Atom-level backbone engineering preserves peptide function while enhancing stability

Peptide therapeutics offer unmatched potency and selectivity but are limited by rapid proteolysis and poor pharmacokinetics. Backbone engineering provides a rational approach to enhance stability while preserving function, yet direct comparisons across strategies remain scarce. Using bradykinin as a model, we systematically evaluated four backbone modifications-D-amino acid substitution, N-methylation, α-methylation, and azapeptide incorporation. Each modification produced distinct outcomes in synthesis, conformation, proteolytic stability, and receptor pharmacology. While D- and N-methyl substitutions yielded high stability, they compromised receptor binding and in vivo function. In contrast, the azapeptide analogue maintained native-like affinity and physiological activity while achieving an enhanced stability profile. These findings highlight the need to balance stability and function in peptide design and position azapeptides as an underexplored class with strong therapeutic potential. More broadly, this study establishes a framework for systematic, data-driven peptide design and optimization.