Pharmacophore-based discovery of FXR agonists. Part I: Model development and experimental validation

Abstract

The farnesoid X receptor (FXR) is involved in glucose and lipid metabolism regulation, which makes it an attractive target for the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. In order to find novel FXR agonists, a structure-based pharmacophore model collection was developed and theoretically evaluated against virtual databases including the ChEMBL database. The most suitable models were used to screen the National Cancer Institute (NCI) database. Biological evaluation of virtual hits led to the discovery of a novel FXR agonist with a piperazine scaffold (compound 19) that shows comparable activity as the endogenous FXR agonist chenodeoxycholic acid (CDCA, compound 2).

Graphical abstract

Figure 1

Number of targets reported per ChEMBL compound.

Figure 2

Molecular property distribution of the ChEMBL database compounds compared with the DrugBank and Virtual DB.

Figure 3

FXR agonists that have been co-crystallized with human FXR and served for binding mode analysis and training compounds for structure-based pharmacophore model generation. The corresponding PDB code is given for each compound and chemical interactions are shown in 2D.

Figure 4

Comparison of FXR ligand binding modes and conformational changes of the binding pocket. (A) Binding modes of 5 (3dct) and its derivatives 14 and 16–18. Hydrophobic areas on the ligands are indicated in yellow, the charged interaction with Arg331 is depicted as red star, and H-bonds with Met265 and Arg331 are highlighted by red arrows. (B) Comparison of binding modes for 11 (black) and 15 using the 3fxv binding pocket. Compound 11 sterically clashes with the binding site in the 3fxv conformation. (C) Compound 13 fitted into the 3dct binding pocket, sharing a large part of it. The acidic functions differ in location and observed interactions. (D) Compound 12 fitted into the 3fxv binding pocket. The difluorophenyl moiety of 12 sterically clashes with the binding pocket conformation from 1fxv.

Figure 5

Pharmacophore models for FXR ligands. Chemical features are color-coded: yellow – hydrophobic, red arrow – H-bond acceptor. (A) Model derived from the binding of 11 to FXR. His294 is shown in ball-and-stick style. (B) Compound 13-based model. His294 and Arg331 are shown in ball-and-stick-style. (C) Compound 5-scaffold model. (D) Compound 12-based model. Tyr369, the H-bonding partner, lies behind the molecule in this perspective; therefore, the H-bond acceptor is not visible. For showing how much the FXR binding pocket changes upon ligand binding compared to 5, His294 and Arg331 are also depicted in ball-and-stick-style.

Figure 6

Reporter gene assay for FXR activation. Application of increasing concentrations (10 nM–100 μM) of the virtual hits 19 (A) and 20 (B), respectively, leads to dose dependent signal increase. FXR activity is expressed as fold difference of relative luciferase units (RLU) represented by mean values ± SEM resulting from triplicate determinations of three independent experiments (^∗P <0.05, ^∗∗P <0.01).

Figure 7

Expression of CYP7A1 mRNA. HepG2 cells treated with 19 and 20 (50 μM) for 6 h in DMEM containing 10% FCS; data expressed as fold of control cells treated with medium/0.1% DMSO (^∗P <0.05, ^∗∗P <0.01, ^∗∗∗P <0.001).

Figure 8

Mapping of compound 19 (black) into the FXR ligand binding site. In comparison to the original ligand, compound 11 (white), 19 established additional hydrogen bonds (arrows) between the hydroxy group and Ser332/Tyr369. The hydrogen bond acceptor (red arrow) with His294 was observed in both compounds 11 and 19, respectively. Hydrogen bond-forming amino acids are depicted in ball-and-stick style. Three hydrophobic interactions (yellow spheres) were predicted to be identical for compounds 11 and 19. The piperazine moiety was not observed to form direct interactions with the receptor and could therefore be regarded as a solubility-enhancing spacer.

Chart 1

Examples for endogenous (1–4) and synthetic (5–10) FXR ligands.

Chart 2

Virtual hits from the models 1osh-1-s and 3bej-1-s that were tested for biological activity in the FXR transactivation assay.