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

Ethnopharmacological Relevance: Hepatitis E virus (HEV), a significant cause of liver diseases, lacks specific treatments. Khaya grandifoliola C.DC (Meliaceae), used in ethnomedicine to treat infections and liver-related ailments, shows promise as an antiviral agent. While its efficacy against hepatitis C virus (HCV) is known, its effects on HEV remain underexplored. Aims of the Study: This study evaluates K. grandifoliola hydro-ethanolic extract (KHE) as a potential source of HEV inhibitor, focusing on the HEV papain-like cysteine protease (HEV-PLpro). Materials and Methods: Phytochemical profiling of KHE was performed using a Waters-Acquity UHPLC-MS system. The recombinant HEV-PLpro, expressed in baculovirus-infected insect cells and purified via nickel-affinity and size-exclusion chromatography, was used to screen KHE 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 compounds with HEV-ORF1 (PDB ID: 6NU9). Additionally, HEV replication inhibition and cytotoxicity were evaluated in Huh7.5 cells using a Gaussia luciferase reporter system and MTT assay, respectively. Results: Eighteen compounds comprising flavonoid-O-glycosides, polyflavonoids, phenolic and terpene glycoside among other were successfully identified. KHE exhibited mixed-type inhibition of HEV-PLpro proteolytic activity, with an IC50 of 20.86 ug/mL, comparable to ribavirin (19.13 ug/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 ug/mL vs. 17.96 ug/mL), with no cytotoxicity at ≤100 ug/mL. Conclusion: Our results demonstrate that KHE exhibits potent anti-HEV activity by inhibiting both viral protease function and replication, likely attributable to its flavonoid-rich composition. Its mixed-type inhibition mechanism and favorable safety profile underscore its potential as promising source of lead candidate for HEV therapeutics. This study bridges traditional medicine and modern pharmacology, supporting further exploration of K. grandifoliola for antiviral drug development.