Deep Alanine Scanning Reveals Potent Multi-alanine-substituted Protein–protein Interaction Inhibitors
semanticscholar(2021)
摘要
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.
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