Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders

Abstract

Retinoic acid receptor-related orphan receptor γ-t (RORγt) and GPBAR1, a transmembrane G-protein-coupled receptor for bile acids, are attractive drug targets to develop clinically relevant small modulators as potent therapeutics for autoimmune diseases. Herein, we designed, synthesized, and evaluated several new bile acid-derived ligands with potent dual activity. Furthermore, we performed molecular docking and MD calculations of the best dual modulators in the two targets to identify the binding modes as well as to better understand the molecular basis of the inverse agonism of RORγt by bile acid derivatives. Among these compounds, 7 was identified as a GPBAR1 agonist (EC₅₀ 5.9 μM) and RORγt inverse agonist (IC₅₀ 0.107 μM), with excellent pharmacokinetic properties. Finally, the most promising ligand displayed robust anti-inflammatory activity in vitro and in vivo in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Figure 1

Chemical structure of 1–21.

Scheme 1. Synthesis of Analogues 1–21

Figure 2

In vitro assays. An overview of the efficacy, potency, and preliminary PK parameters of compounds 3 and 7.

Figure 3

Representation of the MD binding mode of (A) 3 and (B) 7 in RORγ and (E) 3 and (F) 7 in the GPBAR1 homology model. The ligands are represented as pink and gold sticks, respectively, and the interacting residues of the receptor are shown in gray and labeled, with oxygen atoms in red and nitrogens in blue. The receptor is represented as ribbons with its helix labeled. Hydrogens are omitted for the sake of clarity and H-bonds are displayed as dashed lines. Plots of average RMSD calculated on the heavy atoms of the steroidal scaffold of (C) and (G) 3 and (D) and (H) 7.

Figure 4

Electrostatic potential map calculated on GPBAR1. The surface is colored according to the electrostatic potential, where the red color (negative potential) indicates an excess of negative charges and the blue color (positive potential) is an excess of positive charges. The white regions correspond to fairly neutral potentials. The values are expressed in atomic units (a.u).

Figure 5

U937 cells activated with LPS (100 ng/mL) plus TNFα (100 ng/mL) for 24 h, alone or in combination with 3 or 7 at 0.1, 1, 10, 100, and 1000 nM. Dexamethasone (Dex) was used at 5 μM. Quantitative real-time PCR analysis of the expression of proinflammatory genes IL-1β (A and E), TNFα (B and F), IL-6 (C and G), and M1 marker CD11c (D and H). These data are normalized to TBP mRNA expression. Data are derived from five replicates. Results represent the mean ± SEM. ^#p < 0.05 vs NT group and *p < 0.05 vs LPS plus TNFα group. Analysis of variance (ANOVA) was used for statistical comparisons.

Figure 6

Mice treated with TNBS were administered with the vehicle or 7 at doses of 10, 20, or 30 mg/kg/day by gavage from day 0 to day 4. (A) Change in body weight, (B) CDAI score, (C) colon length, (D) ratio between colon weight and colon length, (E) H&E staining on colon sections of the control, TNBS-treated, and TNBS plus various concentrations of 7 treated mice. Results are the mean ± SEM of 5–10 mice per group. *p < 0.05 vs TNBS group and ^#p < 0.05 vs NT group.

Figure 7

C57 male mice treated with TNBS were administered with the vehicle or 7 at doses of 10, 20, or 30 mg/kg/day by gavage from day 0 to day 4. The relative mRNA expression of (A) proinflammatory (IL-1β, IL-6, and TNFα) and (B) anti-inflammatory cytokines (IL-10 and TGFβ), (C) macrophage markers, and (D) T_H17 cell markers in the colon assayed by RTPCR. Data are normalized to the expression of Gapdh, Tbp, and Actb mRNA. Results are the mean ± SEM of 5–10 mice per group. *p < 0.05 vs TNBS group and ^#p < 0.05 vs NT group.

Figure 8

Mice treated with TNBS were administered with the vehicle or 7 30 mg/kg/day, BAR501 30 mg/kg/day, and ML209 20 mg/kg/day by gavage from day 0 to day 4. (A) Change in body weight, (B) CDAI score, (C) ratio between colon weight and colon length, and (D) H&E staining on colon sections of each experimental group. Results are the mean ± SEM of 5–8 mice per group. *p < 0.05 vs TNBS group and ^#p < 0.05 vs NT group.