The discovery of a new potent FXR agonist based on natural product screening.

FXR agonistic activity screening was conducted based on natural product resources containing 38 structurally diverse sesquiterpenoids isolated from Xylopia vielana. Among them, 34 undescribed sesquiterpenoids with 5 different skeleton types were first characterized by HRESIMS, NMR data, ECD calculations and X-ray crystallographic analysis. High-content screening for FXR agonistic activity of these compounds demonstrated that 13 compounds could activate FXR. Then molecular docking results suggested that hydrogen bonding and hydrophobic interactions might contribute to the main interaction of active compounds with FXR. The preliminary structure-activity relationships (SARs) of those isolates were also discussed. The most potent compound 27 significantly elevated the transcriptional activity of the FXR target gene BSEP promoter (EC = 14.26 μM) by a dual-luciferase reporter assay. Western blotting indicated that compound 27 activated the FXR-associated pathway, thereby upregulating SHP and BSEP expression, and downregulating CYP7A1 and NTCP expression. We further revealed that FXR was the target protein of compound 27 through diverse target validation methods, including CETSA, SIP, and DARTS under the intervention of temperature, organic reagents and protease. Pharmacological in vivo experiments showed that compound 27 effectively ameliorated α-naphthyl isothiocyanate (ANIT)-induced cholestasis in mice, as evidenced by the ameliorative histopathology of the liver and the decrease in biochemical markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA). This work showed a practical strategy for the discovery of new FXR agonists from natural products and provided potential insights for sesquiterpenoids as FXR agonist lead compounds.