Amidine isosteric modification tunes proteolytic stability and activity

The therapeutic potential of peptides is severely limited by rapid metabolism mediated by proteases. Traditional stabilization strategies often compromise pharmacological profiles by disrupting native conformation or physicochemical properties. Here we show that backbone amidine substitution offers a minimal, site-specific modification that enhances metabolic stability. Using the pentapeptide Leu-enkephalin as a model, amidine replacement of metabolically labile amides attenuated or blocked proteolysis in a position-dependent manner, with one analog preserving and even enhancing G-protein signaling at the μ-opioid receptor while reducing β-arrestin2 recruitment. Amidines thus provide a modular strategy to rescue promising peptide leads limited by metabolic instability, with the potential of conferring protease resistance without the need for wholesale scaffold redesign.