Abstract
Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.
Highlights
Anti-apoptotic members of the Bcl-2 family are broadly recognized as promising cancer therapeutic targets
We first scored mutations throughout the Bcl-2 homology 3 (BH3) motif using: (1) a position-specific scoring matrix (PSSM) derived from SPOT peptide array data (PSSMSPOT) and (2) STATIUM, a structure-based statistical potential that previously showed good performance evaluating Bcl-2 protein binding to BH3like peptides (Figure 1B,C) (DeBartolo et al, 2014, 2012)
Bfl-1 is implicated in cancer progression, and inhibiting its anti-apoptotic function may be therapeutically beneficial
Summary
Anti-apoptotic members of the Bcl-2 family are broadly recognized as promising cancer therapeutic targets. The appropriate balance of interactions between pro-survival and pro-death Bcl-2 family members in healthy cells is often disrupted in cancer cells, where overexpression of anti-apoptotic Bcl-2 proteins can promote oncogenesis and confer resistance to chemotherapeutic agents (Opferman, 2016). There has been considerable progress developing BH3 mimetic peptides and small molecules to inhibit the function of anti-apoptotic Bcl-2 proteins by blocking their interactions. One outstanding example is the small molecule venetoclax, which targets Bcl-2 and was recently approved by the FDA for treatment of chronic lymphocytic leukemia (Souers et al, 2013; Roberts et al, 2016). A major challenge in developing venetoclax was achieving specificity, which is important because Bcl-2 family members support survival of healthy cells. Highly selective inhibitors of anti-apoptotic proteins are used for profiling experiments that can establish which anti-apoptotic proteins are essential for cancer cell survival in individual patients and predict
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