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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• N-Methylated amino acids (N-MeAAs) are crucial for peptide bioactivity but difficult to incorporate during synthesis.
• Existing ribosome display methods struggle with N-MeAA incorporation due to low EF-Tu affinity for N-methyl-aminoacyl-tRNAs.

Purpose of the Study:

• To overcome limitations in N-MeAA incorporation for de novo peptide discovery.
• To identify novel N-methylated macrocyclic peptides (MCPs) with inhibitory activity against prokaryotic metal-ion-dependent phosphoglycerate mutases (iPGMs).

Main Methods:

• Reconfigured tRNA T-stem regions to enhance EF-Tu affinity for N-methyl-aminoacyl-tRNAs.
• Employed Random nonstandard Peptides Integrated Discovery (RaPID) display with a pool-and-split enrichment strategy.
• Screened a macrocyclic peptide library containing six different N-MeAAs.

Main Results:

• Achieved up to 57% N-methylation, including consecutive N-MeAAs, rivaling natural products.
• Identified potent nanomolar inhibitors of iPGMs with ortholog selectivity influenced by N-methylation.
• Determined co-crystal structures revealing Cys lariat MCPs dependent on cis N-MeAAs for broadened selectivity.

Conclusions:

• The modified RaPID display effectively enables the discovery of N-methylated peptides.
• N-methylation significantly impacts MCP structure, function, and selectivity.
• Identified novel inhibitors, including a metal-ion-independent Staphylococcus aureus iPGM inhibitor, showcasing library diversity.