compound 15b(VUB)

2',4'-dimethoxy-4-hydroxychalcone (15b) has been previously reported as a reversible competitive inhibitor of xanthine oxidase (XOD) with potent activity (> 10-fold vs. allopurinol), but its molecular interactions with the enzyme active site remain uncharacterized. In this research, an integrated approach consisting of multiple spectroscopic methods, isothermal titration calorimetry (ITC), and computer simulations was employed to investigate the binding mode of 15b on XOD. Consequently, ITC data confirmed that the stable 15b-XOD complex was formed spontaneously and driven mainly by hydrophobic interactions with the dissociation constant of the magnitude of 10^-7 mol·L^-1. Three-dimensional fluorescence and circular dichroism spectroscopy indicated that 15b binding induced a conformational transition in the XOD backbone, leading to an increase in global flexibility characterized by enhanced random coil and reduced α-helix/β-sheet. Synchronous fluorescence analysis demonstrated that 15b preferentially perturbed the microenvironment of tyrosine residues over tryptophan residues in XOD. Molecular docking studies (PDB: 1N5X, 2.5 Å) revealed that 15b occupied the xanthine access channel near the molybdenum-pterin (Mo-pt) center, forming critical hydrogen bonds with Arg880 and Thr1010. This binding sterically obstructed substrate xanthine access and subsequent uric acid production. Molecular dynamics simulations indicated that 15b formed a stable binding complex with XOD. Notably, 15b likely bound at the dimer interface and induced inter-subunit proximity, whereas 15b binding preserved the integrity of the surface polarity topology. Furthermore, 15b showed no cytotoxicity in AML-12 hepatocytes at 20 μmol·L^-1(cell viability >90 %), while suppressing uric acid production (> 40 % reduction at 20 μmol·L^-1). These findings may enrich the interaction mechanism of 15b with XOD and also provide a theoretical basis for ligand-guided design of future novel XOD inhibitors.

Hepatic fibrosis poses a significant threat to human health due to excessive extracellular matrix (ECM) deposition leading to liver function damage. Ligand-activated vitamin D receptor (VDR) has been identified as an effective target for hepatic fibrosis, reducing ECM by inhibiting hepatic stellate cell (HSC) activation. Here, a series of novel diphenyl VDR agonists have been rationally designed and synthesized. Among these, compounds 15b, 16i, and 28m showed better transcriptional activity compared to sw-22, which was previously reported to be a potent non-secosteroidal VDR modulator. Moreover, these compounds exhibited outstanding efficacy to inhibit collagen deposition in vitro. In models of CCl₄-induced and bile duct ligation-induced hepatic fibrosis, compound 16i showed the most significant therapeutic effect by ultrasound imaging and histological examination. Moreover, 16i was able to repair liver tissue by reducing the expression levels of fibrosis genes and serum liver function indexes without causing hypercalcemia in mice. In conclusion, compound 16i is a potent VDR agonist with significant anti-hepatic fibrosis action both in vitro and in vivo.