Serum-Stable and Selective Backbone-N-Methylated Cyclic Peptides That Inhibit Prokaryotic Glycolytic Mutases

<i>N</i>-Methylated amino acids (<i>N</i>-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, <i>de novo</i> discovery from ribosome display is limited by poor incorporation of <i>N</i>-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the <i>N</i>-methyl-aminoacyl-tRNA. By reconfiguring the tRNA's T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different <i>N</i>-MeAAs. We have here devised a "pool-and-split" enrichment strategy using the RaPID display and identified <i>N</i>-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% <i>N</i>-methylation with up to three consecutively incorporated <i>N</i>-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by <i>N</i>-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two <i>cis</i> <i>N</i>-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent <i>Staphylococcus aureus</i> iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the <i>N</i>-MeAA MCP library.