Pharmacophore-Based Identification and Molecular Characterization of Potent Neprilysin Inhibitors: Biochemical and...

Neprilysin (NEP), a zinc-dependent metalloprotease involved in the degradation of bioactive peptides, represents a validated target for heart failure therapeutics. In this study, a pharmacophore-based virtual screening approach combined with biochemical and biophysical assays, alongside molecular dynamics (MD) simulations, was employed to identify novel NEP inhibitors. The pharmacophore model Phar-A3D2R1 successfully identified pentagalloylglucose (PGG) and tannic acid as potent inhibitors, with IC₅₀ values of 17.2 ± 1.5 μM and 10.9 ± 0.7 μM, respectively. Local surface plasmon resonance (LSPR) assays confirmed strong binding affinities (KD = 6.2 ± 0.4 μM for PGG and 5.9 ± 0.5 μM for tannic acid). MD simulations revealed stable ligand-enzyme interactions mediated by hydrogen bonding, hydrophobic contacts, electrostatic interactions, and coordination with the catalytic Zn²⁺ ion. Cytotoxicity assessment in HEK293T cells indicated negligible toxicity. These results validate PGG and tannic acid as promising lead compounds for NEP inhibition and provide a basis for further structure-based optimization toward cardiovascular therapeutics.