Mechanistic insights into the inhibition of recombinant hepatitis E virus papain-like cysteine protease by Khaya grandifoliola hydro-Ethanolic extract: UHPLC-MS profiling, enzyme kinetics, computational modeling, and cell-based assays.

Hepatitis E virus (HEV), a significant cause of liver diseases, lacks specific treatments. Khaya grandifoliola used in traditional medicine for liver ailments, shows antiviral potential. While known to act against hepatitis C, its effect on HEV is unclear. This study investigates its hydro-ethanolic extract as a potential HEV inhibitor, targeting the HEV papain-like cysteine protease (HEV-PLpro). KHE's phytochemical profiling was determined using Waters-Acquity UHPLC-MS system. Recombinant HEV-PLpro, expressed in baculovirus-infected insect cells and purified via nickel-affinity and size-exclusion chromatography, was used to screen KHE's antiviral activity. The IC50 and inhibition mechanism were determined using the fluorogenic substrate Z-RLRGG-AMC. Molecular docking and dynamics simulations predicted interactions and analyzed the stability of KHE's compounds with HEV-ORF1 (PDB ID: 6NU9). HEV replication inhibition and cytotoxicity were evaluated in Huh7.5 cells using a Gaussia luciferase reporter system and MTT assay, respectively. Eighteen compounds comprising flavonoid-O-glycosides, phenolic and terpene glycoside were successfully identified. KHE exhibited mixed-type inhibition of HEV-PLpro proteolytic activity, with an IC50 of 20.86 μg/mL, comparable to ribavirin (19.13 μg/mL), acting as competitive inhibitor. Notably, quercetin-3-[rhamnosyl-(1 → 2)-α-L-arabinopyranoside] and quercitrin exhibited stronger binding affinities (-7.68 and - 7.43 kcal/mol) and greater structural stability, robustness, and compactness than ribavirin (-4.87 kcal/mol). In cell-based assays, KHE suppressed HEV replication more effectively than ribavirin (IC₅₀: 12.75 μg/mL vs. 17.96 μg/mL), with no cytotoxicity at ≤100 μg/mL. Our results demonstrate that KHE exhibits potent anti-HEV activity by inhibiting both viral protease function and replication. Its mixed-type inhibition mechanism and favorable safety profile underscore its potential as promising source of lead candidate for HEV therapeutics.