Mechanistic insights into alkaloid-based inhibition of squalene epoxidase: A combined in silico and experimental approach for targeting cholesterol biosynthesis.

Squalene epoxidase (SQLE) is a key enzyme in the cholesterol biosynthesis pathway and an attractive therapeutic target for hypercholesterolemia and antifungal treatment. In this study, we investigated the inhibitory potential of six alkaloids-berberine, evodiamine, harmine, reserpine, matrine, and sanguinarine-against SQLE using a combined in silico and in vitro approach. Molecular docking revealed strong binding affinities ranging from -8.1 to -11.0 kcal/mol, with evodiamine demonstrating the highest affinity, followed by sanguinarine and berberine. 200 ns MD simulations confirmed stable interactions for all alkaloid-enzyme complexes, characterized by low RMSD values, robust hydrogen bonding, and favorable free energy landscapes, as supported by MM/PBSA analysis. Experimental validation through in vitro inhibition assays revealed that evodiamine (IC₅₀ = 2.87 ± 0.08 μM) exhibited potent inhibition comparable to the standard inhibitor TNSCPA (IC₅₀ = 2.65 ± 0.18 μM), while berberine (IC₅₀ = 3.57 ± 0.18 μM) and reserpine (IC₅₀ = 4.91 ± 0.34 μM) showed strong and moderate inhibition, respectively. Harmine, matrine, and sanguinarine were less effective. Enzyme kinetics studies demonstrated that berberine and reserpine act as noncompetitive inhibitors, binding to allosteric sites, whereas evodiamine exhibited competitive inhibition at the active site. ADMET analysis highlighted favorable pharmacokinetic properties for berberine, evodiamine, and sanguinarine, while reserpine and matrine exhibited limited bioavailability due to solubility and size constraints. The unique inhibitory mechanisms observed were consistent with the structural and physicochemical properties of the compounds. These findings establish berberine, evodiamine, and reserpine as promising SQLE inhibitors, with evodiamine emerging as a lead candidate.