Establishing structure–activity relationships is crucial to understand and optimize the activity of peptide-based inhibitors of protein–protein interactions. Single alanine substitutions provide limited information on the residues that tolerate simultaneous modifications with retention of biological activity. To guide optimization of peptide binders, we use combinatorial peptide libraries of over 4,000 variants—in which each position is varied with either the wild-type residue or alanine—with a label-free affinity selection platform to study protein–ligand interactions. Applying this platform to a peptide binder to the oncogenic protein MDM2, several multi-alanine-substituted analogs with picomolar binding affinity were discovered. We reveal a non-additive substitution pattern in the selected sequences. The alanine substitution tolerances for peptide ligands of the 12ca5 antibody and 14-3-3 regulatory protein are also characterized, demonstrating the general applicability of this new platform. We envision that binary combinatorial alanine scanning will be a powerful tool for investigating structure–activity relationships.

Establishing structure–activity relationships is crucial to understand and optimize the activity of peptide-based inhibitors of protein–protein interactions. Single alanine mutagenesis provides limited information toward this goal. To guide multiple simultaneous peptide modifications with retention of biological activity, we used synthetic combinatorial alanine-scanning libraries—in which each position was varied with either the wild type residue or alanine—with an affinity selection platform to study the mutational tolerance of protein–ligand interactions. Applying this platform to a peptide binder to the oncogenic protein MDM2, several multi-alanine-substituted analogs that retained low nanomolar affinity were discovered, including a 13-mer binder with seven alanine substitutions at non-hotspot positions. These binders served as templates for further modifications, generating cysteine-substituted, perfluoroaryl-stapled peptides with sub-nanomolar affinity and ten-fold improved proteolytic stability. The alanine substitution tolerances for peptide ligands of the 12ca5 antibody and 14-3-3 regulatory protein were also reported, demonstrating the general applicability of this new platform. We envision that deep combinatorial alanine scanning will be a powerful tool for structure–activity optimization of potential peptide therapeutics.

Establishing structure–activity relationships is crucial to understand and optimize the activity of peptide-based inhibitors of protein–protein interactions. Single alanine mutagenesis provides limited information toward this goal. To guide multiple simultaneous peptide modifications with retention of biological activity, we used synthetic combinatorial alanine-scanning libraries—in which each position was varied with either the wild type residue or alanine—with an affinity selection platform to study the mutational tolerance of protein–ligand interactions. Applying this platform to a peptide binder to the oncogenic protein MDM2, several multi-alanine-substituted analogs that retained low nanomolar affinity were discovered, including a 13-mer binder with seven alanine substitutions at non-hotspot positions. These binders served as templates for further modifications, generating cysteine-substituted, perfluoroaryl-stapled peptides with sub-nanomolar affinity and ten-fold improved proteolytic stability. The alanine substitution tolerances for peptide ligands of the 12ca5 antibody and 14-3-3 regulatory protein were also reported, demonstrating the general applicability of this new platform. We envision that deep combinatorial alanine scanning will be a powerful tool for structure–activity optimization of potential peptide therapeutics.