Molecular Pharmacology Of Anti-cancer Small Molecule BH3Mimetic S1

The Bcl-2-like protein, as anti-apoptotic members of Bcl-2family is critical for tumor cells evading apoptosis and being immortal. Therefore, the BH3-binding groove govering Bcl-2family protein-protein interaction (PPI) are important targets for small molecule anti-tumor drugs. Previous studies revealed that small molecule inhibitors targeting BH3-binding groove of Bcl-2and Mcl-1protein could antagonize antiapoptotic function of Bcl-2family member in specificity and broad-spectrum manner, by which induce efficient apoptosis in multiple cancer cells. Those small molecule inhibitors are idenfined as BH3mimetics.This study takes a non-DNA intercalative apoptosis-inducing compound S1(8-oxo-3-thiomorpholin-4-yl-8H-acenaphtho [1,2-b] pyrrole-9-carbonitrile) as the research object. By means of protein analysis, cellular biology study, structural biology study and structure-activity study, S1is identified as a Bc1-2/Mcl-1dual inhibitor and exhibiting both in vitro and in vivo anti-tumor effect via targeting BH3binding groove of Bcl-2/Mcl-1to induce Bax/Bak-dependent apoptosis. S1is to data the only BH3mimetic fulfilling both specificity and broad-spectrum need. FP and ITC data show that S1can competitively with BH3peptide bind with both Bcl-2(Ki=310nM) and Mel-1(Ki=58nM). Results of Co-IP, shRNA and FRET experiments determine that S1induces Bax/Bak-dependent apoptosis by direct binding with Bcl-2/Mcl-1protein in multiple cancer cell lines (human breast adenocarcinoma cell line MCF-7, human cervical cancer cell line Hela, human liver cancer cell line SMMC-7721, human colorectal cancer cell line HCT116and human leukemia cell line K562). S1can induce dose and time-dependent disruption of Bcl-2/Bax, Bcl-2/Bim and Mcl-1/Bak interactions, releasing and activating pro-apoptotic protein Bax/Bak, which finally trigger cytochrome c release and caspase activation to execute apoptosis. This molecular mechanism is also the mechanism of anti-tumor effect of S1in H22xenograft mouse model. Protein NMR experiments certify that S1binds in the BH3-binding groove of Mcl-1protein. Based on this, the study combined with fragment-based and structure-activity relationship (SAR) analysis further determines the molecular structural basis by which S1induced apoptosis as a pure BH3mimetic in vitro and in vivo. That is S1specificly binds in the BH3binding groove of Mcl-1, and its carbonyl substitution forms binds hydrogen bonds with R263of Mcl-1. Using S1as a probe, based on its dual inhibition to the two nodes of Bcl-2family network Bcl-2and Mcl-1, this study unveiled a mechanism of Bcl-2network pharmacology regulated by Mcl-1dynamics. The results of apoptosis assay, western blot and Co-IP assays determined that Bim grabbed by S1from Bcl-2, transferred to Mcl-1and stabilized it, resulting in Mcl-1dynamic up-regulation. This further enhanced Mcl-1’s anti-apoptotic function and prompted it constantly grabbing Bak. This increased Mcl-1accounts for cancer cells resistance to Bcl-2specific inhibitor ABT-737, and due to it S1-induced apoptosis is delaied by18hr. Only when Bak was finally grabbed by S1from BH3binding groove of Mcl-1, cancer cell’s fate controlled by Bcl-2family network beginns turned to death. The downstream activated caspase3in turn cleaves Mcl-1, resulting in Mcl-1level decrease and Bak further release. This "cross-talk" mechanism of Bcl-2family network revealed by S1has an important implication for the preclinical and clinical development of the pan-BH3mimetic.As S1is stepping further into preclinal study as an anticancer lead drug, the work aimed to study molecular biomarker in S1induced apoptosis has begain. Bak was determined could serve as the biomarker for the prediction of response to S1.shRNA experiments testified that S1induces apoptosis dependent on Bak, but not Bax. Consistently, Bax-deficient DU145cells are sensitive to S1, whereas Bak-mutant MKN-28cells are significantly more resistant. The in vitro model could be extended to an in vivo mouse xenograft model in which Mcl-1was determined could serve as a biomarker for treatment monitoring of S1. This study will benefit for the future pre-treatment selection and personalized medicine for using S1in clinical.