High-Throughput Screening,Design,Synthesis And Bioactivity Of Small-molecule Inhibitors Of Anti-apoptotic Bcl-2 Proteins

Apoptosis or type ? programmed cell death and autophagy or type ? programmed cell death are two prominent self-destructive processes.Defective apoptotic cell death mechanism is recognized as a hallmark of cancer.However,an autophagy-associated cell death pathway can be seen as a back-up cell death mechanism in cancer cells that are deficient in the apoptosis pathway.Therefore,many attempts have been made to induce autophagy for anticancer therapy.Bcl-2 family members are dual regulators of apoptosis and autophagy.Anti-apoptotic Bcl-2 family members,especially Bcl-2 and Bcl-xL,arc overexpressed in many types of cancers and contribute to tumor initiation,progression and resistance to therapy.Bel-2 and Bcl-xL have also been shown to inhibit autophagy via binding to and antagonizing the BH3-only protein Beclin 1,an essential autophagy stimulator.We hypothesize that compounds inhibiting Bcl-2/xL might be able to disrupt Bcl-2/xL-Beclin 1 interaction,free out Beclin 1,and thus trigger autophagy.In order to identify small molecule Bcl-2/xL inhibitors,a fluorescence polarization-based binding assay has been set up and optimized for high-throughput screening.Using the Bcl-xL protein as the large molecule and the fluorescein-labeled Beclin 1-20-mer peptide as the small molecule,high-throughput screening of 50,316 compounds was carried out with a Z' score of 0.82 ± 0.05,and an outcome of 58 hits.After the structure analysis,three acridinc analogues were unveiled and their binding affinities to Bcl-xL protein were confirmed using the fluorescence polarization assay and the surface plasmon resonance assay(Ki= 0.84 ?M,28.04 ?M or 13.15 ?M).They showed micromolar cytotoxicity and induced autophagy in prostate cancer cells PC-3,PC-3a and DU 145,warranting further medicinal chemistry optimization.To gain primary structure-activity relationships,41 additional acridine analogues were designed,synthesized,and their pharmaceutical activities and mechanism of action were investigated.In the cell viability assay,compounds 7,18,25,29 and 31could effectively inhibit prostatic cancer cell growth and compounds 35 and 41 could inhibit gastric cancer cell growth.Notably,compounds 7 and 8 have nanomolar cytotoxicity.The binding affinities of compounds were confirmed by the surface plasmon resonance assay.Compound 7 is a selective Bcl-xL inhibitor,which has a high binding affinity for Bcl-xL(KD= 6.5 ?M)over Bcl-2(KD= 160 ?M)protein.Compounds 29 and 31 are pan Bcl-2 inhibitors with KD in between 30 ?M and 50 ?M.Furthermore,induction of autophagy was also demonstrated in cancer cells treated with 7,18,25,29,31,35 or 41 by the LC3 conversion immunoblotting and the fluorescence microscopy.Besides,compound 35 could induce both autophagy and apoptosis in gastric cancer cells.In summary,several acridine compounds are discovered and developed as Bcl-2/xL inhibitors.They showed considerable cytotoxicity,potent binding affinities and induced autophagy in cancer cells.The discovery of these small molecule Bcl-2/xL inhibitors as autophagy inducers is not only likely to be a potential strategy for cancer therapy,but it will also facilitate a better understanding of the complicated roles of autophagy in normal physiology and pathophysiology.