Study On The Pharmacodynamics And Mechanism Of A Novel Highly Selective BRD4 Protein Degradation Molecule In AR-positive Castrationresistant Prostate Cancer

Malignant tumors are currently one of the most serious public health problems in the world.The incidence and mortality of cancer are increasing year by year,and they have become the main cause of death in most regions.Among them,prostate cancer is a serious threat to the health of middle-aged and elderly men,and the proportion of global male tumors is increasing year by year.Androgen deprivation therapy（ADT）is the first-line treatment for prostate cancer,but as the treatment progresses,the disease will progress to castration-resistant prostate cancer（CRPC）.The second-generation anti-androgenic drugs,abiraterone and enzalutamide,are currently used in CRPC treatment.Although both show some therapeutic potential,patients will be re-resistant after taking them for a long time.Therefore,it is still very important to develop new treatment strategies and treatment methods for prostate cancer.In prostate cancer with positive androgen receptor（AR）expression,the transcription cofactor BRD4 can interact with AR and affect the transcription of AR’s downstream genes,regulate the expression of MYC,PSA,TMPRSS2-ERG fusion genes,etc.,thereby promoting the proliferation of prostate cancer cells.BRD4 is also closely related to the castration resistance,metastasis and poor prognosis of prostate cancer.Therefore,BRD4 can be used as a potential therapeutic target for AR-positive prostate cancer.However,traditional BRD4inhibitors are often poorly selective,and have varying degrees of inhibitory activity against BET family proteins including BRD2,BRD3 and BRDT,so they are likely to cause a variety of toxic and side effects.The inhibitor’s action time is not long enough,so patients are likely to develop drug resistance.Small molecules that can selectively and continuously down-regulate BRD4 expression level or protein activity are expected to solve the above-mentioned shortcomings of current BRD4 inhibitors.PROTAC（Proteolysis targeting chimeras）is a type of bifunctional molecule containing different ligands at both ends.It connects the target protein ligand with the E3 ubiquitin ligase ligand through a linker of appropriate length,which can connect E3 ubiquitin.The ligase is recruited to the vicinity of the target protein and catalyzes the ubiquitination and degrades the target protein.At present,a large number of literature reports on PROTAC molecules developed for a variety of disease-related proteins.Compared with ordinary small molecule inhibitors,PROTAC molecules have the following advantages:（1）PROTAC work by degrading proteins rather than inhibiting protein functions,so the drug effect is longer and the activity is better than inhibtors;（2）One molecule compound can often induce the degradation of multi-molecule proteins,so the target protein can be fully degraded at a substoichiometric drug concentration,the effective dose of the drug is lower,and the safety is possible better;（3）PROTAC do not need to be combined with the functional domains of the target protein to perform protein degradation,so they can target proteins that could not be targeted before,such as cytoskeleton proteins or transcription factors;（4）The interaction between E3 ubiquitin ligase and target proteins can make PROTAC has higher selectivity than traditional small molecule drugs,so it can avoid the safety problems caused by the off-target effects of traditional drugs to a certain extent.These advantages of PROTAC make it particularly suitable for development as anti-tumor drugs.Our research group was previously committed to the development of BET/PLK1 dual-target inhibitors and their use in the treatment of tumors.A large class of[1,2,4]-triazolo-[4,3-f]-pteridine derivatives had been designed and synthesized in the previous research,and a novel BET/PLK1 dual-target inhibitor WNY0824 was selected from them.WNY0824 can block multiple signaling pathways which regulated by both BET family and PLK1 via simultaneously inhibiting the functions of the BET family and PLK1 proteins.WNY0824exhibits good anti-tumor effects in acute myeloid leukemia and castration-resistant prostate cancer both in vivo and in vitro.In view of the insufficient inhibitory activity of WNY0824on BET and PLK1,this research designed a series PROTACs which based on the molecular structure of WNY0824,in order to find dual-targeting PROTAC molecules with protein degradation activity for both BET and PLK1.However,in the structure-activity relationship study,we unexpectedly found that the PROTAC molecule designed based on WNY0824 can degrade BRD4 protein with high selectivity,but has no significant degradation activity on other proteins of the BET family and PLK1.This research examined the anti-tumor activity and mechanism of the optimal molecule WWL0245 in castration-resistant prostate cancer in detail,and compared its mechanism of action with the BET/PLK1 dual target inhibitor WNY0824.1.The discovery of highly active BRD4-PROTAC molecule WWL0245 which based on the design of BET/PLK1 dual target inhibitorsFirst,we use WNY0824 as the target protein ligand,CRBN ligand thalidomide,lenalidomide and VHL ligand VH032 as the E3 ubiquitin ligase ligand,and use alkyl chains of different lengths or polyethylene glycol chains as linkers to design and synthesize a series of PROTAC molecules.After preliminary screening of PROTACs on cell proliferation inhibitory activity and protein degradation activity,we obtained a number of active molecules that have good proliferation inhibitory activity on MV4-11 and A2780 cells and can significantly degrade BRD4 protein.Among them,the best molecule WWL0245 shows excellent cell proliferation inhibitory activity on a variety of BRD4-dependent tumor cells,while its proliferation inhibitory activity on BRD4-independent tumor cells is poor,suggesting that it may selectively target BRD4.This thesis then select WWL0245 to conduct in-depth pharmacodynamic evaluation and mechanism research on AR-positive and negative castration-resistant prostate cancer cells.2.In vitro pharmacodynamic study of WWL0245 in prostate cancer cellsThis study first investigate the proliferation inhibitory activity of WWL0245 on five prostate cancer cell lines with different AR expressions（including AR-positive CRPC cell lines VCa P,22Rv1,AR-positive hormone-sensitive prostate cancer cell line LNCa P,and AR-negative CRPC cell lines PC-3,DU145）.Similarily to WNY0824,WWL0245 shows strong proliferation inhibitory activity against AR-positive cell lines(IC₅₀ for VCa P,22Rv1,LNCa P respectively 16.0 n M,20.8 n M and 52.9 n M),but the activity against AR-negative cell lines is poor(for PC-3,DU145 IC₅₀ is 2.435μM,>10μM),suggesting that the molecule has potential therapeutic use for AR-positive prostate cancer.WWL0245 can also significantly inhibit 22Rv1 colony formation,even when its concentration ten times lower than that of WNY0824,WWL0245 achieves the same inhibitory effect.WWL0245 and WNY0824 treat tumor cells for three days and then stop the drug treatment and continue to culture cells,after8 days of drug withdrawal,WWL0245 can continue to inhibit the proliferation of AR-positive prostate cancer cells,and its inhibitory effect at a concentration of 100 n M is better than that of WNY0824 at a concentration of 1μM.3.Study on the BRD4 protein degradation activity and selectivity of WWL0245 in prostate cancer cellsThis study further investigate the ability of WWL0245 to degrade BET family and PLK1proteins and to regulate the expression of c-Myc in prostate cancer cells.WWL0245 can degrade BRD4 and down-regulate the expression of c-Myc in a time/concentration-dependent manner.For AR-positive castration-resistant prostate cancer cells 22Rv1 and VCa P,the DC₅₀reach 0.36 n M and 0.07 n M,and Dmax>99%.Although WWL0245 maintains a similar affinity to BRD2/BRD3 as BRD4,it has no significant effect on the protein level of BRD2/BRD3 at the highest concentration of 1μM,suggesting that it has a selective degradation effect on BRD4.WWL0245 also has comparable inhibitory activity to PLK1 as WNY0824,but it has no significant down-regulation effect on PLK1 levels in all test concentration ranges.According to its selectivity for degradation of BET family proteins,we speculate that by selecting appropriate E3 ubiquitin ligase ligands and linkers,multi-target small molecule compounds can be designed and developed into highly selective PROTAC molecules.4.Study on the mechanism of WWL0245 inducing BRD4 degradation and inhibiting the proliferation of AR-positive prostate cancer cellsFinally,this study investigate the specific mechanism of WWL0245 inducing protein degradation and inhibiting cell proliferation,BET/PLK1 inhibitor WNY0824,BET inhibitor JQ1,CRBN ligand lenalidomide,proteasome inhibitor MG-132 and NAE Inhibitor MLN-4924 can reverse the degradation of BRD4 by WWL0245 to varying degrees,indicating that WWL0245 down-regulate BRD4 depends on its combination with BRD4 and CRBN,and also depends on the ubiquitination proteasome system.WWL0245 can significantly down-regulate the protein levels of AR and PSA which are very important in prostate cancer and inhibit the transcription of AR’s downstream genes.It can also induce cell apoptosis in a concentration-dependent manner,and down-regulate PSA protein levels.At the same concentration with WNY0824,the induce effect of WWL0245 is better.In addition,WWL0245 can block the cell cycle in the G0/G1 phase,indicating that it mainly plays its cell cycle arrest effect by degrading BRD4,while WNY0824 blocks the cell cycle in the G2/M phase mainly by inhibiting PLK1,suggesting they may have different mechanisms to their anti-tumor effect.In summary,this study develope a BRD4-PROTAC molecule WWL0245 with high degradation activity and high selectivity through a mild multi-target inhibitor WNY0824,and confirme that it can efficiently degrade BRD4 proteins therby exert better anti-tumor activity than WNY0824 in AR-positive prostate cancer cells.Therefore,WWL0245 is expected to be further developed as a new type of prostate cancer drug candidate.In addition,as a highly selective and active BRD4 protein degradation molecule,WWL0245 can also be used as a good tool molecule to explore the physiological functions of BRD4.