A peptide display system identifies a potent mutant β-melanocyte-stimulating hormone agonist of melanocortin-4...

Non-olfactory G-protein-coupled receptors (GPCRs) regulate vital physiological functions and are targets for ∼34% of US Food and Drug Administration (FDA)-approved drugs. While small-molecule-activated GPCRs are well studied, there is growing interest in peptide GPCRs, particularly the melanocortin-4 receptor (MC4R), a key regulator of energy balance and appetite. Activation of MC4R by β-melanocyte-stimulating hormone (β-MSH) reduces food intake, and pathway dysfunction leads to obesity. However, current methods to study GPCR-peptide interactions are resource intensive and low throughput. To address this, we developed a high-throughput cell surface peptide display platform with a β-arrestin-based MC4R reporter to screen over 2,000 β-MSH point mutants. This approach identified peptide variants that significantly impact MC4R activation, including a novel D5H mutant with enhanced receptor activation. Our results demonstrate a scalable method to directly link GPCR activation to peptide variants, offering insights for therapeutic peptide design.