Selective Targeting of Anti-Apoptotic BFL-1 by Cysteine Reactive Stapled Peptides and Disulfide-Tethering Fragments

Abstract

Cellular homeostasis relies on a critical balance of life and death, and when this balance is disrupted, diseases occur. In particular, excess cellular abundance due to aberrant cell survival results in cancer and autoimmune diseases. As gate-keepers of intrinsic apoptotic pathway, the BCL-2 family of proteins regulate Mitochondrial Outer Membrane Permeabilization (MOMP), the critical decision-point at which cells commit to death. Whereas pro-apoptotic BAX, BAK, and BOK are the effecters of MOMP, six anti-apoptotic proteins including BFL-1 repress pro-apoptotic activity, functions that cancer cells can hijack to maintain aberrant cell survival. In particular, anti- apoptotic BFL-1 promotes the survival, oncogenesis, and chemoresistance of a myriad of cancers and yet no selective BFL-1 inhibitors have been developed to-date. Here, we report two strategies for developing selective covalent inhibitors of anti-apoptotic BFL-1. The first strategy involves appending electrophilic warheads onto the N-terminus of BIM and NOXA BH3 stapled peptides. When compared to their non-covalent analogues, these covalent warhead peptides trigger enhanced apoptosis induction of BFL-1/A1 driven cancers such as melanoma and acute myeloid leukemia. Through solving the crystal structures of apo BFL-1 and BFL-1 in complex with a warhead NOXA stapled peptide, we also determine critical binding determinants and structural consequences of warhead peptide engagement with the BFL-1 binding pocket. Our second strategy for developing covalent inhibitors of BFL-1 comprises conducting a disulfide-tethering fragment screen. We identify an acetyltryptophan-bearing disulfide fragment that alleviates BFL-1 blocked of BAX mediated poration of mouse mitochondria through engagement of the conserved leucine binding pocket. Taken together, our results will inform drug development strategies dedicated to developing covalent inhibitors of the BFL-1 binding groove.