. 81) induced the degradation of HMGCR with DC50 of 0.1 μM. HMGCR was the first ER-localized and polytopic transmembrane protein successfully degraded by PROTAC. It highlighted the potential application of PROTAC technique in treating hypercholesterolemia and CVD.

In 2021, Xiang group developed the VHL-based PROTAC targeting HMGCR.494 By combining VHL with Lovastatin they demonstrated the most potent degrader 246 (Fig. 81) with IC50 of 0.25 µM. Furthermore, they conducted experiments to evaluate the HMGCR degradation potency in vivo and confirmed that the degrader 246 could be the active ingredient to lower cholesterol in vivo. The VHL-based degrader 246 was the first PROTAC targeting HMGCR with oral activity.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR2, previously known as KDR or Flk-1) is a member of VEGFR family of receptor tyrosine kinases (RTK)495,496 and plays an important role in angiogenesis. It is a highly active kinase and can stimulate many signaling pathways and varieties of biological responses. VEGFR2 is the key receptor that mediate the major growth and permeability actions of VEGF. Mice lacking VEGFR2 are unable to develop a vasculature and have rare endothelial cells.497,498 As a member of RTKs, the basic activation principles of VEGFR2 can be separated into three parts. The first procedure is dimerization of receptor monomers mediated by ligand, followed by the transphosphorylation with dimerized receptors and docking of signaling proteins to receptor phosphotyrosines.499,500 Occupancy-based RTK inhibitors are the traditional methods to intervene the pathological angiogenesis. However, owing to the high concentration and potential off-target side effects, it is necessary to develop PROTACs to treat angiogenesis.

In 2020, Zhang group designed several PROTACs based on the previously developed anti-angiogenesis agents.501 They combined their novel potent angiogenesis inhibitors S7 (biphenyl-aryl ureas incorporated with salicyladoxime) and E3 ubiquitin ligases ligand (VH032) with different lengths of dicarboxylic acid. The most potent degrader targeting VEGFR2 were degrader 247 (PROTAC-2, Fig. 81) and 248 (PROTAC-5, Fig. 81) with IC50 of 32.8 μM and 38.7 μM. The degrader 247 (PROTAC-2) could specifically reduce the protein levels of VEGFR2 and normalize the abnormal vessels without significant cytotoxicity.

In the above space, we summarize the main progress in the PROTAC field in the past 2 years. Compared with traditional small-molecule inhibitors, although PROTACs have shown obvious advantages, they also have similar disadvantages as small-molecule inhibitors. First, because PROTAC is developed based on POI inhibitors, it still has a certain degree of off-target effect. Secondly, due to the large molecular weight of PROTAC, it has poor cell-membrane permeability and poor pharmacokinetic(PK) properties, which greatly reduces its biological and therapeutic effects. In addition, although some PROTACs can efficiently induce the degradation of target proteins, their biological effects are so weak that they do not have an effective effect on the disease. Finally, most proteins do not have corresponding small-molecule binders for designing PROTACs, such as most transcription factors, which play an important role in the occurrence and development of diseases. The inhibitors of transcription factors are few, resulting in there is no binder available when designing PROTACs targeting transcription factors. This greatly limits the application of PROTAC technology. Therefore, it is of great significance to integrate other drug design strategies into PROTAC technology to solve the above shortcomings.

In order to solve the problems mentioned above, different types of PROTAC technologies have emerged in recent years, such as Antibody-PROTAC, Aptamer-PROTAC, Dual-target PROTAC, Folate-caged PROTAC, and Transcription factor-PROTACs, etc. Although these technologies are based on PROTACs, they all have advantages that are different from traditional PROTAC technologies. For example, Antibody-PROTAC can overcome the shortcomings of PROTACs off-target effect and weak tissue specificity, Aptamer-PROTAC can improve the cell-membrane permeability and pharmacokinetic(PK) properties of PROTACs. Although these new PROTAC-based technologies achieved degradation on very few proteins, they undoubtedly injected vitality into the development of PROTACs. So we will briefly introduce these new technologies.

Antibody-PROTAC is a new strategy to explore the combination of PROTAC and antibodies to assemble new Antibody-PROTAC conjugates. This technology can achieve the specific degradation of proteins in different cells and tissues, thereby optimizing the therapeutic window and maximizing the treatment window, reducing the side effects of broad-spectrum PROTAC and increasing its potential as a drug or chemical tool. What's more, although examples of intravenous (IV) and oral (PO) administration have been reported, most of the currently reported PROTACs are usually delivered to animals by subcutaneous (SC) or intraperitoneal (IP) administration routes,502,503,504,505 which result in their low bioavailability. Contrastly, the antibody-PROTAC conjugate technology can be used to overcome the potential delivery difficulties of PROTACs.

In 2020, Tate group reported a trastuzumab-PROTAC conjugate 249 (Ab-PROTAC 3, Fig. 82) based on trastuzumab and BRD4 degrader.506 Its antibody connection position is at the end of E3 ligase, and the antibody linker can be hydrolyzed in the cell and then releases active PROTAC and induces the degradation of BRD4 protein. They found that the conjugate 249 (Ab-PROTAC 3) only induced the degradation of BRD4 protein in HER2-positive breast cancer cell lines (SK-BR-3 and BT474), but not in HER2-negative cells (MCF-7 and MDA-MB 231). Significant degradation of BRD4 protein was observed in HER2+ cells incubated with 50 nM and 100 nM conjugate 249 (Ab-PROTAC 3) for 4 hours, but not detected at any concentration in the HER2− cell lines. This study verified the concept of tissue-specific Ab-PROTAC inducing BRD4 protein degradation, overcoming the limitations and selectivity of PROTACs, and laying the foundation for the development of new type PROTAC.

The representative PROTACs of antibody-PROTAC

Researchers from Genentech have also developed a series of Ab-PROTAC conjugates based on different antibodies and BRD4 degraders. In 2020, they reported an antibody-PROTAC conjugate 250 (CLL1-5, Fig. 82) based on CLL1 (C-type lectin-like molecule-1,507 also known as C-type lectin domain family 12 member A(CLEC12A) and the BRD4 degrader developed by themselves. To avoid the aggregation problem caused by high lipophilicity of linker, they linked six BRD4 degraders to CLL1. After antibody-mediated delivery, the higher drug loading of these conjugates could increase the intracellular drug concentration. Afterward, the conjugate 250 (CLL1-5) showed significant antitumor activity in the HL-60 AML xenograft model. All of these results proved that the mechanism of the conjugate 250 (CLL1-5) was the degradation of BRD4 protein induced by PROTAC after antibody-mediated delivery. At the same time, they also reported another antibody-PROTAC conjugate 251 (HER2-14, Fig. 82) based on HER2 and ERa degrader.508 It was subsequently proved that the antibody-PROTAC conjugates could be delivered dependently to MCF-7-neo/HER2 cells, and the conjugate 251 (HER2-14) had the best effect on ERα degradation, with a DC50 of 0.03 µg/mL and a Dmax of 94%. By synthesizing different antibody-PROTAC conjugates and testing their degradation activities, they proved that the linker between the antibody and PROTAC could adjust the stability of the conjugate and regulate the efficiency of releasing the degrader in the cell.

In 2021, they successively reported two types of antibody-PROTAC conjugates based on different BRD4 degraders and antibodies. First, they designed a series of BRD4 protein degraders based on JQ1. On this basis, they used different linkers to connect the degraders to the antibodies to synthesize antibody-PROTAC conjugates. The antibodies they used included STEAP1 (an antibody that recognizes the six transmembrane epithelial antigen of the prostate 1), CLL1, and HER2.509 It was found that in the PC-3-S1 cell line, the conjugate 252 (STEAP1-5a, Fig. 82) showed the best BRD4 degradation activity with a DC50 of 0.67 nM, but its antiproliferative activity was poor, with an IC50 value more than 790 nM. The possible reason may be the poor cell-membrane-permeability properties. Subsequently, on the basis of previous researches, by replacing the structure of the BRD4 degrader and synthesizing a series of antibody-PROTAC conjugates again they found the conjugate 253 (STEAP1-13a, Fig. 82) had good BRD4 protein-degradation activity,510 and its DC50 was 1.4 nM. At the same time, it showed good antiproliferative activity against PC-3-S1 cell line with IC50 of 29 nM. In addition, they also evaluated antiproliferative activity of the conjugate 253 (STEAP1-13a) in PC-3-S1 and HL-60 xenograft models and found that it achieved antigen-dependent antitumor activity in both prostate cancer and AML xenograft models.

In addition to antibody-PROTAC, Aptamer-PROTAC is also a new PROTAC technology that can improve water solubility, membrane permeability, and tumor targeting, which are typical disadvantages of traditional PROTACs.511 Aptamer is single-stranded nucleic acid with complex three-dimensional structures, which mainly include stems, loops, hairpins, and G4 polymers.512 They bind to target proteins with high specificity and affinity through special effects, including hydrogen bonding, van der Waals force, base stacking force and electrostatic effect.513 Due to special properties, aptamers are also called chemical antibodies by researchers. Compared with other targeting carriers, aptamers have the following advantages: (1) good tissue permeability, (2) good in vivo safety, (3) no obvious immunogenicity. They have been widely used in targeted therapy for human tumors.514,515

Inspired by the application of aptamers in targeted tumor therapy, in 2021 Sheng group designed and synthesized the first Aptamer-PROTAC conjugate (APC) by combining the aptamer with the BET protein degrader.516 They used a disulfide bond to connect the effective BET (BRD4) degrader and the nucleoside-dependent aptamer AS to synthesize the APC 254 (APR-Cy3, Fig. 83). The conjugate APC had a good effect on the BET degradation in MCF-7 breast cancer cells. It is the first time this study has proved that aptamer binding could help improve degrader targeting specificity, reduce toxicity, and enhance in vivo antitumor activity and protein-degradation activity. Therefore, the innovative APC technology established in this work may improve the drug similarity of traditional PROTACs and provide a new method for obtaining degraders which have better tumor tissue specificity and clinical efficacy.

The representative PROTACs of aptamer-PROTAC conjugates

In addition to using antibody-PROTAC and Aptamer-PROTAC to solve the targeting specificity and membrane permeability problems of PROTAC, how to improve the antitumor activity of PROTAC is also an urgent problem to be solved. But during the occurrence and development of cancer, there are usually many factors working together, including different kinds of kinases and growth factors, which can act independently or interfere with each other through signal networks.517 In the process of cancer treatment, tumor cells can easily obtain drug resistance by compensatory action or other signal pathways activation.518 Therefore, there are obvious limitations in drug treatment for a single target. In order to overcome the shortcomings, drug development for two or more targets has received more and more attention. Although "cocktail therapy" has achieved good clinical effects, long-term large doses and multiple use of different types of drugs have greatly reduced the compliance and quality of life of patients. Therefore, the development of single-drug-based dual-target drugs has become one of the strategies. The method is mainly to design a single molecule that combined two or more pharmacophores to target two or more antitumor targets at the same time. In the previous studies, there have been a large number of reports showing that the dual inhibitors based on this strategy can achieve good antitumor effects,519,520,521 and it has gradually become an alternative to combination therapy. The currently reported PROTACs are basically degraders designed based on only one protein. Although the one degrader can induce the degradation of different proteins,294,522,523,524,525 it is mostly caused by off-target effects of the protein ligands more than the rational design of multi-target degradation. Therefore, for two abnormal proteins in the same disease, it has become a way to treat the disease with a powerful degrader that is rationally designed based on two different ligands.

In 2021, Li group used the E3 ligase ligand and the EGFR inhibitor Gefitinib and the PARP inhibitor Olaparib to form star-shaped dual-target degraders,526 and then evaluated their degradation activities of the two proteins in H1299 cells. They found that the compound 255 (DP-V-4, Fig. 84) showed the best dual-target degradation activity. Its degradation activity of PARP was slightly better than that of EGFR, and the DC50 of PARP was 0.47 µM. As the concentration increased, its degradation activity gradually increased. Meanwhile, its degradation activity of EGFR was weak and a higher concentration was required to achieve effective degradation. Subsequently, they tested the antiproliferative activity of the compound 255 (DP-V-4) in H1299 cells. Its IC50 was 19.92 µM, which was between EGFR inhibitor Gefitinib (IC50 = 6.56 µM) and PARP inhibitor Olaparib (IC50 = 35.93 µM). The weaker antiproliferative activity of the compound 255 (DP-V-4) may be due to the larger molecular weight of the degrader, which leads to poor solubility and cell permeability.

The representative PROTAC of dual-target PROTACs

Folate-caged PROTACs is another technology that can improve the targeting specificity of PROTACs. Its basic principle is similar to Antibody-PROTAC, which introduces folate groups into PROTAC molecules to achieve release in targeted cells and tissues. Since folate receptor α (FOLR1) is low in normal tissues but is highly expressed in many human cancers, including multiple myelom (MM), lymphoma, and non-small cell lung cancer (NSCLC), folate-conjugating strategy is one of the commonly used drug delivery methods. This strategy has been widely and maturely used in tumor imaging and cancer-targeted drug delivery. At the same time, several FOLR1 targeted drugs have good antitumor effects and are currently in phase II/III clinical trials.527,528,529,530,531,532,533,534 Therefore, the use of folate-conjugating strategy in PROTACs technology (folate-caged PROTACs) to achieve the specific delivery of degraders to cancer cells has become a practical method. In this technology, folate releases active PROTAC by the action of cell endogenous hydrolase, and then the degrader induces the degradation of the target protein (POI). This strategy can eliminate the potential toxicity of degraders in normal tissues.

In 2021, Wei and Jin group reported the first folate-caged PROTAC. To ensure that the design of folate-caged PROTAC was universally applicable, they introduced a folate group on the E3 ubiquitin ligase ligand.535 Based on the reported BRD protein degrader ARV-771, they combined folate to the hydroxyl group of VHL via an ester bond to obtain folate-caged PROTAC 256 (Folate-ARV-771, Fig. 85). Subsequently, they tested the degradation activity of the folate-caged PROTAC 256 (Folate-ARV-771) on BRD4 protein in different cell lines and found that it could introduce the degradation of BRD4 protein effectively, which was equivalent to the degrader ARV-771 in cells that highly express folate, when it also has shown similar antiproliferative activity as ARV-771. Meanwhile, as the expression of folate was less in normal cells, the degradation activity of BRD4 protein and antiproliferative activity were weaker than that of degrader ARV-771. Those experiments have proved that folate-caged PROTAC could exert high-efficiency degradation activity and anti-proliferation activity in tumor cells with high folate expression while its activity was weak in normal cells, which has achieved the purpose of targeting specific cells to generate degradation activity.

The representative PROTACs of Folate-Caged PROTACs

In addition, they also designed other folate-caged PROTAs that target other proteins in order to verify the versatility of the method. They designed folate-caged PROTAC 257 (Folate-MS432, Fig. 85) which was based on the MEK protein degrader MS432, 258 (Folate-MS99, Fig. 85), and 259 (FA-S2-MS4048, Fig. 85) which were based on the ALK protein degrader MS99 and MS4048.536 And then it was also found that the folate-caged PROTACs have similar properties to that of Folate-ARV-771. They could efficiently introduce the degradation of the target proteins and have strong antiproliferative activity in high-expression-folate cell lines whereas having little effect on the target protein in normal cells.

Subsequently, they reported the first case of folate-caged molecular glue 260 (FA-S2-POMA, Fig. 85) that was designed and synthesized based on pomalidomide. They found that in cell lines that highly express folate, the molecular glue 260 (FA-S2-POMA) could efficiently induce the degradation of IKZF3, but its antiproliferative activity (IC50 = 1.0 µM) on MM.1S cells was weaker than that of pomalidomide (IC50 = 58 nM), which may be caused by the incomplete release of the molecular glue caged by folate in the cells.

In summary, they designed a universal folate-caged PROTAC design platform, through which they proved that the strategy could be applied to the protection and targeted delivery of PROTACs and molecular glues. This strategy could realize the selection of degraders for cancer cells and the safety for normal cells, and provide a reference method for avoiding the potential toxicity of protein degraders.

Transcription factors (TFs) are a class of proteins that are related to gene expression and regulation.537 In addition to normal regulatory functions, the cancer dependency map project (DepMap) also finds that TFs are also a class of essential proteins that maintain cancer cell proliferation and tumorigenesis, thus indicating that TFs are potential targets for tumor therapy.538 There are about 1600 TFs discovered and they can be divided into more than a dozen families according to their functions and structures. These TFs are different from traditional kinases as they do not have active pockets or allosteric regulatory site commonly found in kinases or other enzymes, so they are difficult to be targeted by small-molecule inhibitors.539 Among the reported TFs, only a few have small-molecule inhibitors, including NF-κB,540,541 STAT3/5,542,543,544 MYC,545,546, and nuclear receptors AR547,548 and ER.549 Most other TFs cannot be effectively targeted by small-molecule inhibitors, so the development of TFs small-molecule inhibitors is extremely difficult.

Since TFs can bind to specific DNA sequences and regulate the transcription process, different DNA sequences can be used instead of small-molecule inhibitors to target TFs and regulate their biological functions in theory. At present, researchers have experimentally determined more than 600 DNA sequences that can specifically bind to human TFs, which provides great possibilities for the targeted regulation of TFs. Inspired by the PROTAC technology, researchers are thinking about whether the small-molecule ligands that target the protein in the PROTAC can be replaced with the corresponding DNA sequence, so that it can form a TF-PROTAC to target specific TFs and induce their degradation to regulate the level of specific TFs and biological functions.

In 2021, Wei group reported a platform named TF-PROTAC,550 which linked DNA sequences to E3 ligase ligands via a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to selectively induce the degradation of the interested TFs (Fig. 86). They used NF-KB as the first TF to be degraded and designed a series of BCN-modified VHL ligands. At the same time, they generated a single strand DNA oligonucleotide, 5′-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3′ (hereafter termed as NF-κB-ODN)551 that specifically bind to NF-KB, then modified it by azide and obtained N3-NF-κB-ODN. Afterward, they tested the binding of BCN-modified VHL ligands to the modified N3-NF-κB-ODN in cells and found that most VHL ligands could interact well with N3-NF-κB-ODN. By having tested the degradation activity of different VHL ligands to p65 (a subunit of NF-KB) in HeLa cells, they found that dNF-κB #15 and dNF-κB #16 showed the best activity to induce the degradation of p65 and had good antiproliferative activity in tumor cells.

The representative PROTACs of TF-PROTACS

Moreover, they used this technology to design a TF-PROTAC that induced the degradation of E2F. Similar to the above method, they selected a double-strand DNA, in which the sense chain was 5′-CTAGATTTCCCGCG-3′ and the antisense chain was 5′-CTAGCGCGGAAAT-3′ (hereafter named as E2F-ODN)552 that targeted E2F and modified it by azide to obtain N3-E2F-ODN. Then, the degradation activities of different VHL ligands to E2F were tested in HeLa cells, and it was found that dE2F #16 and dE2F #17 showed the best E2F degradation activity and also had good antiproliferative activity in tumor cells. This was the first report of the technology for targeted degradation of TFs. The experimental results have shown this platform is a universal technology for inducing the degradation of TFs and that it has overcome the shortcomings of traditional small-molecule inhibitors and thus provided a possible solution to target the undruggable TFs.

In 2021, Crews group developed a novel targeted TFs degradation technology "oligoTRAFTACs"553 (Fig. 86), which is based on the binding of oligonucleotide sequences to TFs, the binding of E3 ligase ligands to E3 ligase, and the ubiquitination degradation system in vivo. This technology requires the generation of a chimeric oligoTRAFTAC via a copper-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction between an alkyne-oligonucleotide and an azide-containing VHL ligand, which then acts on the target TFs, resulting in the degradation of a transcription factor of interest. In this study, they successfully induced the degradation of c-Myc and brachyury using two different oligoTRAFTACs, and also demonstrated the feasibility of applying oligoTRAFTACs to degrade brachyury in vivo in zebrafish.

Apart from the new technologies based on PROTAC described above, researchers have also developed a series of other new technologies, such as BioPROTACs,554 Covalent PROTACs,555 Photocaged PROTACs,556 Pre-fused PROTACs,557 RNA-PROTAC,558,559,560 Semiconducting polymer nano-PROTACs,561 Trivalent PROTACs212,562, and so on. These innovations can not only induce the degradation of target proteins but also induce the degradation of RNA, make up for the related shortcomings of traditional PROTAC and achieve the control of PROTAC on space and time scales. They have been well applied in a variety of cells, and laid the foundation for the development and maturity of PROTAC technology. Since having been reviewed in literatures, these technologies are only briefly discussed in this article.

In addition to research tools, PROTACs also have great potential applications in disease treatment. It has become a new mode of drug discovery, which has the potential to change traditional drug discovery and may become a new blockbuster therapy.

Update to March 2022, about a dozen PROTACs around the world have entered the clinical development stage (Table 7). Among them, Arvinas’ ARV-110 and ARV-471 have entered clinical phase II stage, which are the fastest clinical progress in PROTAC drugs. Some R&D start-ups have gained the attention of large global pharmaceutical companies such as Roche, Sanofi, Merck, Pfizer, Gilead, etc. In addition, Chinese companies have also actively participated in the development, including BeiGene, Kintor, Haisco, Nuocheng Jianhua, Ascentage, Hengrui, etc.

Based on the application time of clinical trials, there are two applications in 2019, one in 2020, nine in 2021, and eight in 2022. It can be seen that clinical applications are currently in an explosive growth mode. In the future, more and more companies will participate in clinical trials of PROTAC-related drugs.

Based on the targets in clinical trials, AR has the most examples by six, BTK is followed by four, ER and BRD9 are two, respectively, and all the other remaining targets are one. It can be seen that currently pharmaceutical companies are more inclined to targets with relatively high maturity.

Today, PROTAC technology has become a new strategy for new drug research and development, providing new methods for the treatment of diseases. The next few years will be a critical period for the development of PROTACs. More and more PROTACs will enter preclinical and clinical research to further test the therapeutic effect of PROTACs. It is expected that PROTAC technology will provide benefits for human disease treatment and life health in the future.

Since the first case of PROTAC was reported in 2001, PROTAC technology has entered the stage of practical application from concept. Although the field of PROTAC has been rapidly developed in the past 20 years, there are still many challenges to be solved. These challenges mainly come from two aspects, namely PROTAC molecular design and optimization of druggability, and comprehensive evaluation of biological activity.

The first is about the molecular design and druggability of PROTAC, involving target protein ligands, new E3 ligase ligands, and new linking chains.

Design of target protein ligand. Most of the PROTAC molecules that have been reported are kinase degraders, and there are few examples of targeting undruggable targets. PROTAC molecules that target kinases are usually obtained by using existing small-molecule inhibitors as target protein ligands for modification. These inhibitors are designed for target kinase binding pockets. For undruggable targets, including transcription factors, phosphatases, protein–protein interactions, etc., the overall progress is slow due to the lack of effective small-molecule ligands.

New E3 ligase ligand. There are still few E3 ligases that can be used in PROTAC design. How to expand the E3 ubiquitin ligases that can be used in PROTAC technology is also one of the challenges that PROTAC faces.

How to connect POI and E3 ligase ligand. Researchers have realized that the linkers can profoundly affect the activity, selectivity, and druggability of PROTAC molecules. How to efficiently design and connect POI and E3 ligase ligands is also an important issue.

Pharmacological properties. The PROTAC molecule is usually not good enough as a drug due to its large molecular weight. How to optimize quickly is a huge challenge.

PROTAC molecules generally have a "hook" effect. Can a reasonable molecular design be used to weaken or even eliminate this concentration-dependent problem? Obviously, conventional medicinal chemistry screening and optimization methods are not suitable for the rapid development and optimization of PRTOAC molecules, especially for undruggable targets.

Aiming at the above-mentioned problems, AI technology (protein structure prediction), virtual drug screening technology and DEL screening technology, etc. can help develop the corresponding ligands for the target protein and E3 ligase. Based on these screened ligands, it is necessary to develop efficient synthesis methods to quickly and effectively construct large-scale PROTAC molecular libraries characterized by skeletal diversity for high-throughput screening and optimization of molecular druggability. In terms of molecular design, the existing ternary complex structures of PROTAC molecule with POI and E3 ligase proteins are still very few. In the future, more complex information obtained by X-Ray or cryo-electron microscopy will help in better molecular design. In recent years, breakthroughs in the ability to predict the protein and related complex structure by alphafold2 may also contribute to the design of PROTACs. For molecular optimization, increasing the overall rigidity of the molecule and reducing the molecular weight can generally improve the drug-like properties of the hit compounds, such as oral bioavailability, ADME, etc.

The second is about biological activity evaluation. This involves the screening of PROTAC molecules, evaluation of druggability, and pharmacological evaluation.

PROTAC molecular screening. The existing technical methods mainly rely on immunoblotting methods and proteomics methods, which are not only time-consuming, labor-intensive, low-efficiency, and high-cost. In recent years, screening technologies such as fluorescent tags and HiBiT have been gradually introduced. In the future, more new high-throughput and high-sensitivity methods are needed for rapid and accurate assessment.

Evaluation of druggability. In addition to the conventional evaluation indicators such as solubility, in vitro and in vivo activity, toxicity, and other drug-like properties. The role of PROTAC is to catalyze the cycle, so traditional methods cannot accurately assess the properties of PROTAC's PK and PD. For PROTAC molecules, there is a great need to develop PK/PD models that are more consistent with protein degradation as a new drug modality.

How to better understand the degradation activity or degradability, selectivity, possible off-target effects, and pharmacological effects of PROTAC molecules (based on different targets, different cell lines, and different animal models). And how to correspondingly achieve differentiation in clinical treatment.

None of these discussed questions currently have ready-made answers, but we believe that as more and more research progresses, the whole field will be greatly advanced. With the development of more biological, pharmacological, and clinical research, new evaluation methods and systems will gradually be set up to solve these problems. It is believed that in the future, more and more PROTAC molecules will not only be used as tools for basic biological research but will also enter the clinic to solve the actual needs of patients.

Sakamoto, K. M. et al. PROTACs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. USA 98, 8554–8559 (2001).

Article CAS PubMed PubMed Central Google Scholar

Winter, G. E. et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

Article CAS PubMed PubMed Central Google Scholar

Neklesa, T. K. et al. ARV-110: an androgen receptor PROTAC degrader for prostate cancer. Am. Assoc. Cancer Res. 78, 5236 (2018).

Article Google Scholar

Neklesa, T. K. et al. ARV-110: an oral androgen receptor PROTAC degrader for prostate cancer. J. Clin. Oncol. 37, 14–16 (2019).

Halford, B. Arvinas unveils PROTAC structures. Chem. Eng. N. 99, 5 (2021).

Article Google Scholar

Sun, X. Y. et al. PROTACs: great opportunities for academia and industry. Signal Transduct. Target. Ther. 4, 64 (2019).

Article PubMed PubMed Central Google Scholar

Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

Article CAS PubMed Google Scholar

Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat. Rev. Drug Discov. 21, 181–200 (2022).

Article PubMed PubMed Central CAS Google Scholar

Webb, T., Craigon, C. & Ciulli, A. Targeting epigenetic modulators using PROTAC degraders: current status and future perspective. Bioorg. Med. Chem. Lett. 63, 128653 (2022).

Article CAS PubMed Google Scholar

Scholes, N. S., Mayor-Ruiz, C. & Winter, G. E. Identification and selectivity profiling of small-molecule degraders via multi-omics approaches. Cell Chem. Biol. 28, 1048–1060 (2021).

Article CAS PubMed Google Scholar

Dale, B. et al. Advancing targeted protein degradation for cancer therapy. Nat. Rev. Cancer 21, 638–654 (2021).

Article CAS PubMed PubMed Central Google Scholar

He, S. P., Dong, G. Q., Cheng, J. F., Wu, Y. & Sheng, C. Q. Strategies for designing proteolysis targeting chimaeras (PROTACs). Med. Res. Rev. 42, 1280–1342 (2022).

Weng, G. Q. et al. PROTAC-DB: an online database of PROTACs. Nucleic Acids Res. 49, D1381–D1387 (2021).

Article CAS PubMed Google Scholar

Mohler, M. L. et al. An overview of next-generation androgen receptor-targeted therapeutics in development for the treatment of prostate cancer. Int. J. Mol. Sci. 22, 2124–2144 (2021).

Article CAS PubMed PubMed Central Google Scholar

Asangani, I. et al. Using biochemistry and biophysics to extinguish androgen receptor signaling in prostate cancer. J. Biol. Chem. 296, 100240–100257 (2021).

Article CAS PubMed PubMed Central Google Scholar

Han, X. et al. Discovery of ARD-69 as a highly potent proteolysis targeting chimera (PROTAC) degrader of androgen receptor(AR) for the treatment of prostate cancer. J. Med. Chem. 62, 941–964 (2019).

Article CAS PubMed Google Scholar

Zhao, L. J., Han, X., Lu, J. F., Donna, M. E. & Wang, S. M. A highly potent PROTAC androgen receptor(AR) degrader ARD-61 effectively inhibits AR-positive breast cancer cell growth in vitro and tumor growth in vivo. Neoplasia 22, 522–532 (2020).

Article CAS PubMed PubMed Central Google Scholar

Han, X. et al. Discovery of highly potent and efficient PROTAC degraders of androgen receptor(AR) by employing weak binding affinity VHL E3 ligase ligands. J. Med. Chem. 62, 11218–11231 (2019).

Article CAS PubMed Google Scholar

Han, X. et al. Strategies toward discovery of potent and orally bioavailable proteolysis targeting chimera degraders of androgen receptor for the treatment of prostate cancer. J. Med. Chem. 64, 12831–12854 (2021).

Article CAS PubMed PubMed Central Google Scholar

Xiang, W. G. et al. Discovery of ARD-2585 as an exceptionally potent and orally active PROTAC degrader of androgen receptor for the treatment of advanced prostate cancer. J. Med. Chem. 64, 13487–13509 (2021).

Article CAS PubMed PubMed Central Google Scholar

Zhang, X. Y. et al. DCAF11 supports targeted protein degradation by electrophilic proteolysis-targeting chimeras. J. Am. Chem. Soc. 143, 5141–5149 (2021).

Article CAS PubMed PubMed Central Google Scholar

Kim, S. A. et al. A novel cereblon modulator for targeted protein degradation. Eur. J. Med. Chem. 166, 65–74 (2019).

Article CAS PubMed Google Scholar

Akshay, D. T. et al. Design and characterization of cereblon-mediated androgen receptor proteolysis-targeting chimeras. Eur. J. Med. Chem. 208, 112769–112785 (2020).

Article CAS Google Scholar

Chen, L. R. et al. Discovery of A031 as effective proteolysis targeting chimera(PROTAC) androgen receptor(AR) degrader for the treatment of prostate cancer. Eur. J. Med. Chem. 216, 113307–113320 (2021).

Article CAS PubMed Google Scholar

Kim, G. Y. et al. Chemical degradation of androgen receptor (AR) using Bicalutamide analog-thalidomide PROTACs. Molecules 26, 2525–2543 (2021).

Article CAS PubMed PubMed Central Google Scholar

Duncan, E. S. et al. Systematic investigation of the permeability of androgen receptor PROTACs. ACS Med. Chem. Lett. 11, 1539–1547 (2020).

Article CAS Google Scholar

Yang, D. et al. Design, synthesis, and biological evaluation of small molecule PROTACs for potential anticancer effects. Med. Chem. Res. 29, 334–340 (2020).

Article CAS Google Scholar

Geun, T. L. et al. Effects of MTX-23, a novel PROTAC of androgen receptor splice variant-7 and androgen receptor, on CRPC resistant to second-line antiandrogen therapy. Mol. Cancer Ther. 20, 490–499 (2021).

Article Google Scholar

Liang, J. J. et al. Designed, synthesized and biological evaluation of proteolysis targeting chimeras(PROTACs) as AR degraders for prostate cancer treatment. Bioorg. Med. Chem. 45, 116331–116342 (2021).

Article CAS PubMed Google Scholar

Bhumireddy, A. et al. Design, synthesis, and biological evaluation of phenyl thiazole-based AR-V7 degraders. Bioorg. Med. Chem. Lett. 55, 128448 (2022).

Article CAS PubMed Google Scholar

Lavoie, H. & Therrien, M. Regulation of RAFprotein kinases in ERK signalling. Nat. Rev. Mol. Cell Biol. 16, 281–298 (2015).

Article CAS PubMed Google Scholar

Simanshu, D. K., Nissley, D. V. & McCormick, F. RAS proteins and their regulators in human disease. Cell 170, 17–33 (2017).

Article CAS PubMed PubMed Central Google Scholar

Terrell, E. M. & Morrison, D. K. Ras-mediated activation of the Raf family kinases. Cold Spring Harb. Perspect. Med. 9, a033746–a033760 (2019).

Article CAS PubMed PubMed Central Google Scholar

Bollag, G. et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596–599 (2010).

Article CAS PubMed PubMed Central Google Scholar

Rajakulendran, T., Sahmi, M., Lefrancois, M., Sicheri, F. & Therrien, M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature 461, 542–545 (2009).

Article CAS PubMed Google Scholar

Thevakumaran, N. et al. Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation. Nat. Struct. Mol. Biol. 22, 37–43 (2015).

Article CAS PubMed Google Scholar

Chen, H., Chen, F. H., Pei, S. N. & Gou, S. H. pomalidomide hybrids act as proteolysis targeting chimeras: synthesis, anticancer activity and B-Raf degradation. Bioorg. Chem. 87, 191–199 (2019).

Article CAS PubMed Google Scholar

Han, X. R. et al. Discovery of selective small molecule degraders of BRAF-V600E. J. Med. Chem. 63, 4069–4080 (2020).

Article CAS PubMed Google Scholar

Ganna, P. et al. Functional characterization of a PROTAC directed against BRAF mutant V600E. Nat. Chem. Biol. 16, 1170–1178 (2020).

Article CAS Google Scholar

Shanique, A. et al. Mutant-selective degradation by BRAF-targeting PROTACs. Nat. Commun. 12, 920–930 (2021).

Article CAS Google Scholar

Nairn, A. C., Bhagat, B. & Palfrey, H. C. Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues. Proc. Natl Acad. Sci. USA 82, 7939–7943 (1985).

Article CAS PubMed PubMed Central Google Scholar

Ruys, S. P. D. et al. Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase. Biochem. J. 442, 681–692 (2012).

Article CAS Google Scholar

Beckelman, B. C. et al. Genetic reduction of eEF2 kinase alleviates pathophysiology in Alzheimer disease model mice. J. Clin. Invest. 129, 820–833 (2019).

Article PubMed PubMed Central Google Scholar

Rellos, P. et al. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation. PLoS Biol. 8, e1000426 (2010).

Article PubMed PubMed Central CAS Google Scholar

Will, N. et al. Structural dynamics of the activation of elongation factor 2 kinase by Ca2+ Calmodulin. J. Mol. Biol. 430, 2802–2821 (2018).

Article CAS PubMed PubMed Central Google Scholar

Leprivier, G. et al. The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation. Cell 153, 1064–1079 (2013).

Article CAS PubMed PubMed Central Google Scholar

Liu, Y. et al. Designing an eEF2K-targeting PROTAC small molecule that induces apoptosis in MDA-MB-231 cells. Eur. J. Med. Chem. 204, 112505 (2020).

Article CAS PubMed Google Scholar

Sharma, S. V., Bell, D. W., Settleman, J. & Haber, D. A. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 7, 169–181 (2007).

Article CAS PubMed Google Scholar

Krause, D. S. & Etten, R. A. V. Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. 353, 172–187 (2005).

Article CAS PubMed Google Scholar

Nicholson, R. I., Gee, J. M. W. & Harper, M. E. EGFR and cancer prognosis. Eur. J. Cancer 37, 9–15 (2001).

Article Google Scholar

Cheng, M. et al. Discovery of potent and selective epidermal growth factor receptor(EGFR) bifunctional small-molecule degraders. J. Med. Chem. 63, 1216–1232 (2020).

Article CAS PubMed PubMed Central Google Scholar

He, K. L. et al. Discovery and biological evaluation of proteolysis targeting chimeras(PROTACs) as an EGFR degraders based on osimertinib and lenalidomide. Bioorg. Med. Chem. Lett. 30, 127167 (2020).

Article CAS PubMed Google Scholar

Wang, K. & Zhou, H. P. Proteolysis targeting chimera(PROTAC) for epidermal growth factor receptor enhances anti-tumor immunity in non-small cell lung cancer. Drug Dev. Res. 82, 422–429 (2021).

Article CAS PubMed Google Scholar

Zhang, H. et al. Discovery of potent epidermal growth factor receptor(EGFR) degraders by proteolysis targeting chimera(PROTAC). Eur. J. Med. Chem. 189, 112061 (2020).

Article CAS PubMed Google Scholar

Zhao, H. Y. et al. Discovery of potent small molecule PROTACs targeting mutant EGFR. Eur. J. Med. Chem. 208, 112781 (2020).

Article CAS PubMed Google Scholar

Zhang, X. et al. Design and synthesis of selective degraders of EGFRL858R/T790M mutant. Eur. J. Med. Chem. 192, 112199 (2020).

Article CAS PubMed Google Scholar

Qu, X. J. et al. Effective degradation of EGFRL858R+T790M mutant proteins by CRBN based PROTACs through both proteosome and autophagy/lysosome degradation systems. Eur. J. Med. Chem. 218, 113328 (2021).

Article CAS PubMed Google Scholar

Jia, Y., Polunovsky, V., Bitterman, P. B. & Wagner, C. R. Cap-dependent translation initiation factor eIF4E: an emerging anticancer drug target: cap-dependent translation initiation EIF4E inhibition. Med. Res. Rev. 32, 786–814 (2012).

Article CAS PubMed PubMed Central Google Scholar

Lazaris-Karatzas, A., Montine, K. S. & Sonenberg, N. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5’ cap. Nature 345, 544–547 (1990).

Article CAS PubMed Google Scholar

Kerekatte, V. et al. The protooncogene/translation factor eIF4E: a survey of its expression in breast carcinomas. Int. J. Cancer 64, 27–31 (1995).

Article CAS PubMed Google Scholar

Moerke, N. J. et al. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 128, 257–267 (2007).

Article CAS PubMed Google Scholar

Fischer, P. D. et al. A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders. Eur. J. Med. Chem. 219, 113435 (2021).

Article CAS PubMed Google Scholar

Katzenellenbogen, J. A., Mayne, C. G., Katzenellenbogen, B. S., Greene, G. L. & Chandarlapaty, S. Structural underpinnings of oestrogen receptor mutations in endocrine therapy resistance. Nat. Rev. Cancer 18, 377–388 (2018).

Article CAS PubMed PubMed Central Google Scholar

Tryfonidis, K., Zardavas, D., Katzenellenbogen, B. S. & Piccart, M. Endocrine treatment in breast cancer: Cure, resistance and beyond. Cancer Treat. Rev. 50, 68–81 (2016).

Article PubMed Google Scholar

Xu, B. H. et al. Dalpiciclib or placebo plus fulvestrant in hormone receptor-positive and HER2-negative advanced breast cancer: a randomized, phase 3 trial. Nat. Med. 27, 1904–1909 (2021).

Article CAS PubMed Google Scholar

Yi, J. H. et al. Anti-tumor efficacy of fulvestrant in estrogen receptor positive gastric cancer. Sci. Rep. 4, 7592 (2014).

Article CAS PubMed PubMed Central Google Scholar

Dai, Y. X. et al. Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α. Eur. J. Med. Chem. 187, 111967 (2020).

Article CAS PubMed Google Scholar

Gonzalez, T. L. et al. Targeted degradation of activating estrogen receptor α ligand‑binding domain mutations in human breast cancer. Breast Cancer Res. Treat. 180, 611–622 (2020).

Article CAS PubMed Google Scholar

Roberts, B. L. et al. Two-stage strategy for development of proteolysis targeting chimeras and its application for estrogen receptor degraders. ACS Chem. Biol. 15, 1487–1496 (2020).

Article CAS PubMed Google Scholar

Disch, J. S. et al. Bispecific estrogen receptor α degraders incorporating novel binders identified using DNA-encoded chemical library screening. J. Med. Chem. 64, 5049–5066 (2021).

Article CAS PubMed Google Scholar

Kargbo, R. B. PROTAC-mediated degradation of estrogen receptor in the treatment of cancer. ACS Med. Chem. Lett. 10, 1367–1369 (2019).

Article CAS PubMed PubMed Central Google Scholar

Lu, A. S., Rouhimoghadam, M., Arnatt, C. K., Filardo, E. J. & Salem, A. K. Proteolytic targeting chimeras with specificity for plasma membrane and intracellular estrogen receptors. Mol. Pharmaceutics 18, 1455–1469 (2021).

Article CAS Google Scholar

Beenken, A. & Mohammadi, M. The FGF family: biology, pathophysiology and therapy. Nat. Rev. Drug Discov. 8, 235–253 (2009).

Article CAS PubMed PubMed Central Google Scholar

Babina, I. S. & Turner, N. C. Advances and challenges in targeting FGFR signalling in cancer. Nat. Rev. Cancer 17, 318–332 (2017).

Article CAS PubMed Google Scholar

Xie, Y. L. et al. FGF/FGFR signaling in health and disease. Signal Transduct. Target Ther. 5, 181 (2020).

Article CAS PubMed PubMed Central Google Scholar

Xue, W. J., Li, M. T., Chen, L., Sun, L. P. & Li, Y. Y. Recent developments and advances of FGFR as a potential target in cancer. Future Med. Chem. 10, 2109–2126 (2018).

Article CAS PubMed Google Scholar

Katoh, M. Fibroblast growth factor receptors as treatment targets in clinical oncology. Nat. Rev. Clin. Oncol. 16, 105–122 (2019).

Article CAS PubMed Google Scholar

Krook, M. A. et al. Fibroblast growth factor receptors in cancer: genetic alterations, diagnostics, therapeutic targets and mechanisms of resistance. Br. J. Cancer 124, 880–892 (2021).

Article CAS PubMed Google Scholar

Facchinetti, F. et al. Facts and new hopes on selective FGFR inhibitors in solid tumors. Clin. Cancer Res. 26, 764–774 (2020).

Article CAS PubMed Google Scholar

Dai, S. Y., Zhou, Z., Chen, Z. C., Xu, G. Y. & Chen, Y. H. Fibroblast growth factor receptors (FGFRs): structures and small molecule inhibitors. Cells 8, 614 (2019).

Article CAS PubMed Central Google Scholar

Liang, Q. et al. Recent advances of dual FGFR inhibitors as a novel therapy for cancer. Eur. J. Med. Chem. 214, 113205 (2021).

Article CAS PubMed Google Scholar

Du, G. Y. et al. Discovery of a potent degrader for fibroblast growth factor receptor1/2. Angew. Chem. Int. Ed. Engl. 60, 15905–15911 (2021).

Article CAS PubMed Google Scholar

Li, R., Pourpak, A. & Morris, S. W. Inhibition of the insulin-like growth factor-1 receptor(IGF1R) tyrosine kinase as a novel cancer therapy approach. J. Med. Chem. 52, 4981–5004 (2009).

Article CAS PubMed PubMed Central Google Scholar

Yeatman, T. J. A renaissance for SRC. Nat. Rev. Cancer 4, 470–480 (2004).

Article CAS PubMed Google Scholar

Lee, H. J. et al. Development of a 4-aminopyrazolo[3,4-d]pyrimidine-based dual IGF1R/Src inhibitor as a novel anticancer agent with minimal toxicity. Mol. Cancer 17, 50 (2018).

Article PubMed PubMed Central CAS Google Scholar

Manda, S., Lee, N. K., Oh, D. C. & Lee, J. Design, synthesis, and biological evaluation of proteolysis targeting chimeras(PROTACs) for the dual degradation of IGF-1R and Src. Molecules 25, 1948 (2020).

Article CAS PubMed Central Google Scholar

Milburn, M. V. et al. Molecular switch for signal transduction-structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939–945 (1990).

Article CAS PubMed Google Scholar

Ito, Y. et al. Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein. Biochemistry 36, 9109–9119 (1997).

Article CAS PubMed Google Scholar

Scheffzek, K. et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338 (1997).

Article CAS PubMed Google Scholar

Ostrem, J. M. & Shokat, K. M. Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design. Nat. Rev. Drug Discov. 15, 771–785 (2016).

Article CAS PubMed Google Scholar

Kargbo, R. B. PROTAC-mediated degradation of KRAS protein for anticancer therapeutics. ACS Med. Chem. Lett. 11, 5–6 (2019).

Article PubMed PubMed Central CAS Google Scholar

Zeng, M. et al. Exploring targeted degradation strategy for oncogenic KRASG12C. Cell Chem. Biol. 27, 19–31 (2020).

Article CAS PubMed Google Scholar

Bond, M. J. et al. Targeted degradation of oncogenic KRASG12C by VHL-recruiting PROTACs. ACS Cent. Sci. 6, 1367–1375 (2020).

Article CAS PubMed PubMed Central Google Scholar

Li, L. et al. Discovery of KRas G12C-IN-3 and pomalidomide-based PROTACs as degraders of endogenous KRASG12C with potent anticancer activity. Bioorg. Chem. 117, 105447 (2021).

Article CAS PubMed Google Scholar

Yang, F. et al. Efficient targeted oncogenic KRASG12C degradation via first reversible-covalent PROTAC. Eur. J. Med. Chem. 230, 114088 (2022).

Article CAS PubMed Google Scholar

Wei, J. L. et al. Discovery of a first-in-class mitogen-activated protein kinase linase 1/2 degrader. J. Med. Chem. 62, 10897–10911 (2019).

Article CAS PubMed Google Scholar

Hu, J. P. et al. Potent and selective mitogen-activated protein kinase kinase 1/2 (MEK1/2) heterobifunctional small-molecule degraders. J. Med. Chem. 63, 15883–15905 (2020).

Article CAS PubMed PubMed Central Google Scholar

Vollmer, S. et al. Design, synthesis, and biological evaluation of MEK PROTACs. J. Med. Chem. 63, 157–162 (2020).

Article CAS PubMed Google Scholar

Dhanasekaran, R. et al. The MYC oncogene-the grand orchestrator of cancer growth and immune evasion. Nat. Rev. Clin. Oncol. 19, 23–36 (2022).

Article CAS PubMed Google Scholar

Ahmadi, S. E., Rahimi, S., Zarandi, B., Chegeni, R. & Safa, M. MYC: a multipurpose oncogene with prognostic and therapeutic implications in blood malignancies. J. Hematol. Oncol. 14, 121 (2021).

Article CAS PubMed PubMed Central Google Scholar

Duffy, M. J., O’Grady, S., Tang, M. H. & Crown, J. MYC as a target for cancer treatment. Cancer Treat. Rev. 94, 102154 (2021).

Article CAS PubMed Google Scholar

Wang, C., Fang, H., Zhang, J. W. & Gu, Y. Targeting "undruggable" c-Myc protein by synthetic lethality. Front. Med. 15, 541–550 (2021).

Article PubMed Google Scholar

Lier, S. et al. A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer. Bioorg. Chem. 119, 105505 (2022).

Article CAS PubMed Google Scholar

Cuadrado, A. & Nebreda, A. R. Mechanisms and functions of p38 MAPK signalling. Biochem. J. 429, 403–417 (2010).

Article CAS PubMed Google Scholar

Trempolec, N., Dave-Coll, N. & Nebreda, A. R. SnapShot: p38 MAPK substrates. Cell 152, 924–924 (2013).

Article CAS PubMed Google Scholar

Donoghue, C. et al. Optimal linker length for small molecule PROTACs that selectively target p38ɑ and p38β for degradation. Eur. J. Med. Chem. 201, 112451 (2020).

Article CAS PubMed Google Scholar

Chandra, A. et al. The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins. Nat. Cell Biol. 14, 148–158 (2011).

Article PubMed CAS Google Scholar

Ismail, S. A. et al. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo. Nat. Chem. Biol. 7, 942–949 (2011).

Article CAS PubMed Google Scholar

Zimmermann, G. et al. Small molecule inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signalling. Nature 497, 638–642 (2013).

Article CAS PubMed Google Scholar

Winzker, M. et al. Development of a PDEδ-targeting PROTACs that impair lipid metabolism. Angew. Chem. Int. Ed. Engl. 59, 5595–5601 (2020).

Article CAS PubMed PubMed Central Google Scholar

Cheng, J. F., Li, Y., Wang, X., Dong, G. Q. & Sheng, C. Q. Discovery of novel PDEδ degraders for the treatment of KRAS mutant colorectal cancer. J. Med. Chem. 63, 7892–7905 (2020).

Article CAS PubMed Google Scholar

Kontaridis, M. I., Swanson, K. D., David, F. S., Barford, D. & Neel, B. G. PTPN11(Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J. Biol. Chem. 281, 6785–6792 (2006).

Article CAS PubMed Google Scholar

Hanafusa, H., Torii, S., Yasunaga, T. & Nishida, E. Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat. Cell Biol. 4, 850–858 (2002).

Article CAS PubMed Google Scholar

Wang, M. L., Lu, J. F., Wang, M., Yang, C. Y. & Wang, S. M. Discovery of SHP2-D26 as a first, potent, and effective PROTAC degrader of SHP2 protein. J. Med. Chem. 63, 7510–7528 (2020).

Article CAS PubMed Google Scholar

Zheng, M. Z. et al. Novel PROTACs for degradation of SHP2 protein. Bioorg. Chem. 110, 104788 (2021).

Article CAS PubMed Google Scholar

Yang, X. B. et al. Discovery of thalidomide-based PROTAC small molecules as the highly efficient SHP2 degraders. Eur. J. Med. Chem. 218, 113341 (2021).

Article CAS PubMed Google Scholar

Vemulapalli, V. et al. Targeted degradation of the oncogenic phosphatase SHP2. Biochemistry 60, 2593–2609 (2021).

Article CAS PubMed Google Scholar

Cantley, L. C. & Neel, B. G. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl Acad. Sci. USA 96, 4240–4245 (1999).

Article CAS PubMed PubMed Central Google Scholar

Inchul, Y. et al. Discovery of an AKT degrader with prolonged inhibition of downstream signaling. Cell Chem. Biol. 27, 66–73 (2020).

Article CAS Google Scholar

Yu, X. F. et al. Design, synthesis, and evaluation of potent, selective, and bioavailable AKT kinase degraders. J. Med. Chem. 64, 18054–18081 (2021).

Article CAS PubMed Google Scholar

Mossé, Y. P. et al. Identification of ALK as amajor familial neuroblastoma predisposition gene. Nature 455, 930–935 (2008).

Article PubMed PubMed Central CAS Google Scholar

Ren, H. et al. Identification of anaplastic lymphoma kinase as a potential therapeutic target in ovarian cancer. Cancer Res. 72, 3312–3323 (2012).

Article CAS PubMed Google Scholar

Lin, J. J., Riely, G. J. & Shaw, A. T. Targeting ALK: precision medicine takes on drug resistance. Cancer Discov. 7, 137–155 (2017).

Article CAS PubMed PubMed Central Google Scholar

Choi, Y. L. et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N. Engl. J. Med. 363, 1734–1739 (2010).

Article CAS PubMed Google Scholar

Wang, Y. M. et al. Targeted degradation of anaplastic lymphoma kinase by gold nanoparticle-based multi-headed proteolysis targeting chimeras. Colloids Surf. B Biointerfaces 188, 110795–110783 (2020).

Article CAS PubMed Google Scholar

Yan, G. Y. et al. Discovery of a PROTAC targeting ALK with in vivo activity. Eur. J. Med. Chem. 212, 113150–113161 (2021).

Article CAS PubMed Google Scholar

Ren, C. W. et al. Structure-based discovery of SIAIS001 as an oral bioavailability ALK degrader constructed from Alectinib. Eur. J. Med. Chem. 217, 113335–113350 (2021).

Article CAS PubMed Google Scholar

Xie, S. W. et al. Development of Alectinib-based PROTACs as novel potent degraders of anaplastic lymphoma kinase(ALK). J. Med. Chem. 64, 9120–9140 (2021).

Article CAS PubMed Google Scholar

Sun, N. et al. Development of a Brigatinib degrader (SIAIS117) as a potential treatment for ALK positive cancer resistance. Eur. J. Med. Chem. 193, 112190–112204 (2020).

Article CAS PubMed Google Scholar

Ren, C. W. et al. Discovery of a Brigatinib degrader SIAIS164018 with destroying metastasis-related oncoproteins and a reshuffling kinome profile. J. Med. Chem. 64, 9152–9165 (2021).

Article CAS PubMed Google Scholar

Ashkenazi, A., Fairbrother, W. J., Leverson, J. D. & Souers, A. J. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat. Rev. Drug Discov. 16, 273–284 (2017).

Article CAS PubMed Google Scholar

Kaefer, A. et al. Mechanism-based pharmacokinetic/pharmacodynamic meta-analysis of navitoclax (ABT-263) induced thrombocytopenia. Cancer Chemother. Pharmacol. 74, 593–602 (2014).

Article CAS PubMed Google Scholar

Souers, A. J. et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 19, 202–208 (2013).

Article CAS PubMed Google Scholar

Sajid, K. et al. A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity. Nat. Med. 25, 1938–1947 (2019).

Article CAS Google Scholar

Zhang, X. et al. Discovery of PROTAC BCL-XL degraders as potent anticancer agents with low on-target platelet toxicity. Eur. J. Med. Chem. 192, 112186–112208 (2020).

Article CAS PubMed PubMed Central Google Scholar

Zhang, X. et al. Discovery of IAP-recruiting BCL-XL PROTACs as potent degraders across multiple cancer cell lines. Eur. J. Med. Chem. 199, 112397–112408 (2020).

Article CAS PubMed PubMed Central Google Scholar

He, Y. H. et al. Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity. Nat. Commun. 11, 1996–2009 (2020).

Article CAS PubMed PubMed Central Google Scholar

Pratik, P. et al. Discovery of a novel BCL-XL PROTAC degrader with enhanced BCL-2 inhibition. J. Med. Chem. 64, 14230–14246 (2021).

Article CAS Google Scholar

Zhang, X. et al. Utilizing PROTAC technology to address the on-target platelet toxicity associated with inhibition of BCL-XL. Chem. Commun. 55, 14765–14768 (2019).

Article CAS Google Scholar

Chung, C. W. et al. Structural insights into PROTAC-mediated degradation of Bcl-xL. ACS Chem. Biol. 15, 2316–2323 (2020).

Article CAS PubMed Google Scholar

Lv, D. W. et al. Development of a BCL-xL and BCL-2 dual degrader with improved anti-leukemic activity. Nat. Commun. 12, 6896 (2021).

Article CAS PubMed PubMed Central Google Scholar

Talpaz, M. et al. Dasatinib in Imatinib-resistant Philadelphia chromosome-positive leukemias. N. Engl. J. Med. 354, 2531–2541 (2006).

Article CAS PubMed Google Scholar

Reddy, E. P. & Aggarwal, A. K. The ins and outs of bcr-abl inhibition. Genes. Cancer 3, 447–454 (2012).

Article CAS PubMed PubMed Central Google Scholar

Pophali, P. A. & Patnaik, M. M. The role of new tyrosine kinase inhibitors in chronic myeloid leukemia. Cancer J. 22, 40–50 (2016).

Article CAS PubMed PubMed Central Google Scholar

Yang, K. & Fu, L. W. Mechanisms of resistance to Bcr-Abl TKIs and the therapeutic strategies: a review. Crit. Rev. Oncol. Hematol. 93, 277–292 (2015).

Article PubMed Google Scholar

Lai, A. C. et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew. Chem. Int. Ed. Engl. 55, 807–810 (2016).

Article CAS PubMed Google Scholar

George, M. B. et al. Targeting BCR-ABL1 in chronic myeloid leukemia by PROTAC-mediated targeted protein degradation. Cancer Res. 79, 4744–4753 (2019).

Article Google Scholar

George, M. B., Daniel, P. B. & Craig, M. C. Scaffold hopping enables direct access to more potent PROTACs with in vivo activity. Chem. Commun. 56, 6890–6892 (2020).

Article Google Scholar

Yang, Y. Q. et al. A global PROTAC toolbox for degrading BCR-ABL overcomes drug-resistant mutants and adverse effects. J. Med. Chem. 63, 8567–8583 (2020).

Article CAS PubMed Google Scholar

Jiang, L. et al. Design, synthesis, and biological evaluation of Bcr-Abl PROTACs to overcome T315I mutation. Acta Pharm. Sin. B. 11, 1315–1328 (2021).

Article CAS PubMed Google Scholar

Liu, H. X. et al. Discovery of novel BCR-ABL PROTACs based on the cereblon E3 ligase design, synthesis, and biological evaluation. Eur. J. Med. Chem. 223, 113645–113662 (2021).

Article CAS PubMed Google Scholar

Liu, H. X. et al. Construction of an IMiD-based azide library as a kit for PROTAC research. Org. Biomol. Chem. 19, 166–170 (2021).

Article CAS PubMed Google Scholar

Tong, B. et al. A nimbolide-based kinase degrader preferentially degrades oncogenic BCR-ABL. ACS Chem. Biol. 15, 1788–1794 (2020).

Article CAS PubMed PubMed Central Google Scholar

Jin, Y. H. et al. Azo-PROTAC: novel light-controlled small-molecule tool for protein knockdown. J. Med. Chem. 63, 4644–4654 (2020).

Article CAS PubMed Google Scholar

Sulzmaier, F. J., Jean, C. & Schlaepfer, D. D. FAK in cancer: mechanistic findings and clinical applications. Nat. Rev. Cancer 14, 598–610 (2014).

Article CAS PubMed PubMed Central Google Scholar

Cance, W. G., Kurenova, E., Marlowe, T. & Golubovskaya, V. Disrupting the scaffold to improve focal adhesion kinase-targeted cancer therapeutics. Sci. Signal 6, pe10 (2013).

Article PubMed PubMed Central CAS Google Scholar

Frame, M. C., Patel, H., Serrels, B., Lietha, D. & Eck, M. J. The FERM domain: organizing the structure and function of FAK. Nat. Rev. Mol. Cell Biol. 11, 802–814 (2010).

Article CAS PubMed Google Scholar

Kargbo, R. B. Chemically induced degradation of FAK-ALK for application in cancer therapeutics. ACS Med. Chem. Lett. 11, 1367–1368 (2020).

Article CAS PubMed PubMed Central Google Scholar

Law, R. P. et al. Discovery and characterisation of highly cooperative FAK-degrading PROTACs. Angew. Chem. Int. Ed. Engl. 60, 23327–23334 (2021).

Article CAS PubMed Google Scholar

Hainaut, P. & Hollstein, M. P53 and human cancer: the first ten thousand mutations. Adv. Cancer Res. 77, 81–137 (2000).

Article CAS PubMed Google Scholar

Wang, B. et al. Development of selective small molecule MDM2 degraders based on nutlin. Eur. J. Med. Chem. 176, 476–491 (2019).

Article PubMed CAS Google Scholar

Yang, J. L. et al. Simple structural modifications converting a bona fide MDM2 PROTAC degrader into a molecular glue molecule: a cautionary tale in the design of PROTAC degraders. J. Med. Chem. 62, 9471–9487 (2019).

Article CAS PubMed PubMed Central Google Scholar

Qi, Z. W. et al. Design and linkage optimization of ursane-thalidomide-based PROTACs and identification of their targeted-degradation properties to MDM2 protein. Bioorg. Chem. 111, 104901 (2021).

Article CAS PubMed Google Scholar

Wang, B. et al. Development of MDM2 degraders based on ligands derived from Ugi reactions: lessons and discoveries. Eur. J. Med. Chem. 219, 113425 (2021).

Article CAS PubMed Google Scholar

He, S. P. et al. Homo-PROTAC mediated suicide of MDM2 to treat non-small cell lung cancer. Acta Pharm. Sin. B 11, 1617–1628 (2021).

Article CAS PubMed Google Scholar

Lee, H. K. et al. G-749, a novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of acute myeloid leukemia. Blood 123, 2209–2219 (2014).

Article CAS PubMed PubMed Central Google Scholar

Daver, N., Schlenk, R. F., Russell, N. H. & Levis, M. J. Targeting FLT3 mutations in AML: review of current knowledge and evidence. Leukemia 33, 299–312 (2019).

Article CAS PubMed PubMed Central Google Scholar

Weisberg, E. et al. Antileukemic effects of novel first- and second-generation FLT3 Inhibitors: structure-affinity comparison. Genes Cancer 1, 1021–1032 (2010).

Article CAS PubMed PubMed Central Google Scholar

Cao, S. et al. Proteolysis-targeting chimera(PROTAC) modification of Dovitinib enhances the antiproliferative effect against FLT3-ITD-positive acute myeloid leukemia cells. J. Med. Chem. 64, 16497–16511 (2021).

Article CAS PubMed Google Scholar

Chen, Y. et al. Degrading FLT3-ITD protein by proteolysis targeting chimera(PROTAC). Bioorg. Chem. 119, 105508 (2021).

Article PubMed CAS Google Scholar

Ohea, J. J. et al. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu. Rev. Med. 66, 311–328 (2015).

Article CAS Google Scholar

Field, S. D., Arkin, J., Li, J. & Jones, L. H. Selective downregulation of JAK2 and JAK3 by an ATP-competitive pan-JAK inhibitor. ACS Chem. Biol. 12, 1183–1187 (2017).

Article CAS PubMed Google Scholar

Shah, R. R. et al. Hi-JAK-ing the ubiquitin system: the design and physicochemical optimisation of JAK PROTACs. Bioorg. Med. Chem. 28, 115326 (2020).

Article CAS PubMed Google Scholar

Chang, Y. C. et al. Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia. Blood 138, 2313–2326 (2021).

Article CAS PubMed Google Scholar

Johnson, D. E., O’Keefe, R. A. & Grandis, J. R. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat. Rev. Clin. Oncol. 15, 234–248 (2018).

Article CAS PubMed PubMed Central Google Scholar

Beebe, J. D., Liu, J. Y. & Zhang, J. T. Two decades of research in discovery of anticancer drugs targeting STAT3, how close are we? Pharmacol. Ther. 191, 74–91 (2018).

Article CAS PubMed Google Scholar

Zhou, H. B. et al. SD-91 as a potent and selective STAT3 degrader capable of achieving complete and long-lasting tumor regression. ACS Med. Chem. Lett. 12, 996–1004 (2021).

Article CAS PubMed Google Scholar

Zhou, H. B. et al. Structure-based discovery of SD-36 as a potent, selective, and efficacious PROTAC degrader of STAT3 protein. J. Med. Chem. 62, 11280–11300 (2019).

Article CAS PubMed PubMed Central Google Scholar

Clevers, H., Loh, K. M. & Nusse, R. Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346, 1248012 (2014).

Article PubMed CAS Google Scholar

Clevers, H. & Nusse, R. Wnt/beta-catenin signaling and disease. Cell 149, 1192–1205 (2012).

Article CAS PubMed Google Scholar

Kim, S. & Jeong, S. Mutation hotspots in the β-catenin gene: lessons from the human cancer genome databases. Mol. Cell 42, 8–16 (2019).

CAS Google Scholar

Cui, C., Zhou, X., Zhang, W., Qu, Y. & Ke, X. Is β-catenin a druggable target for cancer therapy? Trends Biochem. Sci. 43, 623–634 (2018).

Article CAS PubMed Google Scholar

Li, X. et al. Dithiocarbamate-inspired side chain stapling chemistry for peptide drug design. Chem. Sci. 10, 1522–1530 (2019).

Article CAS PubMed Google Scholar

Liao, H. et al. A PROTAC peptide induces durable β-catenin degradation and suppresses Wnt-dependent intestinal cancer. Cell Discov. 6, 35 (2020).

Article CAS PubMed PubMed Central Google Scholar

Lam, E. W., Brosens, J. J., Gomes, A. R. & Koo, C. Y. Forkhead box proteins: tuning forks for transcriptional harmony. Nat. Rev. Cancer 13, 482–495 (2013).

Article CAS PubMed Google Scholar

Song, X., Kenston, S. S., Zhao, J. S., Yang, D. T. & Gu, Y. L. Roles of FoxM1 in cell regulation and breast cancer targeting therapy. Med. Oncol. 34, 41 (2017).

Article PubMed CAS Google Scholar

Zhang, Y., Qiao, W. B. & Shan, L. Expression and functional characterization of FOXM1 in non-small cell lung cancer. Oncol. Targets Ther. 11, 3385–3393 (2018).

Article Google Scholar

Xu, M. D. et al. A positive feedback loop of lncRNA-PVT1 and FOXM1 facilitates gastric cancer growth and invasion. Clin. Cancer Res. 23, 2071–2080 (2017).

Article CAS PubMed Google Scholar

Pilarsky, C., Wenzig, M., Specht, T., Saeger, H. D. & Grützmann, R. Identification and validation of commonly overexpressed genes in solid tumors by comparison of microarray data. Neoplasia 6, 744–750 (2004).

Article CAS PubMed PubMed Central Google Scholar

Luo, G. S. et al. Targeting of the FOXM1 oncoprotein by E3 ligase-assisted degradation. J. Med. Chem. 64, 17098–17114 (2021).

Article CAS PubMed Google Scholar

Zhong, J. C. & Kenneth, P. M. Recent progress in alpha1-adrenergic receptor research. Acta Pharm. Sin. 26, 1281–1287 (2005).

Article CAS Google Scholar

Dianne, M. P. Structure-function of alpha1-adrenergic receptors. Biochem Pharmacol. 73, 1051–1062 (2006).

Google Scholar

James, R. D. Subtypes of functional alpha1-adrenoceptor. Cell Mol. Life Sci. 67, 405–417 (2010).

Article CAS Google Scholar

Carlos, A. S. R. & André, S. P. Involvement of α1B-adrenoceptors in the anti-immobility effect of imipramine in the tail suspension test. Eur. J. Pharmacol. 750, 39–42 (2015).

Article CAS Google Scholar

Susanna, C., Cosmo, D. D. V., Matilde, C., Stefania, C. & Dario, D. The alpha1-adrenergic receptors in cardiac hypertrophy: signaling mechanisms and functional implications. Cell Signal. 27, 1984–1993 (2015).

Article CAS Google Scholar

Jessica, P., Kenneth, G. & Walter, J. K. G protein-coupled receptor kinases as therapeutic targets in the heart. Nat. Rev. Cardiol. 16, 612–622 (2019).

Article Google Scholar

Yoshiyuki, K., Shoichi, S., Yutaro, H., Gozoh, T. & Kenjiro, K. Subtypes of alpha1-adrenoceptors in BPH: future prospects for personalized medicine. Nat. Clin. Pract. Urol. 6, 44–53 (2009).

Article CAS Google Scholar

Yoshiyuki, K. et al. Quantification of α1-adrenoceptor subtypes by real-time RT-PCR and correlation with age and prostate volume in benign prostatic hyperplasia patients. Prostate 15, 761–767 (2006).

Google Scholar

Stephanie, T. et al. Alpha1-adrenergic receptors activate Ca(2+)-permeable cationic channels in prostate cancer epithelial cells. J. Clin. Invest. 111, 1691–1701 (2003).

Article Google Scholar

Kyprianou, N. & Benning, C. M. Suppression of human prostate cancer cell growth by alpha1-adrenoceptor antagonists doxazosin and terazosin via induction of apoptosis. Cancer Res. 60, 4550–4555 (2000).

CAS PubMed Google Scholar

Li, Z. Z. et al. First small-molecule PROTACs for G protein-coupled receptors: inducing α1A-adrenergic receptor degradation. Acta Pharm. Sin. B. 10, 1669–1679 (2020).

Article CAS PubMed PubMed Central Google Scholar

Dhalluin, C. et al. Structure and ligand of a histone acetyltransferase bromodomain. Nature 399, 491–496 (1999).

Article CAS PubMed Google Scholar

Georg, E. W. et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

Article CAS Google Scholar

Kanak, R. et al. PROTAC-induced BET protein degradation as a therapy for castrationresistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 (2016).

Article CAS Google Scholar

Bai, L. C. et al. Targeted degradation of BET proteins in triple-negative breast cancer. Cancer Res. 77, 2476–2487 (2017).

Article CAS PubMed PubMed Central Google Scholar

Zengerle, M., Chan, K. H. & Ciulli, A. Selective small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem. Biol. 10, 1770–1777 (2015).

Article CAS PubMed PubMed Central Google Scholar

Zhou, B. et al. Discovery of a small-molecule degrader of bromodomain and extra-terminal (BET) proteins with picomolar cellular potencies and capable of achieving tumor regression. J. Med. Chem. 61, 462–481 (2017).

Article PubMed PubMed Central CAS Google Scholar

Qin, C. et al. Discovery of QCA570 as an exceptionally potent and efficacious proteolysis targeting chimera (PROTAC) degrader of the bromodomain and extra-terminal (BET) proteins capable of inducing complete and durable tumor regression. J. Med. Chem. 61, 6685–6704 (2018).

Article CAS PubMed PubMed Central Google Scholar

Mu, X. P., Bai, L. T., Xu, Y. J., Wang, J. Y. & Lu, H. B. Protein targeting chimeric molecules specific for dual bromodomain 4(BRD4) and Polo-like kinase 1(PLK1) proteins in acute myeloid leukemia cells. Biochem. Biophys. Res. Commun. 521, 833–839 (2020).

Article CAS PubMed Google Scholar

Testa, A. et al. Structure-based design of a macrocyclic PROTAC. Angew. Chem. Int. Ed. Engl. 59, 1727–1734 (2020).

Article CAS PubMed Google Scholar

Jiang, F. et al. Discovery of novel small molecule induced selective degradation of the bromodomain and extra-terminal(BET) bromodomain protein BRD4 and BRD2 with cellular potencies. Bioorg. Med. Chem. 28, 115181 (2020).

Article CAS PubMed Google Scholar

Imaide, S. et al. Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity. Nat. Chem. Biol. 17, 1157–1167 (2021).

Article CAS PubMed PubMed Central Google Scholar

Zhang, F. Q. et al. Discovery of a new class of PROTAC BRD4 degraders based on a dihydroquinazolinone derivative and lenalidomide/pomalidomide. Bioorg. Med. Chem. 28, 115228 (2020).

Article CAS PubMed Google Scholar

Zhang, J. et al. Development of small-molecule BRD4 degraders based on pyrrolopyridone derivative. Bioorg. Chem. 99, 103817 (2020).

Article CAS PubMed Google Scholar

Xiang, W. et al. Structure-guided discovery of novel potent and efficacious proteolysis targeting chimera (PROTAC) degrader of BRD4. Bioorg. Chem. 115, 105238 (2021).

Article CAS PubMed Google Scholar

Xue, G., Wang, K., Zhou, D. L., Zhong, H. B. & Pan, Z. Y. Light-induced protein degradation with photocaged PROTACs. J. Am. Chem. Soc. 141, 18370–18374 (2019).

Article CAS PubMed Google Scholar

Li, Z. Z. et al. Development of photocontrolled BRD4 PROTACs for tongue squamous cell carcinoma (TSCC). Eur. J. Med. Chem. 222, 113608 (2021).

Article CAS PubMed Google Scholar

Naro, Y., Darrah, K. & Deiters, A. Optical control of small molecule-induced protein degradation. J. Am. Chem. Soc. 142, 2193–2197 (2020).

Article CAS PubMed PubMed Central Google Scholar

Kounde, C. S. et al. A caged E3 ligase ligand for PROTAC-mediated protein degradation with light. Chem. Commun. 56, 5532–5535 (2020).

Article CAS Google Scholar

Zhang, X. Y. et al. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat. Chem. Biol. 15, 737–746 (2019).

Article CAS PubMed PubMed Central Google Scholar

Li, L. et al. In vivo target protein degradation induced by PROTACs based on E3 ligase DCAF15. Signal Transduct. Target Ther. 5, 129–131 (2020).

Article CAS PubMed PubMed Central Google Scholar

Wei, J. L. et al. Harnessing the E3 ligase KEAP1 for targeted protein degradation. J. Am. Chem. Soc. 143, 15073–15083 (2021).

Article CAS PubMed Google Scholar

Rankovic, Z. et al. Phenyl-Glutarimides: alternative cereblon binders for the design of PROTACs. Angew. Chem. Int. Ed. Engl. 60, 26663–26670 (2021).

Nowak, R. P. et al. Structure-guided design of a "Bump-and-Hole" bromodomain-based degradation tag. J. Med. Chem. 64, 11637–11650 (2021).

Article CAS PubMed Google Scholar

Bond, A. G. et al. Development of BromoTag: a "Bump-and-Hole"-PROTAC system to induce potent, rapid, and selective degradation of tagged target proteins. J. Med. Chem. 64, 15477–15502 (2021).

Article CAS PubMed PubMed Central Google Scholar

Hu, R. et al. Identification of a selective BRD4 PROTAC with potent antiproliferative effects in AR-positive prostate cancer based on a dual BET/PLK1 inhibitor. Eur. J. Med. Chem. 227, 113922–113935 (2021).

Article PubMed CAS Google Scholar

Attar, N. & Kurdistani, S. K. Exploitation of EP300 and CREBBP lysine acetyltransferases by cancer. Cold Spring Harb. Perspect. Med. 7, a026534 (2017).

Article PubMed PubMed Central CAS Google Scholar

Lasko, L. M. et al. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature 550, 128–132 (2017).

Article CAS PubMed PubMed Central Google Scholar

Vannam, R. et al. Targeted degradation of the enhancer lysine acetyltransferases CBP and p300. Cell Chem. Biol. 28, 503–p514 (2021).

Article CAS PubMed Google Scholar

Schulze, J. M., Wang, A. Y. & Kobor, M. S. YEATS domain proteins: a diverse family with many links to chromatin modification and transcription. Biochem. Cell Biol. 87, 65–75 (2009).

Article CAS PubMed Google Scholar

Moustakim, M. et al. Discovery of an MLLT1/3 YEATS domain chemical probe. Angew. Chem. Int. Ed. Engl. 57, 16302–16307 (2018).

Article CAS PubMed PubMed Central Google Scholar

Garnar-Wortzel, L. et al. Chemical inhibition of ENL/AF9 YEATS domains in acute leukemia. ACS Cent. Sci. 7, 815–830 (2021).

Article CAS PubMed PubMed Central Google Scholar

Grozinger, C. M., Hassig, C. & Schreiber, S. L. Three proteins define a class of human histone deacetylases related to yeast hda1p. Proc. Natl Acad. Sci. USA 96, 4868–4873 (1999).

Article CAS PubMed PubMed Central Google Scholar

Boyault, C., Sadoul, K., Pabion, M. & Khochbin, S. HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination. Oncogene 26, 5468–5476 (2007).

Article CAS PubMed Google Scholar

Zhang, X. et al. HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell 27, 197–213 (2007).

Article CAS PubMed PubMed Central Google Scholar

Blander, G. & Guarente, L. The Sir2 family of protein deacetylases. Annu. Rev. Biochem. 73, 417–435 (2004).

Article CAS PubMed Google Scholar

Michan, S. & Sinclair, D. Sirtuins in mammals: insights into their biological function. Biochem. J. 404, 1–13 (2007).

Article CAS PubMed Google Scholar

An, Z. X., Lv, W. X., Su, S., Wu, W. & Rao, Y. Developing potent PROTACs tools for selective degradation of HDAC6 protein. Protein Cell 10, 606–609 (2019).

Article CAS PubMed PubMed Central Google Scholar

Yang, H. Y. et al. Plasticity in designing PROTACs for selective and potent degradation of HDAC6. Chem. Commun. 55, 14848–14851 (2019).

Article CAS Google Scholar

Yang, K. et al. Development of selective histone deacetylase 6(HDAC6) degraders recruiting Von Hippel-Lindau(VHL) E3 ubiquitin ligase. ACS Med. Chem. Lett. 11, 575–581 (2020).

Article PubMed PubMed Central CAS Google Scholar

Yang, K. et al. A cell-based target engagement assay for the identification of cereblon E3 ubiquitin ligase ligands and their application in HDAC6 degraders. Cell Chem. Biol. 27, 866–876 (2020).

Article CAS PubMed PubMed Central Google Scholar

Cao, Z. X. et al. Attenuation of NLRP3 inflammasome activation by indirubin-derived PROTAC targeting HDAC6. ACS Chem. Biol. 16, 2746–2751 (2021).

Article CAS PubMed Google Scholar

Smalley, J. P. et al. PROTAC-mediated degradation of class I histone deacetylase enzymes in corepressor complexes. Chem. Commun. 56, 4476–4479 (2020).

Article CAS Google Scholar

Cao, J. Y. et al. Development of a Bestatin-SAHA hybrid with dual inhibitory activity against APN and HDAC. Molecules 25, 4991 (2020).

Article CAS PubMed Central Google Scholar

Sinatra, L. et al. Hydroxamic acids immobilized on aesins (HAIRs): synthesis of dual-targeting HDAC inhibitors and HDAC degraders(PROTACs). Angew. Chem. Int. Ed. Engl. 59, 22494–22499 (2020).

Article CAS PubMed PubMed Central Google Scholar

Suzuki, T. & Miyata, N. Lysine demethylases inhibitors. J. Med. Chem. 54, 8236–8250 (2011).

Article CAS PubMed Google Scholar

Shi, Y. Histone lysine demethylases: emerging roles in development, physiology and disease. Nat. Rev. Genet. 8, 829–833 (2007).

Article CAS PubMed Google Scholar

Li, K. K., Luo, C., Wang, D. X., Jiang, H. L. & Zheng, Y. G. Chemical and biochemical approaches in the study of histone methylation and demethylation. Med. Res. Rev. 32, 815–867 (2012).

Article CAS PubMed PubMed Central Google Scholar

Iida, T. et al. Design, synthesis, and biological evaluation of lysine demethylase 5C degraders. ChemMedChem 16, 1609–1618 (2021).

Article CAS PubMed Google Scholar

Galluzzi, L., Kepp, O., Heiden, M. G. V. & Kroemer, G. Metabolic targets for cancer therapy. Nat. Rev. Drug Discov. 12, 829–846 (2013).

Article CAS PubMed Google Scholar

Vora, M. et al. Increased nicotinamide phosphoribosyltransferase in rhabdomyosarcomas and leiomyosarcomas compared to skeletal and smooth muscle tissue. Anticancer Res. 36, 503–507 (2016).

CAS PubMed PubMed Central Google Scholar

Chen, W. et al. Dual NAMPT/HDAC inhibitors as a new strategy for multitargeting antitumor drug discovery. ACS Med. Chem. Lett. 9, 34–38 (2017).

Article PubMed PubMed Central CAS Google Scholar

Wu, Y. et al. NAMPT-targeting PROTAC promotes antitumor immunity via suppressing myeloid-derived suppressor cell expansion. Acta Pharm. Sin. B https://doi.org/10.1016/j.apsb.2021.12.017 (2021).

Vougiouklakis, T., Hamamoto, R., Nakamura, Y. & Saloura, V. The NSD family of protein methyltransferases in human cancer. Epigenomics 7, 863–874 (2015).

Article CAS PubMed Google Scholar

Rathert, P. Structure, activity and function of the NSD3 protein lysine methyltransferase. Life 11, 726 (2021).

Article CAS PubMed PubMed Central Google Scholar

Han, X. et al. The role of histone lysine methyltransferase NSD3 in cancer. Onco. Targets Ther. 11, 3847–3852 (2018).

Article PubMed PubMed Central Google Scholar

Xu, C. X. et al. A NSD3-targeted PROTAC suppresses NSD3 and cMyc oncogenic nodes in cancer cells. Cell Chem. Biol. 29, 386–397 (2022).

Article CAS PubMed Google Scholar

Dimou, A. et al. Epigenetics during EMT in lung cancer: EZH2 as a potential therapeutic target. Cancer Treat. Res. Commun. 12, 40–48 (2017).

Article Google Scholar

Kim, K. H. & Roberts, C. W. Targeting EZH2 in cancer. Nat. Med. 22, 128–134 (2016).

Article CAS PubMed PubMed Central Google Scholar

Knutson, S. K. et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat. Chem. Biol. 8, 890–896 (2012).

Article CAS PubMed Google Scholar

Hsu, J. H. et al. EED-targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex. Cell Chem. Biol. 27, 41–46 (2020).

Article CAS PubMed Google Scholar

Potjewyd, F. et al. Degradation of polycomb repressive complex 2 with an EED-targeted bivalent chemical degrader. Cell Chem. Biol. 27, 47–56 (2020).

Article CAS PubMed Google Scholar

Ma, A. Q. et al. Discovery of a first-in-class EZH2 selective degrader. Nat. Chem. Biol. 16, 214–222 (2020).

Article CAS PubMed Google Scholar

Liu, Z. H. et al. Design and synthesis of EZH2-based PROTACs to degrade the PRC2 complex for targeting the noncatalytic activity of EZH2. J. Med. Chem. 64, 2829–2848 (2021). 2021.

Article CAS PubMed Google Scholar

Tu, Y. L. et al. Design, synthesis, and evaluation of VHL-based EZH2 degraders to enhance therapeutic activity against lymphoma. J. Med. Chem. 64, 10167–10184 (2021).

Article CAS PubMed Google Scholar

Lorenzo, A. D. & Bedford, M. T. Histone arginine methylation. FEBS Lett. 585, 2024–2031 (2011).

Article PubMed CAS Google Scholar

Yang, Y. Z. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat. Rev. Cancer 13, 37–50 (2013).

Article CAS PubMed Google Scholar

Stopa, N., Krebs, J. E. & Shechter, D. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond. Cell Mol. Life Sci. 72, 2041–2059 (2015).

Article CAS PubMed PubMed Central Google Scholar

Richters, A. Targeting protein arginine methyltransferase 5 in disease. Future Med. Chem. 9, 2081–2098 (2017).

Article CAS PubMed Google Scholar

Shen, Y. D. et al. Discovery of first-in-class protein arginine methyltransferase 5 (PRMT5) degraders. J. Med. Chem. 63, 9977–9989 (2020).

Article CAS PubMed PubMed Central Google Scholar

Imai, S. et al. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800 (2000).

Article CAS PubMed Google Scholar

Zhou, W. H. et al. The SIRT2 deacetylase stabilizes slug to control malignancy of basal-like breast cancer. Cell Rep. 17, 1302–1317 (2016).

Article CAS PubMed PubMed Central Google Scholar

Hong, J. Y. et al. Simultaneous inhibition of SIRT2 deacetylase and defatty-acylase activities via a PROTAC strategy. ACS Med. Chem. Lett. 11, 2305–2311 (2020).

Article CAS PubMed PubMed Central Google Scholar

Dou, Y. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat. Struct. Mol. Biol. 13, 713–719 (2006).

Article CAS PubMed Google Scholar

Chacón Simon, S. et al. Discovery of WD repeat-containing protein 5 (WDR5)-MYC inhibitors using fragment-based methods and structure-based design. J. Med. Chem. 63, 4315–4333 (2020).

Article PubMed PubMed Central CAS Google Scholar

Aho, E. R. et al. Displacement of WDR5 from chromatin by a WIN site inhibitor with picomolar affinity. Cell Rep. 26, 2916–2928 (2019).

Article CAS PubMed PubMed Central Google Scholar

Dölle, A. et al. Design, synthesis, and evaluation of WD-repeat-containing protein 5(WDR5) degraders. J. Med. Chem. 64, 10682–10710 (2021).

Article PubMed CAS Google Scholar

Yu, X. F. et al. A selective WDR5 degrader inhibits acute myeloid leukemia in patient-derived mouse models. Sci. Transl. Med. 13, eabj1578 (2021).

Article CAS PubMed PubMed Central Google Scholar

Tomotoshi, M., Zhang, D. W. & Hideyuki, S. Aurora A—a guardian of poles. Nat. Rev. Cancer 5, 42–50 (2005).

Article CAS Google Scholar

Dhanasekaran, K., Sallekoppal, B. B. P., Jayasha, S., Shipra, A. & Tapas, K. K. Biology of Aurora A kinase: implications in cancer manifestation and therapy. Med. Res. Rev. 31, 757–793 (2011).

Article CAS Google Scholar

Portella, G., Passaro, C. & Chieffi, P. Aurora B: a new prognostic marker and therapeutic target in cancer. Curr. Med. Chem. 18, 482–496 (2011).

Article CAS PubMed Google Scholar

Bikash, A. et al. PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase. Nat. Chem. Biol. 16, 1179–1188 (2020).

Article CAS Google Scholar

Katherine, A. D. et al. Mapping the degradable kinome provides a resource for expedited degrader development. Cell 183, 1714–1731 (2020).

Article CAS Google Scholar

Richard, W. et al. Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool. Commun. Biol. 4, 64–80 (2021).

CAS Google Scholar

Zur, A. & Brandeis, M. Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis. EMBO J. 20, 792–801 (2001).

Article CAS PubMed PubMed Central Google Scholar

Zhou, Z. et al. Insights into APC/C: from cellular function to diseases and therapeutics. Cell Div. 11, 9 (2016).

Article PubMed PubMed Central CAS Google Scholar

Wang, L. et al. Targeting Cdc20 as a novel cancer therapeutic strategy. Pharmacol. Ther. 151, 141–151 (2015).

Article CAS PubMed PubMed Central Google Scholar

Chi, J. J. et al. A novel strategy to block mitotic progression for targeted therapy. EBioMedicine 49, 40–54 (2019).

Article PubMed Google Scholar

Ying, M. D. et al. Ubiquitin-dependent degradation of CDK2 drives the therapeutic differentiation of AML by targeting PRDX2. Blood 131, 2698–2711 (2018).

Article CAS PubMed Google Scholar

Berthet, C., Aleem, E., Coppola, V., Tessarollo, L. & Kaldis, P. Cdk2 knockout mice are viable. Curr. Biol. 13, 1775–1785 (2003).

Article CAS PubMed Google Scholar

Takada, M. et al. FBW7 loss promotes chromosomal instability and tumorigenesis via cyclin E1/CDK2-mediated phosphorylation of CENP-A. Cancer Res. 77, 4881–4893 (2017).

Article CAS PubMed PubMed Central Google Scholar

Tadesse, S., Caldon, E. C., Tilley, W. & Wang, S. D. Cyclin-dependent kinase 2 inhibitors in cancer therapy: an update. J. Med. Chem. 62, 4233–4251 (2019).

Article CAS PubMed Google Scholar

Fujimoto, T., Anderson, K., Jacobsen, S. E. W., Nishikawa, S. I. & Nerlov, C. Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. EMBO J. 26, 2361–2370 (2007).

Article CAS PubMed PubMed Central Google Scholar

Zhou, F. et al. Development of selective mono or dual PROTAC degrader probe of CDK isoforms. Eur. J. Med. Chem. 187, 111952 (2020).

Article CAS PubMed Google Scholar

Wang, L. G. et al. Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy. Nat. Chem. Biol. 17, 567–575 (2021).

Article CAS PubMed Google Scholar

Hati, S. et al. AZD5438-PROTAC: a selective CDK2 degrader that protects against cisplatin- and noise-induced hearing loss. Eur. J. Med. Chem. 226, 113849 (2021).

Article CAS PubMed Google Scholar

Teng, M. X. et al. Development of CDK2 and CDK5 Dual Degrader TMX-2172. Angew. Chem. Int. Ed. Engl. 59, 13865–13870 (2020).

Article CAS PubMed PubMed Central Google Scholar

Vermeulen, K., Bockstaele, D. R. V. & Berneman, Z. N. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 36, 131–149 (2003).

Article CAS PubMed PubMed Central Google Scholar

Otto, T. & Sicinski, P. Cell cycle proteins as promising targets in cancer therapy. Nat. Rev. Cancer 17, 93–115 (2017).

Article CAS PubMed PubMed Central Google Scholar

Yang, C. et al. Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence. Oncogene 36, 2255–2264 (2017).

Article CAS PubMed Google Scholar

Zhao, B. S. & Burgess, K. PROTACs suppression of CDK4/6, crucial kinases for cell cycle regulation in cancer. Chem. Commun. 55, 2704–2707 (2019).

Article CAS Google Scholar

Jiang, B. S. et al. Development of dual and selective degraders of cyclin-dependent kinases 4 and 6. Angew. Chem. Int. Ed. Engl. 58, 6321–6326 (2019).

Article CAS PubMed PubMed Central Google Scholar

Brand, M. et al. Homolog-selective degradation as a strategy to probe the function of CDK6 in AML. Cell Chem. Biol. 26, 300–306 (2019).

Article CAS PubMed Google Scholar

Su, S. et al. Potent and preferential degradation of CDK6 via proteolysis targeting chimera degraders. J. Med. Chem. 62, 7575–7582 (2019).

Article CAS PubMed PubMed Central Google Scholar

Garrido-Castro, A. C. & Goel, S. CDK4/6 inhibition in breast cancer: mechanisms of response and treatment failure. Curr. Breast Cancer Rep. 9, 26–33 (2017).

Article CAS PubMed PubMed Central Google Scholar

Yang, C. et al. Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence. Oncogene 36, 2255–2264 (2016).

Article PubMed PubMed Central CAS Google Scholar

Dominici, M. D. et al. Selective inhibition of Ph-positive ALL cell growth through kinase-dependent and -independent effects by CDK6-specific PROTACs. Blood 135, 1560–1573 (2020).

Article PubMed PubMed Central Google Scholar

Steinebach, C. et al. Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders. Chem. Sci. 11, 3474–3486 (2020).

Article CAS PubMed PubMed Central Google Scholar

Wei, M. M. et al. First orally bioavailable prodrug of proteolysis targeting chimera(PROTAC) degrades cyclin-dependent kinases 2/4/6 in vivo. Eur. J. Med. Chem. 209, 112903 (2021).

Article CAS PubMed Google Scholar

Malumbres, M. & Barbacid, M. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 9, 153–166 (2009).

Article CAS PubMed Google Scholar

Ball, B. & Wahab, O. A. Activating p53 and inhibiting super enhancers to cure leukemia. Trends Pharmacol. Sci. 39, 1002–1004 (2018).

Article CAS PubMed PubMed Central Google Scholar

Hoodless, L. J. et al. Genetic and pharmacological inhibition of CDK9 drives neutrophil apoptosis to resolve inflammation in zebrafish in vivo. Sci. Rep. 5, 36980 (2016).

Article PubMed PubMed Central CAS Google Scholar

Cassaday, R. D. et al. A phase II, single-arm, open-label, multicenter study to evaluate the efficacy and safety of P276-00, a cyclin-dependent kinase inhibitor, in patients with relapsed or refractory mantle cell lymphoma. Clin. Lymphoma, Myeloma Leuk. 15, 392–397 (2015).

Article Google Scholar

Robb, C. M. et al. Chemically induced degradation of CDK9 by a proteolysis targeting chimera (PROTAC). Chem. Commun. 53, 7577–7580 (2017).

Article CAS Google Scholar

King, H. M. et al. Aminopyrazole based CDK9 PROTAC sensitizes pancreatic cancer cells to venetoclax. Bioorg. Med. Chem. Lett. 43, 128061 (2021).

Article CAS PubMed Google Scholar

Qiu, X. Q. et al. Discovery of selective CDK9 degraders with enhancing antiproliferative activity through PROTAC conversion. Eur. J. Med. Chem. 211, 113091 (2021).

Article CAS PubMed Google Scholar

Wei, D. et al. Discovery of potent and selective CDK9 degraders for targeting transcription regulation in triple-negative breast cancer. J. Med. Chem. 64, 14822–14847 (2021).

Article CAS PubMed Google Scholar

Liang, S. J. et al. CDK12: a potent target and biomarker for human cancer therapy. Cells 9, 1483 (2020).

Article CAS PubMed Central Google Scholar

Chen, H. H., Wang, Y. C. & Fann, M. J. Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation. Mol. Cell Biol. 26, 2736–2745 (2006).

Article CAS PubMed PubMed Central Google Scholar

Iniguez, A. B. et al. EWS/FLI confers tumor cell synthetic lethality to CDK12 inhibition in Ewing sarcoma. Cancer Cell 33, 202–216 (2018).

Article CAS PubMed PubMed Central Google Scholar

Dubbury, S. J., Boutz, P. L. & Sharp, P. A. CDK12 regulates DNA repair genes by suppressing intronic polyadenylation. Nature 564, 141–145 (2018).

Article CAS PubMed PubMed Central Google Scholar

Zhang, T. H. et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 12, 876–884 (2016).

Article CAS PubMed PubMed Central Google Scholar

Jiang, B. S. et al. Discovery and resistance mechanism of a selective CDK12 degrader. Nat. Chem. Biol. 17, 675–683 (2021).

Article CAS PubMed PubMed Central Google Scholar

Niu, T. et al. Noncovalent CDK12/13 dual inhibitors-based PROTACs degrade CDK12-Cyclin K complex and induce synthetic lethality with PARP inhibitor. Eur. J. Med. Chem. 228, 114012 (2022).

Article CAS PubMed Google Scholar

Schmidt, M. et al. Regulation of G2/M transition by inhibition of WEE1 and PKMYT1 kinases. Molecules 22, 2045 (2017).

Article PubMed Central CAS Google Scholar

Fu, S. Q. et al. Strategic development of AZD1775, a Wee1 kinase inhibitor, for cancer therapy. Expert Opin. Investig. Drugs 27, 741–751 (2018).

Article CAS PubMed Google Scholar

Li, Z. N. et al. Development and characterizationof a Wee1 kinase degrader. Cell Chem. Biol. 27, 57–65 (2020).

Article CAS PubMed Google Scholar

Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).

Article CAS PubMed Google Scholar

Ito, T. & Handa, H. Cereblon as a primary target of IMiDs. Rinsho. Ketsueki. 60, 1013–1019 (2019).

PubMed Google Scholar

Ito, T. & Handa, H. Molecular mechanisms of thalidomide and its derivatives. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 96, 189–203 (2020).

Article CAS PubMed PubMed Central Google Scholar

Chamberlain, P. & Cathers, B. E. Cereblon modulators: Low molecular weight inducers of protein degradation. Drug Discov. Today Technol. 31, 29–34 (2019).

Article PubMed Google Scholar

Steinebach, C. et al. Homo-PROTACs for the chemical knockdown of cereblon. ACS Chem. Biol. 13, 2771–2782 (2018).

Article CAS PubMed Google Scholar

Kim, H. K. et al. Cereblon in health and disease. Pflug. Arch. 468, 1299–1309 (2016).

Article CAS Google Scholar

Steinebach, C. et al. PROTAC-mediated crosstalk between E3 ligases. Chem. Commun. 55, 1821–1824 (2019).

Article CAS Google Scholar

Girardini, M., Maniaci, C., Hughes, S. J., Testa, A. & Ciulli, A. Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs. Bioorg. Med. Chem. 27, 2466–2479 (2019).

Article CAS PubMed PubMed Central Google Scholar

Kim, K. et al. Disordered region of cereblon is required for efficient degradation by proteolysis-targeting chimera. Sci. Rep. 9, 19654 (2019).

Article CAS PubMed PubMed Central Google Scholar

Powell, C. E. et al. Selective degradation-inducing probes for studying cereblon(CRBN) biology. RSC Med. Chem. 12, 1381–1390 (2021).

Article CAS PubMed Google Scholar

Schreiner, P. et al. Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction. Nature 453, 548–552 (2008).

Article CAS PubMed PubMed Central Google Scholar

Sanchez-Pulido, L., Kong, L. & Ponting, C. P. A common ancestry for BAP1 and Uch37 regulators. Bioinformatics 28, 1953–1956 (2012).

Article CAS PubMed Google Scholar

Osei-Amponsa, V. et al. Impact of losing hRpn13 Pru or UCHL5 on proteasome clearance of ubiquitinated proteins and RA190 cytotoxicity. Mol. Cell Biol. 40, e00122–20 (2020).

Article CAS PubMed PubMed Central Google Scholar

Lu, X. X. et al. Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma. Nat. Commun. 12, 7318 (2021).

Article CAS PubMed PubMed Central Google Scholar

Skaar, J. R., Pagan, J. K. & Pagano, M. SCF ubiquitin ligase-targeted therapies. Nat. Rev. Drug Discov. 13, 889–903 (2014).

Article CAS PubMed PubMed Central Google Scholar

Bulatov, E. & Ciulli, A. Targeting cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation. Biochem. J. 467, 365–386 (2015).

Article CAS PubMed Google Scholar

Maniaci, C. et al. Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat. Commun. 8, 830 (2017).

Article PubMed PubMed Central CAS Google Scholar

Jin, X. et al. An atypical E3 ligase zinc finger protein 91 stabilizes and activates NF-κB inducing kinase via Lys63-linked ubiquitination. J. Biol. Chem. 285, 30539–30547 (2010).

Article CAS PubMed PubMed Central Google Scholar

Hanafi, M., Chen, X. D. & Neamati, N. Discovery of a Napabucasin PROTAC as an effective degrader of the E3 ligase ZFP91. J. Med. Chem. 64, 1626–1648 (2021).

Article CAS PubMed PubMed Central Google Scholar

Hendriks, R. W., Yuvaraj, S. & Kil, L. P. Targeting Bruton tyrosine kinase in B cell malignancies. Nat. Rev. Cancer 14, 219–232 (2014).

Article CAS PubMed Google Scholar

Woyach, J. A. et al. Resistance mechanisms for the Bruton tyrosine kinase inhibitor ibrutinib. N. Engl. J. Med. 370, 2286–2294 (2014).

Article PubMed PubMed Central CAS Google Scholar

Sun, Y. H. et al. PROTAC-induced BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell malignancies. Cell Res. 28, 779–781 (2018).

Article CAS PubMed PubMed Central Google Scholar

Sun, Y. H. et al. Degradation of Bruton tyrosine kinase mutants by PROTACs for potential treatment of ibrutinib-resistant non-Hodgkin lymphomas. Leukemia 33, 2105–2110 (2019).

Article PubMed Google Scholar

Buhimschi, A. D. et al. Targeting the C481S ibrutinib-resistance mutation in Bruton tyrosine kinase using PROTAC-mediated degradation. Biochemistry 57, 1273564–1273575 (2018).

Article CAS Google Scholar

Huang, H. T. et al. A chemoproteomic approach to query the degradable kinome using a multi-kinase degrader. Cell Chem. Biol. 25, 88–99 (2018).

Article CAS PubMed Google Scholar

Zorba, A. et al. Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc. Natl Acad. Sci. USA 115, E7285–E7292 (2018).

Article PubMed PubMed Central CAS Google Scholar

Liu, S. D. et al. Targeted selective degradation of Bruton tyrosine kinase by PROTACs. Med. Chem. Res. 29, 802–808 (2020).

Article CAS Google Scholar

Figueroa, S. J., Buhimschi, A. D., Toure, M., Hines, J. & Crews, C. M. Design, synthesis and biological evaluation of Proteolysis Targeting Chimeras(PROTACs) as a BTK degraders with improved pharmacokinetic properties. Bioorg. Med. Chem. Lett. 30, 126877–126889 (2020).

Article CAS Google Scholar

Zhao, Y. P. et al. Discovery of novel BTK PROTACs for B-Cell lymphomas. Eur. J. Med. Chem. 225, 113820–113822 (2021).

Article CAS PubMed Google Scholar

Yang, Z. M. et al. Merging PROTAC and molecular glue for degrading BTK and GSPT1 proteins concurrently. Cell Res. 31, 1315–1318 (2021).

Article CAS PubMed Google Scholar

Schiemer, J. et al. Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes. Nat. Chem. Biol. 17, 152–160 (2021).

Article CAS PubMed Google Scholar

Xue, G. et al. Protein degradation through covalent inhibitor-based PROTACs. Chem. Commun. 56, 1521–1524 (2020).

Article CAS Google Scholar

Gabizon, R. et al. Efficient targeted degradation via reversible and irreversible covalent PROTACs. J. Am. Chem. Soc. 142, 11734–11742 (2020).

Article CAS PubMed PubMed Central Google Scholar

Guo, W. H. et al. Enhancing intracellular accumulation and target engagement of PROTACs with reversible covalent chemistry. Nat. Commun. 11, 4268–4283 (2020).

Article PubMed PubMed Central CAS Google Scholar

Wendt, E. & Keshav, S. CCR9 antagonism: potential in the treatment of inflammatory bowel disease. Clin. Exp. Gastroenterol. 8, 119–130 (2015).

PubMed PubMed Central Google Scholar

Oswald, C. et al. Intracellular allosteric antagonism of the CCR9 receptor. Nature 540, 462–465 (2016).

Article CAS PubMed Google Scholar

Huber, M. E. et al. A chemical biology toolbox targeting the intracellular binding site of CCR9: fluorescent ligands, new drug leads and PROTACs. Angew. Chem. Int. Ed. Engl. 61, e202116782 (2022).

Article CAS PubMed Google Scholar

Muramatsu, T. Basigin(CD147), a multifunctional transmembrane glycoprotein with various binding partners. J. Biochem. 159, 481–490 (2016).

Article CAS PubMed Google Scholar

Wang, Y. et al. A chimeric antibody targeting CD147 inhibits hepatocellular carcinoma cell motility via FAK-PI3K-Akt-Girdin signaling pathway. Clin. Exp. Metastasis 32, 39–53 (2015).

Article PubMed CAS Google Scholar

Zhou, Y. Q. et al. Chemical proteomics reveal CD147 as a functional target of pseudolaric acid B in human cancer cells. Chem. Commun. 53, 8671–8674 (2017).

Article CAS Google Scholar

Zhou, Z. et al. Targeted degradation of CD147 proteins in melanoma. Bioorg. Chem. 105, 104453 (2020).

Article CAS PubMed Google Scholar

Schug, T. T. et al. Overcoming retinoic acid-resistance of mammary carcinomas by diverting retinoic acid from PPARβ/δ to RAR. Proc. Natl Acad. Sci. USA 105, 7546–7551 (2008).

Article CAS PubMed PubMed Central Google Scholar

Kizaki, M. et al. Retinoid resistance in leukemic cells. Leuk. Lymphoma 25, 427–434 (1997).

Article CAS PubMed Google Scholar

Uhrig, M. et al. Upregulation of CRABP1 in human neuroblastoma cells overproducing the Alzheimer-typical Abeta42 reduces their differentiation potential. BMC Med. 6, 38 (2008).

Article PubMed PubMed Central CAS Google Scholar

Lind, G. E. et al. ADAMTS1, CRABP1, and NR3C1 identified as epigenetically deregulated genes in colorectal tumorigenesis. Cell Oncol. 28, 259–272 (2006).

CAS PubMed PubMed Central Google Scholar

Itoh, Y., Ishikawa, M., Naito, M. & Hashimoto, Y. Protein knockdown using methyl bestatin-ligand hybrid molecules: design and synthesis of inducers of ubiquitination-mediated degradation of cellular retinoic acid-binding proteins. J. Am. Chem. Soc. 132, 5820–5826 (2010).

Article CAS PubMed Google Scholar

Ohoka, N. et al. Development of small molecule chimeras that recruit AhR E3 ligase to target proteins. ACS Chem. Biol. 14, 2822–2832 (2019).

Article CAS PubMed Google Scholar

Hoter, A., Rizk, S. & Naim, H. Y. The multiple roles and therapeutic potential of molecular chaperones in prostate cancer. Cancers 11, 1194 (2019).

Article CAS PubMed Central Google Scholar

Kamal, A. et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 425, 407–410 (2003).

Article CAS PubMed Google Scholar

Li, L., Wang, L., You, Q. D. & Xu, X. L. Heat shock protein 90 inhibitors: an update on achievements, challenges, and future directions. J. Med. Chem. 63, 1798–1822 (2020).

Article CAS PubMed Google Scholar

Liu, Q. Y. et al. Discovery of BP3 as an efficacious proteolysis targeting chimera(PROTAC) degrader of HSP90 for treating breast cancer. Eur. J. Med. Chem. 228, 114013 (2022).

Article CAS PubMed Google Scholar

Platten, M., Wick, W. & Eynde, B. V. Tryptophan catabolism in cancer: beyond IDO and tryptophan depletion. Cancer Res. 72, 5435–5440 (2012).

Article CAS PubMed Google Scholar

Munn, D. H. et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 281, 1191–1193 (1998).

Article CAS PubMed Google Scholar

Zamanakou, M., Germenis, A. E. & Karanikas, V. Tumor immune escape mediated by indoleamine 2,3-dioxygenase. Immunol. Lett. 111, 69–75 (2007).

Article CAS PubMed Google Scholar

Hu, M. X. et al. Discovery of the first potent proteolysis targetingchimera(PROTAC) degrader of indoleamine2,3-dioxygenase 1. Acta Pharm. Sin. B 10, 1943–1953 (2020).

Article CAS PubMed PubMed Central Google Scholar

Edwards, P. A., Kast, H. R. & Anisfeld, A. M. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis. J. Lipid Res. 43, 2–12 (2002).

Article CAS PubMed Google Scholar

Zhu, Y. F. & Li, Y. S. Liver X receptors as potential therapeutic targets in atherosclerosis. Clin. Invest. Med. 32, 383–394 (2009).

Article Google Scholar

Kick, E. K. et al. Discovery of highly potent liver X receptor β agonists. ACS Med. Chem. Lett. 7, 1207–1212 (2016).

Article CAS PubMed PubMed Central Google Scholar

Xu, H. Q. et al. Development of agonist-based PROTACs targeting liver X receptor. Front. Chem. 9, 674967 (2021).

Article CAS PubMed PubMed Central Google Scholar

Dagogo-Jack, I. & Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nat. Rev. Clin. Oncol. 15, 81–94 (2018).

Article CAS PubMed Google Scholar

Bucala, R. & Donnelly, S. C. Macrophage migration inhibitory factor: a probable link between inflammation and cancer. Immunity 26, 281–285 (2007).

Article CAS PubMed Google Scholar

Xiao, Z. P. et al. Proteolysis targeting chimera (PROTAC) for macrophage migrationInhibitory factor (MIF) has anti-proliferative activity in lung cancer cells. Angew. Chem. Int. Ed. Engl. 60, 17514–17521 (2021).

Article CAS PubMed PubMed Central Google Scholar

Kleine, H. et al. Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation. Mol. Cell 32, 57–69 (2008).

Article CAS PubMed Google Scholar

Rouleau, M., Patel, A., Hendzel, M. J., Kaufmann, S. H. & Poirier, G. G. PARP inhibition: PARP1 and beyond. Nat. Rev. Cancer 10, 293–301 (2010).

Article CAS PubMed PubMed Central Google Scholar

Durkacz, B. W., Omidiji, O., Gray, D. A. & Shall, S. ADP-ribose)n participates in DNA excision repair. Nature 283, 593–596 (1980).

Gibson, B. A. & Kraus, W. L. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat. Rev. Mol. Cell Biol. 13, 411–424 (2012).

Article CAS PubMed Google Scholar

Wang, S. et al. Uncoupling of PARP1 trapping and inhibition using selective PARP1 degradation. Nat. Chem. Biol. 15, 1223–1231 (2019).

Article CAS PubMed PubMed Central Google Scholar

Zhang, Z. M. et al. Identification of probe-quality degraders for Poly(ADP-ribose) polymerase-1(PARP-1). J. Enzym. Inhib. Med. Chem. 35, 1606–1615 (2020).

Article CAS Google Scholar

Cao, C. G. et al. Discovery of SK-575 as a highly potent and efficacious proteolysis-targeting chimera degrader of PARP1 for treating cancers. J. Med. Chem. 63, 11012–11033 (2020).

Article CAS PubMed Google Scholar

Leutert, M., Pedrioli, D. M. L. & Hottiger, M. O. Identification of PARP-specific ADP-ribosylation targets reveals a regulatory function for ADP-ribosylation in transcription elongation. Mol. Cell 63, 181–183 (2016).

Article CAS PubMed Google Scholar

Wigle, T. J. et al. Targeted degradation of PARP14 using a heterobifunctional small molecule. Chembiochem 22, 2107–2110 (2021).

Article CAS PubMed Google Scholar

Zhao, L., Liu, Y. W., Sun, X. G., He, M. Y. & Ding, Y. Q. Overexpression of T lymphoma invasion and metastasis 1 predict renal cell carcinoma metastasis and overall patient survival. J. Cancer Res. Clin. Oncol. 137, 393–398 (2011).

Article CAS PubMed Google Scholar

Sharpe, A. H., Wherry, E. J., Ahmed, R. & Freeman, G. J. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat. Immunol. 8, 239–245 (2007).

Article CAS PubMed Google Scholar

Cheng, B. B., Ren, Y. C., Cao, H. & Chen, J. J. Discovery of novel resorcinol diphenyl ether-based PROTAC-like molecules as dual inhibitors and degraders of PD-L1. Eur. J. Med. Chem. 199, 112377 (2020).

Article CAS PubMed Google Scholar

Wang, Y. B. et al. In vitro and in vivo degradation of programmed cell death ligand 1(PD-L1) by a proteolysis targeting chimera(PROTAC). Bioorg. Chem. 111, 104833 (2021).

Article CAS PubMed Google Scholar

Renner, A. G. et al. Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells. Blood 114, 659–662 (2009).

Article CAS PubMed Google Scholar

Brandwein, J. M. Targeting polo-like kinase 1 in acute myeloid leukemia. Ther. Adv. Hematol. 6, 80–87 (2015).

Article CAS PubMed PubMed Central Google Scholar

Mao, F. et al. PLK1 inhibition enhances the efficacy of bet epigenetic reader blockade in castration-resistant prostate cancer. Mol. Cancer Ther. 17, 1554–1565 (2018).

Article CAS PubMed PubMed Central Google Scholar

Bourguet, W. et al. Synthesis of a biospecific adsorbent for the purification of the three human retinoic acid receptors by affinity chromatography. Biochem. Biophys. Res. Commun. 187, 711–716 (1992).

Article CAS PubMed Google Scholar

Itoh, Y., Kitaguchi, R., Ishikawa, M., Naito, M. & Hashimoto, Y. Design, synthesis and biological evaluation of nuclear receptor-degradation inducers. Bioorg. Med. Chem. 19, 6768–6778 (2011).

Article CAS PubMed Google Scholar

Davis, J. T. G-quartets 40 years later: from 5’-GMP to molecular biology and supramolecular chemistry. Angew. Chem. Int. Ed. Engl. 43, 668–698 (2004).

Article CAS PubMed Google Scholar

Brázda, V., Hároníková, L., Liao, J. & Fojta, M. DNA and RNA quadruplex-binding proteins. Rev. Int. J. Mol. Sci. 15, 17493–17517 (2014).

Article CAS Google Scholar

Creacy, S. D. et al. G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates. J. Biol. Chem. 283, 34626–34634 (2008).

Article CAS PubMed PubMed Central Google Scholar

Patil, K. N. et al. G4-PROTAC: targeted degradation of a G-quadruplex binding protein. Chem. Commun. 57, 12816–12819 (2021).

Article CAS Google Scholar

Girardin, S. E. et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872 (2003).

Article CAS PubMed Google Scholar

Shaw, P. J. et al. Signaling via the RIP2 adaptor protein in central nervous system-infiltrating dendritic cells promotes inflammation and autoimmunity. Immunity 34, 75–84 (2011).

Article CAS PubMed PubMed Central Google Scholar

Mares, A. et al. Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2. Commun. Biol. 3, 140 (2020).

Article CAS PubMed PubMed Central Google Scholar

Golas, M. M., Sander, B., Will, C. L., Luhrmann, R. & Stark, H. Molecular architecture of the multiprotein splicing factor SF3b. Science 300, 980–984 (2003).

Article CAS PubMed Google Scholar

Eskens, F. A. et al. Phase I pharmacokinetic and pharmacodynamic study of the first-in-class spliceosome inhibitor E7107 in patients with advanced solid tumors. Clin. Cancer Res. 19, 6296–6304 (2013).

Article CAS PubMed Google Scholar

Gama-Brambila, R. A. et al. PROTAC targets splicing factor 3B1. Cell Chem. Biol. 28, 1616–1627 (2021).

Article CAS PubMed Google Scholar

Lin, L., Yee, S. W., Kim, R. B. & Giacomini, K. M. SLC transporters as therapeutic targets: emerging opportunities. Nat. Rev. Drug Discov. 14, 543–560 (2015).

Article CAS PubMed PubMed Central Google Scholar

César-Razquin, A. et al. A call for systematic research on solute carriers. Cell 162, 478–487 (2015).

Article PubMed CAS Google Scholar

Bensimon, A. et al. Targeted degradation of SLC transporters reveals amenability of multi-pass transmembrane proteins to ligand-induced proteolysis. Cell Chem. Biol. 27, 728–739 (2020).

Article CAS PubMed PubMed Central Google Scholar

Pierre, R. S. & Kadoch, C. Mammalian SWI/SNF complexes in cancer: emerging therapeutic opportunities. Curr. Opin. Genet. Dev. 42, 56–67 (2017).

Article CAS Google Scholar

Shi, J. W. et al. Role of SWI/SNF in acute leukemia maintenance and enhancermediated Myc regulation. Genes Dev. 27, 2648–2662 (2013).

Article CAS PubMed PubMed Central Google Scholar

Papillon, J. P. N. et al. Discovery of orally active inhibitors of brahma homolog (BRM)/SMARCA2 ATPase activity for the treatment of brahma related gene 1 (BRG1)/SMARCA4-mutant cancers. J. Med. Chem. 61, 10155–10172 (2018).

Article CAS PubMed Google Scholar

Farnaby, W. et al. BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat. Chem. Biol. 15, 672–680 (2019).

Article CAS PubMed PubMed Central Google Scholar

Xiao, L. B. et al. Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer. Nature 601, 434–439 (2022).

Article CAS PubMed Google Scholar

Xu, J. M., Wu, R. C. & O’Malley, B. W. Normal and cancer-related functions of the p160 steroid receptor co-activator(SRC) family. Nat. Rev. Cancer 9, 615–630 (2009).

Article CAS PubMed PubMed Central Google Scholar

Qin, L., Liu, Z. L., Chen, H. W. & Xu, J. M. The steroid receptor coactivator-1 regulates twist expression and promotes breast cancer metastasis. Cancer Res. 69, 3819–3827 (2009).

Article CAS PubMed PubMed Central Google Scholar

Rohira, A. D. et al. Targeting SRC coactivators blocks the tumor-initiating capacity of cancer stem-like cells. Cancer Res. 77, 4293–4304 (2017).

Article CAS PubMed PubMed Central Google Scholar

Lee, Y. et al. Targeted degradation of transcription coactivator SRC-1 through the N-degron pathway. Angew. Chem. Int. Ed. Engl. 59, 17548–17555 (2020).

Article CAS PubMed Google Scholar

Slominski, A., Zmijewski, M. A. & Pawelek, J. L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions. Pigment. Cell Melanoma Res. 25, 14–27 (2012).

Article CAS PubMed Google Scholar

Pillaiyar, T., Manickam, M. & Namasivayam, V. Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. J. Enzym. Inhib. Med. Chem. 32, 403–425 (2017).

Article CAS Google Scholar

Haudecoeur, R. et al. 2-Hydroxypyridine-N-oxide-embedded aurones as potent human tyrosinase inhibitors. ACS Med. Chem. Lett. 8, 55–60 (2016).

Article PubMed PubMed Central CAS Google Scholar

Fu, D. Q. et al. Design, synthesis and biological evaluation of tyrosinase-targeting PROTACs. Eur. J. Med. Chem. 226, 113850 (2021).

Article CAS PubMed Google Scholar

Rao, P. P. N., Kabir, S. N. & Mohamed, T. Nonsteroidal anti-inflammatory drugs (NSAIDs): progress in small molecule drug development. Pharm. (Basel) 3, 1530–1549 (2010).

Article CAS Google Scholar

Al-Horani, R. A. & Kar, S. Potential anti-SARS-CoV-2 therapeutics that target the post-entry stages of the viral life cycle: a comprehensive. Viruses 12, 1092 (2020).

Article CAS PubMed Central Google Scholar

Desantis, J. et al. Indomethacin-based PROTACs as pan-coronavirus antiviral agent. Eur. J. Med. Chem. 226, 113814 (2021).

Article CAS PubMed PubMed Central Google Scholar

Shaheer, M., Singh, R. & Sobhia, M. E. Protein degradation: a novel computational approach to design protein degrader probes for main protease of SARS-CoV-2. J. Biomol. Struct. Dyn. 30, 1–13 (2021).

Dokmanovic, M., Clarke, C. & Marks, P. A. Histone deacetylase inhibitors: overview and perspectives. Mol. Cancer Res. 5, 981–989 (2007).

Article CAS PubMed Google Scholar

Leus, N. G. J. et al. HDAC 3 selective inhibitor RGFP966 demonstrates antiinflammatory properties in RAW 264.7 macrophages and mouse precisioncut lung slices by attenuating NF-κB p65 transcriptional activity. Biochem. Pharmacol. 108, 58–74 (2016).

Article CAS PubMed PubMed Central Google Scholar

Cao, F. Y. et al. Induced protein degradation of histone deacetylases 3(HDAC3) by proteolysis targeting chimera(PROTAC). Eur. J. Med. Chem. 208, 112800 (2020).

Article CAS PubMed Google Scholar

Xiao, Y. F. et al. Discovery of histone deacetylase 3(HDAC3)-specific PROTACs. Chem. Commun. 56, 9866–9869 (2020).

Article CAS Google Scholar

Rittchen, S. & Heinemann, A. Therapeutic potential of hematopoietic prostaglandin D2 synthase in allergic inflammation. Cells 8, 619 (2019).

Article CAS PubMed Central Google Scholar

Thurairatnam, S. Hematopoietic prostaglandin D synthase inhibitors. Prog. Med. Chem. 51, 97–133 (2012).

Article CAS PubMed Google Scholar

Yokoo, H. et al. Development of a hematopoietic prostaglandin D synthase-degradation inducer. ACS Med. Chem. Lett. 12, 236–241 (2021).

Article CAS PubMed PubMed Central Google Scholar

Yokoo, H. et al. Discovery of a highly potent and selective degrader targeting hematopoietic prostaglandin D synthase via in silico design. J. Med. Chem. 64, 15868–15882 (2021).

Article CAS PubMed Google Scholar

Barton, G. M. & Medzhitov, R. Toll-like receptor signaling pathways. Science 300, 1524–1525 (2003).

Article CAS PubMed Google Scholar

Cohen, P. The TLR and IL-1 signalling network at a glance. J. Cell Sci. 127, 2383–2390 (2014).

CAS PubMed PubMed Central Google Scholar

Ferrao, R. et al. IRAK4 dimerization and trans-autophosphorylation are induced by myddosome assembly. Mol. Cell 55, 891–903 (2014).

Article CAS PubMed PubMed Central Google Scholar

Lin, S. C., Lo, Y. C. & Wu, H. Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465, 885–890 (2010).

Article CAS PubMed PubMed Central Google Scholar

Fu, L. Q. et al. Discovery of highly potent and selective IRAK1 degraders to probe scaffolding functions of IRAK1 in ABC DLBCL. J. Med. Chem. 64, 10878–10889 (2021).

Article CAS PubMed Google Scholar

Su, L. C., Xu, W. D. & Huang, A. F. IRAK family in inflammatory autoimmune diseases. Autoimmun. Rev. 19, 102461 (2020).

Article CAS PubMed Google Scholar

Kobayashi, K. et al. IRAK-M is a negative regulator of toll-like receptor signaling. Cell 110, 191–202 (2002).

Article CAS PubMed Google Scholar

Rhyasen, G. W. & Starczynowski, D. T. IRAK signalling in cancer. Br. J. Cancer 112, 232–237 (2015).

Article CAS PubMed Google Scholar

Kesselring, R. et al. IRAK-M expression in tumor cells supports colorectal cancer progression through reduction of antimicrobial defense and stabilization of STAT3. Cancer Cell 29, 684–696 (2016).

Article CAS PubMed Google Scholar

Zhang, Y. et al. Neutrophils deficient in innate suppressor IRAK-M enhances anti-tumor immune responses. Mol. Ther. 28, 89–99 (2020).

Article CAS PubMed Google Scholar

Degorce, S. L. et al. Discovery of proteolysis-targeting chimera molecules that selectively degrade the IRAK3 pseudokinase. J. Med. Chem. 63, 10460–10473 (2020).

Article CAS PubMed Google Scholar

Chaudhary, D., Robinson, S. & Romero, D. L. Recent advances in the discovery of small molecule inhibitors of interleukin-1 receptor-associated kinase 4(IRAK4) as a therapeutic target for inflammation and oncology disorders. J. Med. Chem. 58, 96–110 (2015).

Article CAS PubMed Google Scholar

Mullard, A. IRAK4 degrader to take on innate immunity. Nat. Biotechnol. 38, 1221–1223 (2020).

Article CAS PubMed Google Scholar

Nunes, J. et al. Targeting IRAK4 for degradation with PROTACs. ACS Med. Chem. Lett. 10, 1081–1085 (2019).

Article CAS PubMed PubMed Central Google Scholar

Zhang, J. et al. Assessing IRAK4 functions in ABC DLBCL by IRAK4 kinase inhibition and protein degradation. Cell Chem. Biol. 27, 1500–1509 (2020).

Article CAS PubMed Google Scholar

Chen, Y. et al. Design, synthesis, and biological evaluation of IRAK4-targeting PROTACs. ACS Med. Chem. Lett. 12, 82–87 (2021).

Article PubMed CAS Google Scholar

Nichols, W. C. et al. Genetic screening for a single common LRRK2 mutation in familial Parkinson disease. Lancet 365, 410–412 (2005).

CAS PubMed Google Scholar

Thaler, A., Ash, E., Gan-Or, Z., Orr-Urtreger, A. & Giladi, N. The LRRK2 G2019S mutation as the cause of Parkinson disease in Ashkenazi Jews. J. Neural Transm.(Vienna) 116, 1473–1482 (2009).

Article CAS Google Scholar

Konstantinidou, M. et al. The tale of proteolysis targeting chimeras(PROTACs) for Leucine-Rich Repeat Kinase 2(LRRK2). ChemMedChem 16, 959–965 (2021).

Article CAS PubMed Google Scholar

Schwab, A. D. et al. Immunotherapy for Parkinson disease. Neurobiol. Dis. 137, 104760 (2020).

Article CAS PubMed PubMed Central Google Scholar

Kargbo, R. B. PROTAC compounds targeting α‑Synuclein protein for treating neurogenerative disorders: Alzheimer and Parkinson diseases. ACS Med. Chem. Lett. 11, 1086–1087 (2020).

Article CAS PubMed PubMed Central Google Scholar

Jeremic, D., Jiménez-Díaz, L. & Navarro-López, J. D. Past, present and future of therapeutic strategies against amyloid-β peptides in Alzheimer disease: a systematic review. Ageing Res. Rev. 72, 101496 (2021).

Article CAS PubMed Google Scholar

Congdon, E. E. & Sigurdsson, E. M. Tau-targeting therapies for Alzheimer disease. Nat. Rev. Neurol. 14, 399–415 (2018).

Article CAS PubMed PubMed Central Google Scholar

Wang, W. J. et al. A novel small-molecule PROTAC selectively promotes tau clearance to improve cognitive functions in Alzheimer-like models. Theranostics 11, 5279–5295 (2021).

Article CAS PubMed PubMed Central Google Scholar

Martin-Zanca, D., Hughes, S. H. & Barbacid, M. A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. Nature 319, 743–748 (1986).

Article CAS PubMed Google Scholar

Segal, R. A. Selectivity in neurotrophin signaling: theme and variations. Annu. Rev. Neurosci. 26, 299–330 (2003).

Article CAS PubMed Google Scholar

Chen, L. Q. et al. Discovery of first-in-class potent and selective tropomyosin receptor kinase degraders. J. Med. Chem. 63, 14562–14575 (2020).

Article CAS PubMed Google Scholar

Ryden, M. et al. Expression of mRNA for the neurotrophin receptor TrkC in neuroblastomas with favourable tumour stage and good prognosis. Br. J. Cancer 74, 773–779 (1996).

Article CAS PubMed PubMed Central Google Scholar

Jiang, Z. et al. Targeted maytansinoid conjugate improves therapeutic index for metastatic breast cancer cells. Bioconjugate Chem. 29, 2920–2926 (2018).

Article CAS Google Scholar

Zhao, B. S. & Burgess, K. TrkC-targeted kinase inhibitors and PROTACs. Mol. Pharm. 16, 4313–4318 (2019).

Article CAS PubMed PubMed Central Google Scholar

Marraffini, L. A. CRISPR-Cas immunity in prokaryotes. Nature 526, 55–61 (2015).

Article CAS PubMed Google Scholar

Haft, D. H., Selengut, J., Mongodin, E. F. & Nelson, K. E. A guild of 45 CRISPR-associated(Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60 (2005).

Article PubMed PubMed Central CAS Google Scholar

Garcia-Doval, C. & Jinek, M. Molecular architectures and mechanisms of Class2 CRISPR-associated nucleases. Curr. Opin. Struct. Biol. 47, 157–166 (2017).

Article CAS PubMed Google Scholar

Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).

Article CAS PubMed PubMed Central Google Scholar

Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

Article CAS PubMed PubMed Central Google Scholar

Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).

Article CAS PubMed PubMed Central Google Scholar

Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

Article PubMed CAS Google Scholar

Li, Y., Li, S. Y., Wang, J. & Liu, G. Z. CRISPR/Cas systems towards next-generation biosensing. Trends Biotechnol. 37, 730–743 (2019).

Article PubMed CAS Google Scholar

Wolter, F. & Puchta, H. The CRISPR/Cas revolution reaches the RNA world:Cas13, a new Swiss Army knife for plant biologists. Plant J. 94, 767–775 (2018).

Article CAS PubMed Google Scholar

Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).

Article CAS PubMed Google Scholar

Gama-Brambila, R. A. et al. A chemical toolbox for labeling and degrading engineered cas proteins. JACS Au. 1, 777–785 (2021).

Article CAS PubMed PubMed Central Google Scholar

Lu, X. Y. et al. Feeding induces cholesterol biosynthesis via the mTORC1-USP20-HMGCR axis. Nature 588, 479–484 (2020).

Article CAS PubMed Google Scholar

Vallianou, N. G., Kostantinou, A., Kougias, M. & Kazazis, C. Statins and cancer. Anticancer Agents Med. Chem. 14, 706–712 (2014).

Article CAS PubMed Google Scholar

Kita, T., Brown, M. S. & Goldstein, J. L. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J. Clin. Invest. 66, 1094–1100 (1980).

Article CAS PubMed PubMed Central Google Scholar

LaRosa, J. C. et al. Intensive lipid lowering with atorvastatin in patients with stable coronary disease. N. Engl. J. Med. 352, 1425–1435 (2005).

Article CAS PubMed Google Scholar

Preiss, D. et al. Risk of incident diabetes with intensive-dose compared with moderate-dose statin therapy: a meta-analysis. JAMA 305, 2556–2564 (2011).

Article CAS PubMed Google Scholar

Neklesa, T. K., Winkler, J. D. & Crews, C. M. Targeted protein degradation by PROTACs. Pharmacol. Ther. 174, 138–144 (2017).

Article CAS PubMed Google Scholar

Li, M. X. et al. Degradation versus inhibition: development of proteolysis-targeting chimeras for overcoming Statin-induced compensatory upregulation of 3‑hydroxy-3-methylglutaryl coenzyme A reductase. J. Med. Chem. 63, 4908–4928 (2020).

Article CAS PubMed Google Scholar

Luo, G. S. et al. Discovery of an orally active VHL-recruiting PROTAC that achieves robust HMGCR degradation and potent hypolipidemic activity in vivo. Acta Pharm. Sin. B 11, 1300–1314 (2021).

Article CAS PubMed Google Scholar

Blume-Jensen, P. & Hunter, T. Oncogenic kinase signalling. Nature 411, 355–365 (2001).

Article CAS PubMed Google Scholar

Yancopoulos, G. D. et al. Vascular-specific growth factors and blood vessel formation. Nature 407, 242–248 (2000).

Article CAS PubMed Google Scholar

Shalaby, F. et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62–66 (1995).

Article CAS PubMed Google Scholar

Eriksson, U. & Alitalo, K. Structure, expression and receptor-binding properties of novel vascular endothelial growth factors. Curr. Top. Microbiol. Immunol. 237, 41–57 (1999).

CAS PubMed Google Scholar

Rahimi, N. VEGFR-1 and VEGFR-2: two non-identical twins with a unique physiognomy. Front. Biosci. 11, 818–829 (2006).

Article CAS PubMed PubMed Central Google Scholar

Hubbard, S. R., Mohammadi, M. & Schlessinger, J. Autoregulatory mechanisms in protein-tyrosine kinases. J. Biol. Chem. 273, 11987–11990 (1998).

Article CAS PubMed Google Scholar

Shan, Y. Y. et al. Part 11: Development of PROTACs based on active molecules with potency of promoting vascular normalization. Eur. J. Med. Chem. 205, 112654 (2020).

Article CAS PubMed Google Scholar

Zhou, B. et al. Discovery of a small-molecule degrader of bromodomain and extra-terminal(BET) proteins with picomolar cellular potencies and capable of achieving tumor regression. J. Med. Chem. 61, 462–481 (2018).

Article CAS PubMed Google Scholar

Sun, X. Y. et al. A chemical approach for global protein knockdown from mice to non-human primates. Cell Discov. 5, 10 (2019).

Article PubMed PubMed Central CAS Google Scholar

Saenz, D. T. et al. Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary(s) AML cells. Leukemia 31, 1951–1961 (2017).

Article CAS PubMed PubMed Central Google Scholar

Raina, K. et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 (2016).

Article CAS PubMed PubMed Central Google Scholar

Maneiro, M. et al. Antibody-PROTAC conjugates enable HER2-dependent targeted protein degradation of BRD4. ACS Chem. Biol. 15, 1306–1312 (2020).

Article CAS PubMed PubMed Central Google Scholar

Pillow, T. H. et al. Antibody conjugation of a chimeric BET degrader enables in vivo activity. ChemMedChem 15, 17–25 (2020).

Article CAS PubMed Google Scholar

Dragovich, P. S. et al. Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha(ERα). Bioorg. Med. Chem. Lett. 30, 126907–126914 (2020).

Article CAS PubMed Google Scholar

Dragovich, P. S. et al. Antibody-mediated delivery of chimeric BRD4 degraders. Part 1: exploration of antibody linker, payload loading, and payload molecular properties. J. Med. Chem. 64, 2534–2575 (2021).

Article CAS PubMed Google Scholar

Dragovich, P. S. et al. Antibody-mediated delivery of chimeric BRD4 degraders. Part 2: improvement of in vitro antiproliferation activity and in vivo antitumor efficacy. J. Med. Chem. 64, 2576–2607 (2021).

Article CAS PubMed Google Scholar

Raina, K. & Crews, C. M. Targeted protein knockdown using small molecule degraders. Curr. Opin. Chem. Biol. 39, 46–53 (2017).

Article CAS PubMed PubMed Central Google Scholar

Bunka, D. H. J. & Stockley, P. G. Aptamers come of age-at last. Nat. Rev. Microbiol. 4, 588–596 (2006).

Article CAS PubMed Google Scholar

Kalra, P., Dhiman, A., Cho, W. C., Bruno, J. G. & Sharma, T. K. Simple methods and rational design for enhancing aptamer sensitivity and specificity. Front. Mol. Biosci. 5, 41 (2018).

Article PubMed PubMed Central CAS Google Scholar

Zhou, J. H. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nat. Rev. Drug Discov. 16, 181–202 (2016).

Article PubMed PubMed Central CAS Google Scholar

Thiel, K. W. & Giangrande, P. H. Intracellular delivery of RNA-based therapeutics using aptamers. Ther. Deliv. 1, 849–861 (2010).

Article CAS PubMed Google Scholar

He, S. P. et al. Aptamer-PROTAC conjugates(APCs) for tumor-specific targeting in breast cancer. Angew. Chem. Int. Ed. Engl. 60, 2–9 (2021).

CAS Google Scholar

Veeken, J. V. D. et al. Crosstalk between epidermal growth factor receptor and insulin-like growth factor-1 receptor signaling: implications for cancer therapy. Curr. Cancer Drug Targets 9, 748–760 (2009).

Article PubMed Google Scholar

Anighoro, A. et al. Polypharmacology: Challenges and opportunities in drug discovery. J. Med. Chem. 57, 7874–7887 (2014).

Article CAS PubMed Google Scholar

Tang, C. et al. Novel bioactive hybrid compound dual targeting estrogen receptor and histone deacetylase for the treatment of breast cancer. J. Med. Chem. 58, 4550–4572 (2015).

Article CAS PubMed Google Scholar

Ota, Y. et al. Targeting cancer with PCPA-Drug conjugates: LSD1 inhibition-triggered release of 4-Hydroxytamoxifen. Angew. Chem. Int. Ed. Engl. 55, 16115–16118 (2016).

Article CAS PubMed Google Scholar

He, M. et al. Design, synthesis and biological evaluation of novel dual-acting modulators targeting both estrogen receptor α(ERα) and lysine-specific demethylase 1(LSD1) for treatment of breast cancer. Eur. J. Med. Chem. 195, 112281 (2020).

Article CAS PubMed Google Scholar

Mu, X. P., Bai, L. T., Xu, Y. J., Wang, J. Y. & Lu, H. B. Protein targeting chimeric molecules specific for dual bromodomain 4(BRD4) and Polo-like kinase 1(PLK1) proteins in acute myeloidleukemia cells. Biochem. Biophys. Res. Commun. 521, 833–839 (2019).

Article PubMed CAS Google Scholar

Fung, L. et al. Bioavailable dual-protein degraders of CK1α and transcriptional kinase CDK9 as potential therapeutics for hematological malignancies. Blood 134, 4643–4644 (2019).

Article Google Scholar

Zoppi, V. et al. Iterative design and optimization of initially inactive proteolysis targeting chimeras(PROTACs) identify VZ185 as a potent, fast, and selective von Hippel-Lindau (VHL) based dual degrader probe of BRD9 and BRD7. J. Med. Chem. 62, 699–726 (2019).

Article CAS PubMed Google Scholar

Wang, Z. Q. et al. Proteolysis targeting chimeras for the selective degradation of Mcl-1/Bcl-2 derived from nonselective target binding ligands. J. Med. Chem. 62, 8152–8163 (2019).

Article CAS PubMed Google Scholar

Zheng, M. Z. et al. Rational design and synthesis of novel dual PROTACs for simultaneous degradation of EGFR and PARP. J. Med. Chem. 64, 7839–7852 (2021).

Article CAS PubMed Google Scholar

Scaranti, M., Cojocaru, E., Banerjee, S. & Banerji, U. Exploiting the folate receptor α in oncology. Nat. Rev. Clin. Oncol. 17, 349–359 (2020).

Article PubMed Google Scholar

Numasawa, K. et al. A fluorescent probe for rapid, high-contrast visualization of folate-receptor-expressing tumors in vivo. Angew. Chem., Int. Ed. Engl. 59, 6015–6020 (2020).

Article CAS Google Scholar

Yang, Z. et al. Folate-based near-infrared fluorescent theranostic gemcitabine delivery. J. Am. Chem. Soc. 135, 11657–11662 (2013).

Article CAS PubMed Google Scholar

Low, P. S. & Kularatne, S. A. Folate-targeted therapeutic and imaging agents for cancer. Curr. Opin. Chem. Biol. 13, 256–262 (2009).

Article CAS PubMed Google Scholar

Leamon, C. P. & Reddy, J. A. Folate-targeted chemotherapy. Adv. Drug Deliv. Rev. 56, 1127–1141 (2004).

Article CAS PubMed Google Scholar

Leamon, C. P. et al. Folate targeting enables durable and specific antitumor responses from a therapeutically null tubulysin B analogue. Cancer Res. 68, 9839–9844 (2008).

Article CAS PubMed Google Scholar

Sega, E. I. & Low, P. S. Tumor detection using folate receptor-targeted imaging agents. Cancer Metastasis Rev. 27, 655–664 (2008).

Article CAS PubMed Google Scholar

Ocak, M. et al. Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model. Mol. Pharm. 12, 542–553 (2015).

Article CAS PubMed Google Scholar

Liu, J. et al. Cancer selective target degradation by folate-caged PROTACs. J. Am. Chem. Soc. 143, 7380–7387 (2021).

Article CAS PubMed PubMed Central Google Scholar

Chen, H., Liu, J., Kaniskan, H. Ü., Wei, W. Y. & Jin, J. Folate-guided protein degradation by immunomodulatory imide drug-based molecular glues and proteolysis targeting chimeras. J. Med. Chem. 64, 12273–12285 (2021).

Article CAS PubMed Google Scholar

Mitchell, P. J. & Tjian, R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371–378 (1989).

Article CAS PubMed Google Scholar

Tsherniak, A. et al. Defining a cancer dependency map. Cell 170, 564–576 (2017).

Article CAS PubMed PubMed Central Google Scholar

Lambert, S. A. et al. The human transcription factors. Cell 172, 650–665 (2018).

Article CAS PubMed Google Scholar

Gilmore, T. D. & Herscovitch, M. Inhibitors of NF-κB signaling: 785 and counting. Oncogene 25, 6887–6899 (2006).

Article CAS PubMed Google Scholar

Gupta, S. C., Sundaram, C., Reuter, S. & Aggarwal, B. B. Inhibiting NF-κB activation by small molecules as a therapeutic strategy. Biochim. Biophys. Acta 1799, 775–787 (2010).

Article CAS PubMed PubMed Central Google Scholar

Furqan, M. et al. STAT inhibitors for cancer therapy. J. Hematol. Oncol. 6, 90 (2013).

Article PubMed PubMed Central CAS Google Scholar

Schust, J., Sperl, B., Hollis, A., Mayer, T. U. & Berg, T. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem. Biol. 13, 1235–1242 (2006).

Article CAS PubMed Google Scholar

Song, H., Wang, R. X., Wang, S. M. & Lin, J. A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc. Natl Acad. Sci. USA 102, 4700–4705 (2005).

Article CAS PubMed PubMed Central Google Scholar

Yin, X. Y., Giap, C., Lazo, J. S. & Prochownik, E. V. Low molecular weight inhibitors of Myc-Max interaction and function. Oncogene 22, 6151–6159 (2003).

Article CAS PubMed Google Scholar

Han, H. Y. et al. Small-molecule MYC inhibitors suppress tumor growth and enhance immunotherapy. Cancer Cell 36, 483–497 (2019).

Article CAS PubMed PubMed Central Google Scholar

Watson, P. A., Arora, V. K. & Sawyers, C. L. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat. Rev. Cancer 15, 701–711 (2015).

Article CAS PubMed PubMed Central Google Scholar

Wong, Y. N. S., Ferraldeschi, R., Attard, G. & Bono, J. Evolution of androgen receptor targeted therapy for advanced prostate cancer. Nat. Rev. Clin. Oncol. 11, 365–376 (2014).

Article CAS PubMed Google Scholar

Riggs, B. L. & Hartmann, L. C. Selective estrogen-receptor modulators-mechanisms of action and application to clinical practice. N. Engl. J. Med. 348, 618–629 (2003).

Article CAS PubMed Google Scholar

Liu, J. et al. TF-PROTACs enable targeted degradation of transcription factors. J. Am. Chem. Soc. 143, 8902–8910 (2021).

Article CAS PubMed PubMed Central Google Scholar

Govan, J. M., Lively, M. O. & Deiters, A. Photochemical control of DNA decoy function enables precise regulation of nuclear factor κB activity. J. Am. Chem. Soc. 133, 13176–13182 (2011).

Article CAS PubMed PubMed Central Google Scholar

Morishita, R. et al. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc. Natl Acad. Sci. USA 92, 5855–5859 (1995).

Article CAS PubMed PubMed Central Google Scholar

Samarasinghe, K. T. G. et al. OligoTRAFTACs: a generalizable method for transcription factor degradation. Preprint at bioRxiv https://doi.org/10.1101/2021.12.20.473482 (2021).

Lim, S. H. et al. bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA). Proc. Natl Acad. Sci. USA 117, 5791–5800 (2020).

Article CAS PubMed PubMed Central Google Scholar

Gabizon, R. & London, N. The rise of covalent proteolysis targeting chimeras. Curr. Opin. Chem. Biol. 62, 24–33 (2021).

Article CAS PubMed Google Scholar

He, M., Lv, W. X. & Rao, Y. Opportunities and challenges of small molecule induced targeted protein degradation. Front. Cell Dev. Biol. 9, 685106 (2021).

Article PubMed PubMed Central Google Scholar

Chen, J. J. et al. Enhanced protein degradation by intracellular delivery of pre-fused PROTACs using lipid-like nanoparticles. J. Control Release 330, 1244–1249 (2020).

Article PubMed PubMed Central CAS Google Scholar

Costales, M. G., Suresh, B., Vishnu, K. & Disney, M. D. Targeted degradation of a hypoxia-associated noncoding RNA enhances the selectivity of a small molecule interacting with RNA. Cell Chem. Biol. 26, 1180–1186 (2019).

Article CAS PubMed PubMed Central Google Scholar

Ghidini, A., Cléry, A., Halloy, F., Allain, F. H. T. & Hall, J. RNA-PROTACs: degraders of RNA-binding. proteins Angew. Chem. Int. Ed. Engl. 60, 3163–3169 (2021).

Article CAS PubMed Google Scholar

Li, X. Y., Pu, W. C., Chen, S. & Peng, Y. Therapeutic targeting of RNA-binding protein by RNA-PROTAC. Mol. Ther. 29, 1940–1942 (2021).

Article CAS PubMed PubMed Central Google Scholar

Zhang, C. et al. Semiconducting polymer nano-PROTACs for activatable photo-immunometabolic cancer therapy. Nat. Commun. 12, 2934 (2021).

Article CAS PubMed PubMed Central Google Scholar

Huang, Y. F. et al. Design, synthesis, and evaluation of trivalent PROTACs having a functionalization site with controlled orientation. Bioconjug. Chem. 33, 142–151 (2022).

Article CAS PubMed Google Scholar

Download references

This work was supported by the National Natural Science Foundation of China (#82125034, 81773567), National Major Scientific and Technological Project for #2020YFE0202200, #2021YFA1300200, and #2021YFA1302100), Fellowship of China Postdoctoral Science Foundation (No. 2021M691832).

These authors contributed equally: Ming He, Chaoguo Cao, Zhihao Ni

Ministry of Education (MOE) Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, 100084, Beijing, P. R. China

Ming He, Chaoguo Cao, Zhihao Ni, Yongbo Liu, Peilu Song, Shuang Hao, Yuna He, Xiuyun Sun & Yu Rao

Tsinghua-Peking Center for Life Sciences, 100084, Beijing, P. R. China

Chaoguo Cao

School of Pharmaceutical Sciences, Zhengzhou University, 450001, Zhengzhou, China

Yu Rao

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

Y.R. designed the project, reviewed and revised the manuscript. M.H., C.C., and Z.N. summarized the literature, related structures, and data. M.H., C.C., Z.N., Y.L., P.S., S.H., and Y.H. wrote part of the manuscript and structures. X.S. polished the language of the manuscript. M.H. and C.C. helped organize the manuscript, and M.H. reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.

Correspondence to Yu Rao.

The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

He, M., Cao, C., Ni, Z. et al. PROTACs: great opportunities for academia and industry (an update from 2020 to 2021). Sig Transduct Target Ther 7, 181 (2022). https://doi.org/10.1038/s41392-022-00999-9

Download citation

Received: 18 January 2022

Revised: 25 March 2022

Accepted: 12 April 2022

Published: 09 June 2022

DOI: https://doi.org/10.1038/s41392-022-00999-9

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative