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.
Fig. 4The representative PROTACs targeting AR
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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.
Table 1 The summary and comparison of PROTACs targeting ARFull size table
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.
Fig. 5The representative PROTACs targeting BRAF and BRAFV600E
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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.
Fig. 6The representative PROTAC targeting eEF2K
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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.
Fig. 7The representative PROTACs targeting EGFR
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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.
Table 2 The summary and comparison of PROTACs targeting EGFRFull size table
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.
Fig. 8The representative PROTACs targeting eIF4E
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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.
Fig. 9The representative PROTACs targeting ER
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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.
Fig. 10The representative PROTAC targeting FGFR1/2
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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.
Fig. 11The representative PROTACs targeting IGF-1R and Src
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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.
Fig. 12The representative PROTACs targeting KRASG12C
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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.
Fig. 13The representative PROTACs targeting MEK
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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.
Fig. 14The representative PROTAC targeting Myc
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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.
Fig. 15The representative PROTACs targeting p38
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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.
Fig. 16The representative PROTACs targeting PDEδ
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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.
Fig. 17The representative PROTACs targeting SHP2
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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.
Fig. 18The representative PROTACs targeting AKT
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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.
Fig. 19The representative PROTACs targeting ALK
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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.
Fig. 20The representative PROTACs targeting Bcl-xl
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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.
Table 3 The summary and comparison of PROTACs targeting Bcl-xlFull size table
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.
Fig. 21The representative PROTACs targeting BCR-ABL
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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.
Table 4 The summary and comparison of PROTACs targeting BCR-ABLFull size table
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.
Fig. 22The representative PROTACs targeting FAK
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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.
Fig. 23The representative PROTACs targeting MDM2
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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.
Fig. 24The representative PROTACs targeting FLT3
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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.
Fig. 25The representative PROTACs targeting JAK
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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.
Fig. 26The representative PROTAC targeting STAT3
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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.
Fig. 27The representative PROTAC targeting β-catenin
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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.
Fig. 28The representative PROTAC targeting FOXM1
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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.
Fig. 29The representative PROTAC targeting α1A-AR
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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.
Fig. 30The representative PROTACs targeting BRD
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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.
Table 5 The summary and comparison of PROTACs targeting BRDFull size table
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.
Fig. 31The representative PROTAC targeting CBP and p300
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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.
Fig. 32The representative PROTAC targeting ENL
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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.
Fig. 33The representative PROTACs targeting HDAC
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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.
Fig. 34The representative PROTAC targeting KDM5C
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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.
Fig. 35The representative PROTAC targeting NAMPT
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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.
Fig. 36The representative PROTAC targeting NSD3
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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.
Fig. 37The representative PROTACs targeting PRC2 (EZH2, EED)
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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.
Fig. 38The representative PROTAC targeting PRMT5
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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.
Fig. 39The representative PROTAC targeting SIRT2
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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.
Fig. 40The representative PROTACs targeting WDR5
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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.
Fig. 41The representative PROTACs targeting Aurora A
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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.
Fig. 42The representative PROTAC targeting Cdc20
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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.
Fig. 43The representative PROTACs targeting CDK2
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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.
Fig. 44The representative PROTAC targeting CDK2/5
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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.
Fig. 45The representative PROTACs targeting CDK2/4/6
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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.
Fig. 46The representative PROTACs targeting CDK9
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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.
Fig. 47The representative PROTACs targeting CDK12
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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.
Fig. 48The representative PROTAC targeting Wee1
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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.
Fig. 49The representative PROTACs targeting CRBN
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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.
Fig. 50The representative PROTAC targeting hRpn13Pru
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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.
Fig. 51The representative PROTAC targeting VHL
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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.
Fig. 52The representative PROTAC targeting ZFP91
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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.
Fig. 53The representative PROTACs targeting BTK
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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.
Table 6 The summary and comparison of PROTACs targeting BTKFull size table
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.
Fig. 54The representative PROTAC targeting CCR9
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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.
Fig. 55The representative PROTAC targeting CD147
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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.
Fig. 56The representative PROTAC targeting CRABP
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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.
Fig. 57The representative PROTAC targeting HSP90
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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.
Fig. 58The representative PROTAC targeting IDO1
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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
Fig. 59The representative PROTAC targeting LXR-β
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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.
Fig. 60The representative PROTAC targeting MIF
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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.
Fig. 61The representative PROTACs targeting PARP1
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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.
Fig. 62The representative PROTAC targeting PARP14
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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.
Fig. 63The representative PROTACs targeting PD-L1
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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.
Fig. 64The representative PROTAC targeting PLK1
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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.
Fig. 65The representative PROTAC targeting RAR
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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.
Fig. 66The representative PROTAC targeting RHAU
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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.
Fig. 67The representative PROTAC targeting RIPK2
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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.
Fig. 68The representative PROTAC targeting SF3B1
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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.
Fig. 69The representative PROTAC targeting SLC
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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.
Fig. 70The representative PROTACs targeting SMARCA2/4
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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.
Fig. 71The representative PROTAC targeting SRC-1
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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.
Fig. 72The representative PROTAC targeting tyrosinase
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