Discovery of a small-molecule pBcl-2 inhibitor that overcomes pBcl-2-mediated resistance to apoptosis.

Although the role of Bcl-2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT-737 and S1. We identified an S1 analogue, S1-16, as a small-molecule inhibitor of pBcl-2. S1-16 efficiently kills EEE-Bcl-2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)-expressing HL-60 cells and high endogenously expressing pBcl-2 cells, by disrupting EEE-Bcl-2 or native pBcl-2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1-16 binds to the BH3 binding groove of EEE-Bcl-2 (K-d=0.38 M by ITC; IC50=0.16 M by ELISA), as well as nonphosphorylated Bcl-2 (npBcl-2; K-d=0.38 M; IC50=0.12 M). However, ABT-737 and S1 had much weaker affinities to EEE-Bcl-2 (IC50=1.43 and >10 M, respectively), compared with npBcl-2 (IC50=0.011 and 0.74 M, respectively). The allosteric effect on BH3 binding groove by Bcl-2 phosphorylation in the loop region was illustrated for the first time.