NMR-Guided Fragment Screening Identifies Privileged Noncanonical Amino Acids for mRNA Display.

Fragment-based strategies have transformed small-molecule drug discovery, yet analogous approaches for selecting noncanonical amino acids (ncAAs) in peptide drug discovery remain largely unexplored. Here we introduce an NMR-guided screening workflow that identifies individual ncAAs as "fragments" to identify privileged monomers prior to peptide library construction. Using the p53-MDM2 interaction as a benchmark system, we screened a panel of tryptophan analogs and identified 6-bromo and 6-chloro tryptophan (6Br-Trp and 6Cl-Trp) as a higher-affinity fragments relative to native Trp. Incorporation of 6Br-Trp into a model peptide improved binding to MDM2, demonstrating that amino acid affinity translates directly to peptide-level binding. We further show that 6Br-Trp and 6Cl-Trp can be efficiently charged onto tRNA and incorporated into peptides using the PURE translation system. In an mRNA display selection, peptides containing 6Br-Trp and 6Cl-Trp exhibited enhanced enrichment compared to their Trp-containing counterparts. These results show ncAA fragment screening to be a front-end strategy for guiding monomer selection in high-throughput peptide discovery via mRNA display. ncAA screening also provides a route to bias peptide libraries toward more productive chemical space to accelerate the identification of high-affinity ligands for challenging protein targets.