Novel selective small-molecule inhibitors of Mcl-1 protein

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

DESCRIPTION (provided by applicant): The anti-apoptotic protein Mcl-1 (myeloid cell leukemia), a member of the Bcl-2 family proteins, is an important survival factor for many cancers and is a promising therapeutic target. There are however no known specific small-molecule inhibitors of Mcl-1. Mcl-1 is a homologous protein related to other anti-apoptotic proteins such as Bcl-2 and Bcl-xL, but it has a distinctly different structure and exhibits selective binding to the pro-apoptotic BH3-only proteins. This suggests that specific targeting of the Mcl-1 protein is possible and that drugs specific to Mcl-1 can be developed. ABT- 737, a potent small-molecule inhibitor designed to bind to Bcl-2 and Bcl-xL fails to bind to Mcl-1 and does not induce apoptosis in cancer cells with high levels of Mcl-1 but is effective when the Mcl-1 expression is knocked down, indicating the critical importance of Mcl-1 in maintaining cell survival when Bcl-2 and Bcl-xL are inhibited. Targeting Mcl-1 is therefore an important strategy for the development of a new class of anticancer drugs which can overcome apoptosis resistance. We have identified by high throughput screening over 20 novel, small-molecule inhibitors of Mcl-1. Using complementary biochemical, biophysical, functional and cellular based assays we have evaluated and characterized their potency, specificity, and mechanism of action. The most promising compounds, 59, 62 and 65 have diverse chemical scaffolds. We have shown that these small molecule inhibitors bind to the BH3 binding site in Mcl-1 with IC50 values of 1200 nM, 200 nM and 500 nM, respectively, and compete with BH3 peptides derived from Bid, Bim, or Noxa proteins. They bind to the Mcl-1 protein selectively over two other Bcl- 2 family members, Bcl-2 and Bcl-xL. We conclusively confirmed by NMR spectroscopy that these lead compounds bind to the same BH3 domain of Mcl-1 as the Bim BH3 peptide. These lead compounds antagonize Mcl-1 on the functional level and they induce release of cytochrome c, inhibit cell growth and induce apoptosis in melanoma and lymphoma cancer cell lines with high levels of Mcl-1 showing selectivity over normal cells. We have tested several synthetic and commercially available analogues of 59 and 62, and established initial structure-activity relationships. One of the compounds tested (77), an analogue of 59, has improved binding affinity to Mcl-1 protein, as well as potent activity in inhibition of cell growth in cancer cells. Our results suggest that it is possible to develop small-molecule inhibitors specific for the Mcl-1 protein. We propose in this application to further optimize these lead compounds through a structure-based strategy and integrating experimental structural biology, computational modeling, design and synthesis, and biochemical and biological testing.