Exploiting the “Hot-Spots” of Hsp70<b>–</b>Bim Protein<b>–</b>Protein Interaction to Optimize the 1-Oxo-1<i>H</i>-phenalene-2,3-dicarbonitrile Analogues as Specific Hsp70<b>–</b>Bim Inhibitors

Abstract

Selectively targeting the cancer-specific protein<b>-</b>protein interaction (PPI) between Hsp70 and Bim has been discovered as a promising strategy for treating chronic myeloid leukemia (CML). The first Hsp70<b>-</b>Bim PPI inhibitor, <b>S1g-2</b>, has been identified to overcome the on-target toxicity of known Hsp70 inhibitors when it induces apoptosis of CML cells. Herein, we carried out a hit-to-lead optimization of <b>S1g-2</b>, yielding <b>S1g-10</b>, which exhibited a 10-fold increase in Hsp70/Bim suppressing potency. Furthermore, <b>S1g-10</b> not only exhibited a 5- to 10-fold stronger antitumor activity in the sub-μM range against CML cells than <b>S1g-2</b> in vitro, but it also overcame BCR-ABL-independent tyrosine kinase inhibitor resistance in CML in vivo depending on the Hsp70<b>-</b>Bim signaling pathway. Moreover, through structure<b>-</b>activity relationship analysis, TROSY-HSQC NMR, molecular dynamics simulation, and point mutation validation, two hydrophobic pockets composed of eight key residues were demonstrated to produce predominant interactions with either Bim or <b>S1g-10</b>, regarded as the "hot-spots" in the Hsp70<b>-</b>Bim PPI interface.