Selective Covalent Targeting of BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors to Reactivate Apoptosis in Cancer

Abstract

Apoptosis is a highly conserved form of programmed cell death that is essential for organismal development and homeostasis. BCL-2 family proteins regulate mitochondrial apoptosis through homo- and hetero-oligomeric protein interactions. Anti-apoptotic proteins inhibit cell death by trapping the critical α-helical BH3 domain of pro-apoptotic proteins in a surface groove, thereby blocking their conformational activation and mitochondrial poration. Cancer cells hijack this system by overexpressing anti-apoptotic members, resulting in cellular immortality and treatment resistance. Thus, drugging BCL-2 family anti-apoptotic proteins is a high-priority therapeutic goal. Venetoclax is a BCL-2 inhibitor that selectively induces apoptosis in BCL-2-dependent cancers, and it is inspiring the design of next-generation compounds that target individual, subsets, or all anti-apoptotic proteins. BFL-1/A1 is an anti-apoptotic protein implicated in the pathogenesis and chemoresistance of melanoma, lymphoma, and leukemia, but it remains undrugged. As an alternative to small-molecule development, we applied hydrocarbon stapling to transform natural BH3 sequences into α-helical peptide probes and prototype therapeutics to respectively dissect and target the BCL-2 family signaling network. Motivated by the natural juxtaposition of two unique cysteines at the binding interface between the NOXA BH3 helix and the BFL-1/A1 binding pocket, we developed stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1/A1 through covalent targeting. The cysteine-reactive stapled peptides demonstrate exquisite selectivity for BFL-1/A1 as well as the capacity to trigger more rapid and potent apoptosis of BFL-1-driven cancers compared to their non-reactive analogs. Thus, we present design principles and proof-of-concept validation for covalent stapled peptide inhibitors as a novel therapeutic modality to reactivate apoptosis in cancer.