Discovery and characterization of new small-molecule inhibitors of Mcl-1 by high-throughput screening

4148 Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are key regulators of the apoptotic pathway and their increased expression in human malignancies is associated with disease progression, tumor resistance to chemotherapy, radiation and poor clinical outcome. Recently,a novel Bcl-2/Bcl-xL/Bcl-w inhibitor (ABT-737) has been developed, which is highly potent in killing tumor cells displayinghigh levels of Bcl-2 and dependent upon Bcl-2 forsurvival. However, ABT-737 exhibits a relatively low affinityfor Mcl-1 and is considerably less efficient in killing tumor cellsexpressing high Mcl-1 levels. Several groups provide strong evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and one approach for overcoming this resistance could be the combination of ABT-737 with agentscapable of down-regulating/inhibiting Mcl-1.
 Here, we describe a high throughput screening approach that led to the identification of several chemical classes, small molecule Mcl-1 inhibitors. The compounds were characterized in a number of assays including in vitro binding using fluorescence polarization based assay, SPR solution competitive assay, heteronuclear single quantum correlation (HSQC) NMR spectroscopy, pull-down assay, functional assay for cytochrome c and Smac release and a cell-based viability assay with human cancer cells.
 The most potent of the inhibitors identified was compound 65, which binds to Mcl-1 protein with IC50 of 0.3 μM, showing >100-fold selectivity over Bcl-xL and 60-fold selectivity over Bcl-2 protein. 15N HSQC spectra conclusively showed that compound 65 interacts with the BH3 domain in Mcl-1 protein and many residues in the BH3 binding groove of Mcl-1 protein were affected by 65. Furthermore, pull-down assay demonstrates that the compound 65 is able in dose dependent manner to disrupt interactions between endogenous Mcl-1 protein and biotin labeled Noxa and Bid BH3 peptides, but not between Bid BH3 peptide and other members of Bcl-2 family (Bcl-xL and Bcl-2). Functional studies showed that compound 65 effectively antagonizes recombinant Mcl-1 protein and promotes release of cytochrome c and Smac from isolated mitochondria. Compound 65 displayed cytotoxicity with IC50 from 3 μM to 8 μM against a number of human cancer cell lines with high level of Mcl-1 protein. Collectively, these data indicate that 65 represents a novel and bone fide small-molecule inhibitor of Mcl-1 and a promising lead compound for further optimization toward developing a new class of anticancer drugs by targeting the Mcl-1 protein.