From Multi-Species Screening to Targeted Investigation: Discovery of ACE Inhibitory Peptides in Gigantidas platifrons via Peptidomics, Virtual Screening, and Molecular Dynamics Simulations

Deep-sea mollusks represent untapped resources for searching novel biologically active peptides effectual against many chronic diseases. Here we presented the identification of four novel angiotensin I-converting enzyme (ACE) inhibitory peptides from the deep-sea mollusk <i>Gigantidas platifrons</i> by using a combined approach of peptidomics and virtual screening. Fifteen protein hydrolysates from five deep-sea macroorganisms were prepared using three different proteases and were determined for their ACE inhibitory activities. Pepsin hydrolysate of <i>G. platifrons</i> protein (GPp) demonstrated the highest inhibition rate against ACE at 400 μg/mL. Then, targeted investigation was conducted on the GPp with peptidomic profiling; more than 3000 peptides were de novo identified, which were then subject to virtual screening using the docking software Smina. Subsequently, 29 peptides were selected and synthesized based on the affinity threshold and the interactions with ACE active sites. More than 58% peptides were biologically active, showing more than 50% inhibition to ACE at 400 μM. Four peptides, LAAHFAR, YAAPYR, NGAGPYGRP, and FTTFGK, exhibited low micromolar inhibition. The most potent peptide, LAAHFAR with an IC₅₀ of 6.01 ± 1.06 μM, was subject to molecular dynamics simulations for revealing atomistic interaction analysis. LAAHFAR forms comprehensively stable hydrogen bonds with the classic active site of ACE, and its N terminal arginine residue is anchored by additional hydrogen bonding to Cys370, Asp377, and Thr372. This study highlights deep-sea mollusks as an important source of novel ACE inhibitory peptides, contributing to the development of new therapeutic ingredients or functional food agents against hypertension.