Design,Synthesis And Biological Activity Study Of Protein Degradation Targeted Chimera For Targeted Ubiquitin Degradation Of PLK1 And BRD4 Proteins

Proteolysis-targeting chimeras?PROTAC?is a bifunctional heterozygous compound,which binds target proteins on one side and E3 ligase on the other,so that target proteins can bind to E3 ligase,ubiquitinate target proteins and be degraded by proteomics.The experimental results show that,compared with traditional protein inhibitors,PROTAC have broad prospects in the development of therapeutics.Recent studies have shown thalidomide and its derivatives can bind to CRBN protein.CRBN can bind to damaged DNA binding protein 1?DDB1?,Cullin-4A?CUL4A?,cullins 1 regulator?Roc1?to form E3 ubiquitin ligase complex CRL4.The complex can be used to label specific proteins by ubiquitination,followed by hydrolysis of targeted proteins.BI2536 is a double-target protease inhibitor of PLK1(IC₅₀:0.83 nM)and BRD4(IC₅₀:25 nM),and its structure-activity relationship proves that its amide bonding site is the active modification site.In this project,BI2536analogues were selected as the binding sites between PROTAC and target proteins PLK1 and BRD4,thalidomide analogues as the binding sites between PROTAC and E3 ligase,and different lengths and types of linking chains were selected to connect the two,and PROTAC BP series compounds were designed and synthesized.By studying the inhibitory activity of PLK1 and BRD4 protein,the proliferation inhibitory activity of human lung cancer cell A549 and human myeloid monocyte leukemia cell MV4-11,and the effect of BP-3 on apoptosis of MV4-11 cells,the degradation ability of BRD4 and PLK1 protein and the expression of C-Myc protein in MV4-11 cells were investigated,and the anti-tumor mechanism of PROTAC was studied.The pharmacokinetic characteristics of compound BP-3 in rats were also studied.The inhibition of PLK1 and BRD4 proteins by the compounds was studied by ELISA.The results showed that the synthesized BP compounds exhibited good inhibitory activity against BRD4 and PLK1 proteins.The structure-activity relationship showed that the inhibitory activity of compound R to BRD4 and PLK1protein(BRD4:IC₅₀=12.3 nM;PLK1:IC₅₀=8.9 nM)was the strongest when the structure of compound R was benzyl bromide and the link chain of BI2536derivative to thalidomide analogue was PEG and n=3.The inhibitory activity to BRD4 and PLK1 protein decreased when the link chain length increased or replaced with alkyl chain.The results of CCK8 cell experiment showed that the synthesized BP compounds exhibited good inhibitory activity on the proliferation of human lung cancer A549 cells and human myelomonocytic leukemia MV4-11 cells.The inhibitory activities of compounds BP-2,BP-3 and BP-4 on cell proliferation were better than those of control compound BI2536.Compound BP-3 had the strongest inhibitory activity on A549(IC₅₀=10.8 nM)and MV4-11(IC₅₀=7.6 nM).Flow cytometry showed that apoptotic rates of MV4-11 cells induced by BP-3were 6.2%,26.3%and 33.3%respectively at 0,50 and 100 nM.It was proved that BP-3 could induce apoptosis of MV4-11 cells,and the apoptotic activity of BP-3was better than that of the control BI2536.Studies on the effects of MV4-11 cell cycle showed that BP-3 blocked the cell cycle in G0/G1 phase.The expression of apoptosis-related protein in MV4-11 cells showed that BP-3 induced apoptosis of MV4-11 cells may be related to PARP cleavage,caspase-3 activation and Bcl-2down-regulation.Western-blot results showed that BP-3 could degrade BRD4 protein at 20-30nM,PLK1 protein at 40-50 nM,and C-Myc protein expression in downstream pathway of BRD4 could be reduced,while C-Myc protein expression disappeared at40-50 nM.It is proved that BP-3 has the ability to degrade BRD4 and PLK1 proteins,and because of the degradation of BRD4 protein,the downstream channel C-Myc protein expression decreases and disappears,and its protein degradation ability is dose-dependent.When BP-3 was treated with MV4-11 cells at 50 nM for 12 hours,it caused significant degradation of BRD4 and PLK1.It was proved that BP-3 has strong and rapid degradation ability to BRD4 and PLK1 proteins in MV4-11 cells.In vivo experiments with MV4-11 cells in SCID mice demonstrated that both BP-3 and BI2536 significantly inhibited tumor growth,but BP-3 inhibition was superior to BI2536 at the same dose.At the same time,the expression of BRD4,PLK1 and C-Myc protein in tumor tissues showed that BP-3 can inhibit tumor growth by degrading PLK1 and BRD4 proteins and decreasing C-Myc protein expression.At the same time,a high performance liquid chromatography?HPLC?method for the determination of BP-3 in blood was established.The results showed that the method was specific and accurate.The plasma sample was treated by precipitation method.The method was simple and could satisfy the pharmacokinetic study of BP-3.In conclusion,the vivo and vitro bioactivity experiments demonstrated that PROTAC BP-3 designed and synthesized in this study can rapidly and efficiently degrade BRD4 and PLK1 proteins,and reduce the expression of C-Myc protein,and showed potent MV4-11 tumor growth inhibition ability.