Stapled BAD BH3 Helices Effectively Target BCL-2 Mutants That Cause Venetoclax Resistance

Background and Rationale: BCL-2 is the founding member of a family of proteins that regulate apoptosis by controlling the integrity of the mitochondrial outer membrane. BCL-2 intercepts and traps the critical BH3 helices of BAX and BAK in a groove on the protein surface, blocking BAX/BAK-mediated apoptosis. Venetoclax is a selective small molecule inhibitor that binds within the BCL-2 groove and restores the apoptotic pathway by neutralizing the capacity of BCL-2 to inhibit BAX and BAK, effectively “inhibiting an inhibitor” of apoptosis (Souers et al., Nat Med 2013). As the clinical use of venetoclax has expanded, resistance mutations in BCL-2 have arisen that thwart venetoclax binding yet preserve native BH3 interactions, thus re-establishing apoptotic blockade (Blombery et al., Blood 2020). Here, we harnessed the capacity to stabilize the secondary structure of native, bioactive alpha-helices by all-hydrocarbon stapling (Walensky et al., Science 2004) to develop and interrogate BH3 helices that bind venetoclax-resistant mutants of BCL-2 with high affinity and restore BCL-2 inhibition.

Results and Conclusions: We screened a library of “stapled” BH3 peptides modeled after the BAD BH3-only protein, which selectively engages the anti-apoptotic proteins BCL-2, BCL-X_L, and BCL-w. We identified lead alpha-helical constructs that retain the ability to potently target and functionally inhibit BCL-2 irrespective of venetoclax resistance mutations. We performed a series of structural analyses that revealed both the conformational consequences of BCL-2 mutagenesis and the mechanistic basis for retention of high-affinity binding by lead stapled BAD BH3 peptides. These compounds provide prototypes for the development of next-generation BCL-2 inhibitors that overcome resistance to small molecule treatment.

No relevant conflicts of interest to declare.