Integrating Computational Modeling and Experimental Assay to Discover New Potent ACE-Inhibitory Peptides

Human angiotensin-I-converting enzyme (ACE) is an important target of antihypertensive therapy, which possesses a bulky, hydrophobic pocket that is physicochemically compatible with a wide variety of peptide substrates and small-molecule ligands. Rational design of potent ACE inhibitors has long been an attractive topic in the chemical, biological and medical communities. In the present study, an integrative protocol is described to optimize and modify peptides bound with ACE based on their complex three-dimensional structures. The protocol combines a number of sophisticated computational methods including molecular dynamics (MD) simulation, quantitative structure-activity relationship (QSAR), molecular mechanics/Poisson-Boltzmann surface area (MM-PB/SA) and quantum mechanics/molecular mechanics (QM/MM) to discover new potent ACE-inhibitory peptides. With this strategy hundreds of potential peptides are generated virtually, from which several promising candidates are synthesized and assayed in vitro using a standard Fmoc -protected amino acid solid phase synthesis and spectrophotometric method, respectively. Six peptides are found to have potency against ACE, four of which (LVY, VLKP, MLPVY and LKIPLY) show satisfactory inhibitory capability (pIC50 =5.84, 5.27, 5.40 and 5.57, respectively). Subsequently, the complex structures of tripeptide LVY and hexapeptide LKIPLY with ACE are computationally modeled and their binding free energies are estimated as - 34.2 and - 57.8 kcal/mol, respectively, by using a rigorous QM/MM scheme. Intensive steric collisions are observed around the C-terminus of peptide ligands in the bound state. Based upon the findings the C-terminal residues of the two peptides are modified to small, hydrophobic amino acids Val and Ala, resulting in mutated peptides LVA and LKIPVA, respectively. Consequently, a significant improvement in ACE-inhibitory activity is observed for the LKIPVA mutant (pIC50 increases from 5.57 to 6.07), whereas only a modest activity change was associated with the LVA mutant (pIC50 decreases from 5.84 to 5.80).