Unraveling molecular mechanisms of β-glucuronidase inhibition by flavonoids from Centaurea scoparia: integrated in silico and in vitro insights

Abstract

Investigating the detailed molecular mechanisms of β-glucuronidase inhibition is critical for pioneering new therapeutic solutions and driving progress in pharmaceutical research. The inhibitory potential of four flavonoid aglycones derived from Centaurea scoparia against β-glucuronidase was thoroughly examined using a combination of in vitro and in silico methodologies. The in vitro assays revealed that compounds 1 and 3 had the strongest inhibitory effects, demonstrated by their low IC₅₀ values of 3.16 ± 0.34 and 3.82 ± 0.1 μM, respectively. The results of the enzyme kinetics assay revealed that compounds 2 and 3, and the reference drug EGCG displayed a mixed inhibition mode. Conversely, compound 1 was found to operate via a noncompetitive inhibition mechanism, as evidenced by the intersection patterns in the Lineweaver–Burk plots. The outcomes from the docking analysis are consistent with the in vitro inhibitory efficacy assays, showing that compounds 1 and 3 possess the lowest binding affinities (−8.6 and −9.0 kcal mol⁻¹, respectively). Isolated phytochemicals demonstrated substantial polar and hydrophobic interactions with the residues within the enzyme's binding site. We investigated the interaction dynamics of isolated compounds with β-glucuronidase using a 100 ns molecular dynamics (MD) simulation. The analysis of various MD parameters indicated that compounds 1 and 3 showed stable trajectories and significant energy stabilization when bound to β-glucuronidase. Furthermore, compounds 1 and 3 exhibited the most favorable average Coulombic short-range interaction energies, recorded at −86.53 ± 11 kJ mol⁻¹ and −98.04 ± 17 kJ mol⁻¹, respectively. These compounds also demonstrated the lowest average Lennard–Jones short-range interaction energies, measured at −84.56 ± 14 kJ mol⁻¹ and −106.02 ± 4.6 kJ mol⁻¹, respectively. The results of MM/PBSA calculations revealed binding free energies of −10.99 ± 2.07, −11.39 ± 2.71, −26.77 ± 3.83, and 0.20 ± 0.32 kJ mol⁻¹ for isolated compounds 1–4 with the target enzyme, respectively. These computational results support the experimental data, suggesting that compounds 1 and 3 could be potent inhibitors of β-glucuronidase.