Structural Insights into Chemoresistance Mutants of BCL-2 and their Targeting by Stapled BAD BH3 Helices

BCL-2 is a central regulator of apoptosis that inhibits cell death by sequestering pro-apoptotic BH3 alpha-helices within a hydrophobic surface groove. While venetoclax, a BH3-mimetic drug, has transformed the treatment of BCL-2–driven malignancies, its efficacy is increasingly limited by acquired resistance mutations that disrupt binding yet preserve anti-apoptotic function—a remarkable structural adaptation. Here, we employed hydrocarbon-stapled alpha-helices derived from the BAD BH3 motif as conformation-sensitive molecular probes to investigate this therapeutic challenge. The stapled peptides not only retain high-affinity binding to all BCL-2 variants, but also show enhanced potency to select venetoclax-resistant mutants. Structural analyses, including X-ray crystallography and hydrogen-deuterium exchange mass spectrometry (HDX MS), revealed the ability of stapled helices to restore native-like BH3 engagement by reverting the conformational consequences of resistance mutations. Notably, we identified a serendipitous interaction between the α3–α4 hairpin of BCL-2 and the hydrocarbon staple that compensates for altered groove conformation and contributes to mutant binding affinity. Together, these findings offer mechanistic insights into BCL-2 drug resistance and reveal a blueprint for designing next-generation inhibitors that overcome this clinically significant barrier to durable treatment responses.