The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathologic cell survival remains a formidable but pressing challenge. Precisely tailored compounds would serve as molecular probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of Stabilized Alpha-Helix of BCL-2 domains (SAHBs), we determined that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallography and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK, and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature’s solution to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-x L vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x L selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bclx L , Bcl-2, Bcl-w and Bfl-1, whereas Bcl-x L selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-x L binders.

Mcl-1 is an antiapoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the a-helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl-2 family function. We report the crystal structure of human Mcl-1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl-1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine-to-alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix a3 accommodating an isoleucine-to-tyrosine mutation at this same position. We surveyed the variation in available Mcl-1 and Bcl-x L structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3-only proteins with Mcl-1. With the antiapoptotic Bcl-2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl-1.

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