<i>De Novo</i> Discovery of α,α-Disubstituted α-amino Acid-Containing α-helical Peptides as Competitive PPARγ PPI Inhibitors

α,α-Disubstituted α-amino acids (dαAAs) are important building blocks for peptidomimetics as they are strong inducers of helicity and protect against proteolytic degradation. However, <i>de novo</i> discovery of dαAA-containing peptides with genetically encoded libraries is limited due to their poor incorporation efficiency. Here, we report the optimized ribosomal incorporation of multiple achiral dαAAs into peptide libraries and their application to high-throughput (>10¹² members) affinity selection against the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ). This dαAA-based screening methodology discovered potent linear and macrocyclic α-helical peptides with low-to-sub nanomolar binding affinities. Hit peptides were proteolytically stable in serum and cell permeable, allowing for <i>in cellulo</i> antagonism of PPARγ. X-ray crystallography revealed that dαAA-containing peptides bound at the α-helical protein-protein interaction (PPI) interface via an α-helical conformation. This work validates the potential of a dαAA-based, α-helical discovery platform, providing access to new chemical and conformational space to <i>de novo</i> identify novel α-helical peptidomimetics.