Probing The Spatial Orientation Of BH3 Groove Of Mcl-1 And Bcl-2 Protein By Novle Acenaphtho-heterocycle Molecules

Pan Bcl-2 protein inhibitors namely bind to at least Bcl-2 and Mcl-1 proteins has been proved the most potential targeted anti-cancer drug candidate molecule. This study focused on S1 (3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile), a small molecule of Bcl-2/Mcl-1 protein dual inhibitor. Compared to the difference affinities of a number of S1 derivatives toward Bcl-2 and Mcl-1 though the structure-activity relationship, probing the spatial orientation of BH3 domain of Bcl-2 and Mcl-1, which guided to design and synthetize more efficient antitumor drug candidate molecules of pan Bcl-2 inhibitors.Small molecule S1 had been verified an efficient specific Bcl-2/Mcl-1 dual inhibitor. Functional and structure studies shown that S1 occupied the BH3 groove of Mcl-1/Bcl-2, a hydrogen bonding network could be formed between carbonyl group of S1 and R263 of Mcl-1 and R146 of Bcl-2, respectively. The thiomorpholine of S1 occupied the p2 pockets of Mcl-1/Bcl-2 and the cyano extended into the p4 pockets of Bcl-2/Mcl-1.Under this guidance, firstly, we designed two series compounds (A and B) by replacing the thiomorpholine of S1 with various steric hindrance groups and explored spatial orientation of the p2 pocket of Bcl-2/Mcl-1 proteins. The results of fluorescence resonance energy (FP) and test cytotoxic test (MTT) shown that for Mcl-1, the Ki value of para-substituted 2f was 122 nM, which is a little enhanced than meta-substituted 2e (Ki=375 nM); but for Bcl-2, compared to 2f (=304 nM), a 50-fold decrease was found for 2e (Ki=16.6μM), revealed most of the affinities have been lost, which illustrated that the p2 pocket of Mcl-1 is relatively wider than that of Bcl-2. Additionally, the Ki value of para-substituted 4g contained with tert-amyl group was 355 nM toward Bcl-2, but 1.38μM toward Mcl-1, which illustrated that the p2 pocket of Mcl-1 is shorter than that of Bcl-2. A novel dual inhibitor 4h was obtained based on these new findings that it exhibited higher affinities toward Mcl-1 and Bcl-2 (Ki=24 and 158 nM), more efficiently than S1, as well as nanomalar cytotoxicity activity against multiple cancer cell lines.Further, applied fragment-based drug design approach, we designed compounds series C by molecular growth at the position of cyano of S1 and probed the space structure of the p4 pocket of Mcl-1 and Bcl-2 protein. ELSIA and structure-activity relationship analysis combined with protein structure analysis showed that the enhancement of binging ability about 20-30 times for Bcl-2, but only 7-10 times for Mcl-1 in the optimization process, which illustrated that the p4 pocket of Mcl-1 is more exposed than Bcl-2. So, ocuption the p4 pocket of Bcl-2 may contribute to enhance the affinity much more than that of Mcl-1. Consequently, a novel dual inhibitor 7b was obtained and that exhibited nanomalar affinities toward Mcl-1/Bcl-2 (IC50=15 and 9 nM), significantly better than SI, especially toward Bcl-2.