Licraside as novel potent FXR agonist for relieving cholestasis: structure-based drug discovery and biological evaluation studies

Abstract

Cholestasis is a common clinical disease caused by a disorder in bile acids (BAs) homeostasis, which promotes its development. The Farnesoid X receptor (FXR) plays a critical role in regulating BAs homeostasis, making it an essential target for cholestasis treatment. Although several active FXR agonists have been identified, effective drugs for cholestasis are still lacking. To address this, a molecular docking-based virtual screening method was used to identify potential FXR agonists. A hierarchical screening strategy was employed to improve the screening accuracy, and six compounds were selected for further evaluation. Dual-luciferase reporter gene assay was used to demonstrate FXR activation by the screened compounds, and their cytotoxicity was then evaluated. Among the compounds, licraside showed the best performance and was selected for in vivo evaluation using an ANIT-induced cholestasis animal model. Results demonstrated that licraside significantly reduced biliary TBA, serum ALT, AST, GGT, ALP, TBIL, and TBA levels. Liver histopathological analysis showed that licraside also had a therapeutic effect on ANIT-induced liver injury. Overall, these findings suggest that licraside is an FXR agonist with potential therapeutic effects on cholestasis. This study provides valuable insights into the development of novel lead compounds from traditional Chinese medicine for cholestasis treatment.

Keywords: FXR; agonist; biological evaluation; cholestasis; licraside; virtual screening.

FIGURE 1

(A) Redocking analysis: the superposition of the docked and the co-crystalized poses (33Y in PDB file). Violet: the co-crystalized pose; yellow: the docked pose (B) Binding mode of compound 2 in active sites (residues within 3 Å and 2D-interaction was shown).

FIGURE 2

The 2D structures of six candidate compounds.

FIGURE 3

Effect of compounds intervention on cell viability of HepG2 cell (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001; data is expressed as mean ± SD).

FIGURE 4

(A) The relative luciferase activities of six candidate compounds were evaluated at a single concentration of 20 µM (B) The relative luciferase activities of compound 2 in a concentration-dependent manner (*p < 0.05, **p < 0.01, ***p < 0.001 vs control group; data is expressed as mean ± SD.). (C) Western blot analysis for FXR, SHP and BSEP expressions after treatment 24 h in HepG2 cells (n = 3, data is shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group.).

FIGURE 5

Animal experiment results (A) Experimental protocol: the scheme outlining the design and timeline of the animal experiments (B) Body weight: the body weight of mice in each experimental group. Data are shown as mean ± SD. with n = 8 per group (C) TBA level in bile: the levels of TBA in each experimental group (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ANIT group, n = 8).

FIGURE 6

Serum biochemical indicators of ANIT-induced cholestasis animal model (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ANIT group, n = 3).

FIGURE 7

The effect of licraside intervention on the histopathology of liver in ANIT-induced in-trahepatic cholestasis mice (20 ×), control group (control), ANIT group (model), OCA group (OCA), low-, medium- and high-dose licraside groups (L, M, H).