Discovery of high affinity and specificity stapled peptide Bcl-xL inhibitors using bacterial surface display

Intracellular protein-protein interactions are involved in many different diseases, making them prime targets for therapeutic intervention. Several diseases are characterized by their overexpression of Bcl-xL, an anti-apoptotic B cell lymphoma 2 (Bcl-2) protein expressed on mitochondrial membranes. Bcl-xL overexpression inhibits apoptosis, and selective inhibition of Bcl-xL has the potential to increase cancer cell death while leaving healthy cells comparatively less affected. However, high homology between Bcl-xL and other Bcl-2 proteins has made it difficult to selectively inhibit this interaction by small molecule drugs. We engineered stapled peptides, a chemical modification that can improve cell penetration, protease stability, and conformational stability, towards the selective inhibition of Bcl-xL. To accomplish this task, we built a focused combinatorial mutagenesis library of peptide variants on the bacterial cell surface, used copper catalyzed click chemistry to form stapled peptides, and sorted the library for high binding to Bcl-xL and minimal binding towards other Bcl-2 proteins. We characterized the sequence and staple placement trends that governed specificity and identified molecules with [~]10 nM affinity to Bcl-xL and greater than 100-fold selectivity versus other Bcl-2 family members on and off the cell surface. We confirmed the mechanism of action of these peptides is consistent with apoptosis biology through mitochondrial outer membrane depolarization assays (MOMP). Overall, high affinity (10 nM Kd) and high specificity (100-fold selectivity) peptides were developed to target the Bcl-xL protein. These results demonstrate that stapled alpha helical peptides are promising candidates for the specific treatment of cancers driven by Bcl-2 dysregulation.