Discovery and mechanistic benchmarking of ACE inhibitory Di- and tripeptides

Synthetic angiotensin-converting enzyme (ACE) inhibitors demonstrate clinical efficacy, but their adverse effects stimulate interest in natural, food-derived alternatives. We employed an integrated computational framework to systematically screen all possible di- and tripeptides for ACE inhibitory potential, followed by in vitro experimental validation. Computational screening, benchmarked against clinical drugs, revealed that aromatic residues, particularly tryptophan (W), phenylalanine (F), and tyrosine (Y), dominate high-affinity sequences, with binding primarily driven by van der Waals and hydrophobic interactions. Structural analysis demonstrated that the N-terminal WW motif is critical for optimal binding. DFT calculations showed that high-affinity peptides possessed narrow HOMO-LUMO gaps (4.72-4.96 eV), high polarizability (> 500 a.u.), and low chemical hardness. Experimental dose-response assays of the top-ranked candidates validated the computational pipeline, demonstrating a strong rank-order agreement between predicted binding affinities and measured inhibitory potency. Notably, the tryptophan-rich peptides WW, WWY, and WWF exhibited IC50 values of 0.108, 0.134, and 0.136 mM, respectively, underscoring the dominant role of tryptophan in ACE inhibition. These findings establish a validated computational framework and actionable design principles for peptide-based drug discovery, highlighting tryptophan-rich peptides as promising lead molecules of ACE inhibitory candidates for further investigation.