Phytochemical inhibitors of squalene epoxidase: Integrated In silico and In vitro mechanistic insights for targeting cholesterol biosynthesis

Squalene epoxidase (SQLE) is a critical enzyme in the sterol biosynthesis pathway and a promising therapeutic target for various diseases. This study investigates the inhibitory potential of six phytochemicals, amentoflavone, dihydromyricetin, withaferin A, ursolic acid, paeonol, and maslinic acid, against SQLE through an integrated approach combining in silico predictions and experimental validation. Computational analyses, including molecular docking, molecular dynamics (MD) simulations, Potential energy landscape (PEL), MM/PBSA analysis, and ADMET profiling, identified amentoflavone, dihydromyricetin, and withaferin A as the most promising inhibitors, with high binding affinities and stable interactions within the SQLE binding site. Among these, amentoflavone exhibited the strongest binding affinity (−10.4 kcal/mol), binding free energy (−42.01 ± 2.78 kcal/mol), and stability during a 300 ns MD simulation, supported by favorable MD trajectory and interaction energy profiles. Experimental in vitro assays further validated these findings, showing that all tested compounds exhibited inhibitory activity against SQLE, with amentoflavone demonstrating the lowest IC₅₀ (1.92 ± 0.28 μM), confirming its role as a potent inhibitor. Enzyme kinetics studies revealed that maslinic acid and ursolic acid exhibited noncompetitive inhibition, while withaferin A, paeonol, and amentoflavone acted as competitive inhibitors. Dihydromyricetin demonstrated a mixed inhibition mode due to its dual interaction with the enzyme's active and allosteric sites. The pharmacokinetic analysis indicated that all compounds exhibited drug-like properties, with varying ADMET profiles influencing their potential as therapeutic candidates. This study highlights the therapeutic potential of amentoflavone, withaferin A, and dihydromyricetin as potent SQLE inhibitors, underscoring the value of integrated in silico and in vitro approaches in drug discovery.