Deciphering molecular mechanisms underlying the inhibition of β-glucuronidase by xanthones from Centaurium spicatum

Herein, we scrutinized the inhibitory potential of five xanthones and a flavonoid, sourced from Centaurium spicatum, against β-glucuronidase activity. The results showed that gentisin and azaleatin emerged as the most potent inhibitors, with significantly lower IC₅₀ values of 0.96 ± 0.10 and 0.57 ± 0.04 μM, respectively. The evaluation of enzyme kinetics unveiled that the isolated xanthones manifested inhibition of β-glucuronidase through a mixed inhibition mode, whereas azaleatin exhibited a noncompetitive inhibition mechanism. The findings from molecular docking analysis unveiled that the compounds under investigation, particularly azaleatin, displayed comparatively diminished binding affinities towards β-glucuronidase. Furthermore, the tested drugs were shown to occupy a common binding site as the employed reference drug. Our comprehensive Molecular Dynamics (MD) simulations analysis revealed consistent trajectories for the investigated drugs, wherein azaleatin and gentisin demonstrated notable stabilization of energy levels. Analysis of various MD parameters revealed that drugs with the lowest IC₅₀ values maintained relatively stable interactions with β-glucuronidase. These drugs were shown to exert notable alterations in their conformation or flexibility upon complexation with the target enzyme. Conversely, the flexibility and accessibility of β-glucuronidase was reduced upon drug binding, particularly with azaleatin and gentisin, underscoring the stability of the drug-enzyme complexes. Analysis of Coul-SR and LJ-SR interaction energies unveiled consistent and stable interactions between certain isolated drugs and β-glucuronidase. Azaleatin notably displayed the lowest average Coul-SR interaction energy, suggesting strong electrostatic interactions with the enzyme's active site and significant conformational variability during simulation. Remarkably, LJ-SR interaction energies across different xanthones complexes were more negative than their Coul-SR counterparts, emphasizing the predominant role of van der Waals interactions, encompassing attractive dispersion and repulsive forces, in stabilizing the drug-enzyme complexes rather than electrostatic interactions.