Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist - PubMed (original) (raw)

Comparative Study

doi: 10.1124/mol.110.064501. Epub 2010 Jul 14.

Daniela Passeri, Francesca De Franco, Gianmario Ciaccioli, Loredana Donadio, Giorgia Rizzo, Stefano Orlandi, Bahman Sadeghpour, Xiaoxin X Wang, Tao Jiang, Moshe Levi, Mark Pruzanski, Luciano Adorini

Affiliations

Comparative Study

Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist

Giovanni Rizzo et al. Mol Pharmacol. 2010 Oct.

Abstract

Two dedicated receptors for bile acids (BAs) have been identified, the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, which represent attractive targets for the treatment of metabolic and chronic liver diseases. Previous work characterized 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid (INT-747), a potent and selective FXR agonist, as well as 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (INT-777), a potent and selective TGR5 agonist. Here we characterize 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt (INT-767), a novel semisynthetic 23-sulfate derivative of INT-747. INT-767 is a potent agonist for both FXR (mean EC(50), 30 nM by PerkinElmer AlphaScreen assay) and TGR5 (mean EC(50), 630 nM by time resolved-fluorescence resonance energy transfer), the first compound described so far to potently and selectively activate both BA receptors. INT-767 does not show cytotoxic effects in HepG2 cells, does not inhibit cytochrome P450 enzymes, is highly stable to phase I and II enzymatic modifications, and does not inhibit the human ether-a-go-go-related gene potassium channel. In line with its dual activity, INT-767 induces FXR-dependent lipid uptake by adipocytes, with the beneficial effect of shuttling lipids from central hepatic to peripheral fat storage, and promotes TGR5-dependent glucagon-like peptide-1 secretion by enteroendocrine cells, a validated target in the treatment of type 2 diabetes. Moreover, INT-767 treatment markedly decreases cholesterol and triglyceride levels in diabetic db/db mice and in mice rendered diabetic by streptozotocin administration. Collectively, these preclinical results indicate that INT-767 is a safe and effective modulator of FXR and TGR5-dependent pathways, suggesting potential clinical applications in the treatment of liver and metabolic diseases.

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Figures

Fig. 1.

Fig. 1.

INT-767 is a potent FXR agonist. A, ligand-dependent recruitment of Src-1 peptide assessed by AlphaScreen assay. hFXR-LBD-GST was incubated with increasing concentrations of the indicated ligands in the presence of biotinylated Src-1 peptide. The AlphaScreen signal increases when the complex receptor-coactivator is formed. EC50 values were 7 μM, 76 nM, and 7 nM for CDCA, INT-747, and INT-767, respectively. The results show mean ± S.D. of triplicate samples from a representative experiment of 10 performed. B, transactivation assay on HepG2 cells performed by transient transfection of full-length FXR and the canonical FXRE containing 3 inverted repeats (IR1). Vector only, empty vector-transfected cells treated with 10 μM concentrations of the indicated compounds. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. C, transactivation assay in HEK293T cells by using Gal4-FXR-LBD and (UAS)5-Luc system. Vector only: empty vector-transfected cells treated with 10 μM INT-767. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. D to G, regulation of FXR target genes assessed by quantitative RT-PCR. INT-767 increases Bsep, Ost, and Shp and downregulates Cyp7α1 mRNA expression. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. *, p < 0.05; **, p < 0.001 versus vector only or control, cells stimulated with medium only.

Fig. 2.

Fig. 2.

INT-767 is a potent TGR5 agonist. A, NCI-H716 cells were stimulated with increasing concentrations of INT-767 and cAMP levels measured by TR-FRET. INT-767 induces a robust increase of cAMP (EC50 = 0.68 μM). LCA and INT-777 were used as positive controls. The results show mean ± S.D. of triplicate samples from a representative experiment of 10 performed. The Z′ factor of 0.75 indicates that the assay is robust and suitable for high-throughput screening. B, HEK293T cells were transiently transfected with TGR5 or vector only, and cAMP levels were measured by TR-FRET. INT-767 induces TGR5-dependent cAMP production. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. C, transactivation assay performed in HEK293 cells transiently transfected with TGR5 or vector only and the CRE-Luc reporter plasmid. Cells transfected with vector only were stimulated with 100 μM INT-767. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. D, c-fos expression determined by quantitative RT-PCR in NCI-H716 cells treated with the indicated concentrations of test compounds for 1 h. *, p < 0.0001 versus NT, cells stimulated with medium only.

Fig. 3.

Fig. 3.

Cytotoxicity, potential for drug-drug interaction, metabolic stability, and hERG activity of INT-767. A, ATP quantification in HepG2 cells treated with the indicated concentrations of test compounds for 4 h. INT-767 shows no cytotoxic effect. Control refers to cells treated with vehicle alone. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. B, INT-767 does not inhibit CYP3A4. Purified CYP3A4 enzyme and its substrate, 7-benzyloxy-4-trifluoromethyl coumarin, were incubated with the indicated concentrations of the reference inhibitor ketoconazole or INT-767. Although ketoconazole inhibits CYP3A4 with an IC50 of 36 nM, INT-767 does not induce measurable inhibition. The results show mean ± S.D. of triplicate samples from a representative experiment of two performed. C, metabolic stability of INT-767. Liver microsomes and S9 fraction were incubated with increasing concentrations of INT-767, and the compound remaining was measured by high-performance liquid chromatography-MS/MS analysis. The half-life (50% of compound remaining) of INT-767 was 909 min in liver microsomes (top right) and 492 min in the S9 fraction (bottom right), indicating that this compound is very stable to phase I and II enzymatic modifications. Testosterone and 7-hydroxycoumarin were used as positive controls. The results show mean of triplicate samples from a representative experiment of two performed. D, INT-767 does not inhibit human ERG K+ channel. Predictor hERG membranes were incubated with increasing concentrations of E-4031 or tamoxifen, potent and moderate hERG channel inhibitors, respectively, or INT-767. E-4031 and tamoxifen inhibit hERG channel with an IC50 of 4 nM and 3 μM, respectively, whereas INT-767 does not induce measurable inhibition. The results show mean ± S.D. of triplicate samples from a representative experiment of two performed.

Fig. 4.

Fig. 4.

INT-767 enhances adipogenesis in 3T3-L1 preadipocytes. A, qRT-PCR (top) and Western blot (bottom) showing FXR induction at mRNA and protein level during Mix-induced differentiation of 3T3-L1 preadipocytes. HepG2 and NCI are cellular extracts from HepG2 and NCI-H716 cells used as positive control for FXR expression. The results are from a representative experiment of two performed. B, ORO staining. 3T3-L1 cells were treated with vehicle alone (NT), differentiation Mix (Mix), or Mix in combination with INT-747 or INT-767 and stained with ORO 7 days after induction of differentiation. Cells treated with INT-767 show larger cytoplasm and lipid droplets compared with cells treated with Mix alone. The results are from a representative experiment of three performed. C, modulation of adipocyte-related genes detected by qRT-PCR. INT-767 increases adiponectin and FABP4 expression by approximately 3-fold and Glut4 and PPARγ mRNA expression by approximately 2-fold. The results show mean ± S.D. of triplicate samples from a representative experiment of two performed. D, adiponectin, leptin, and resistin levels in culture supernatants from 3T3-L1 adipocytes stimulated to differentiate for the indicated time periods with or without INT-767. The results show mean ± S.D. of triplicate samples from a representative experiment of two performed. *, p < 0.01 versus NT; **, p < 0.05 versus Mix.

Fig. 5.

Fig. 5.

FXR-dependent adipocyte differentiation induced by INT-767. A, ORO staining, at days 4 and 7 of culture, of 3T3-L1 cells not transfected (WT) or transfected with specific FXR siRNA and differentiated with Mix alone or containing 1 μM INT-767. INT-767 increases Mix-induced differentiation in FXR-dependent manner. Controls are cells cultured without differentiation Mix. White arrows indicate lipid droplets. The results are from a representative experiment of three performed. B, lipid quantification at day 7 by Adipored assay of 3T3-L1 cells treated as in A. Values represent mean ± S.D. of six determinations, obtained from two separate experiments performed in triplicate. C, FXR mRNA expression of 3T3-L1 cells treated as in A. Nontargeting siRNA was used as negative control to distinguish sequence-specific silencing from nonspecific effects. *,p < 0.01 versus control; **,p < 0.05 versus Mix; °,p < 0.05 versus WT.

Fig. 6.

Fig. 6.

TGR5-dependent GLP-1 secretion induced by INT-767 in human intestinal cells NCI-H716. A, detection of the active form of GLP-1 in supernatants of NCI-H716 cells treated with the indicated compounds. INT-767 induces secretion of the active form of GLP-1. The results show mean ± S.D. of triplicate samples from a representative experiment of two performed. B, detection of the active form of GLP-1 in supernatants of NCI-H716 cells transfected with vector alone (Vector) or overexpressing TGR5 (TGR5) and treated with the indicated compounds. C, detection of the active form of GLP-1 in supernatants of NCI-H716 cells transfected with nontargeting siRNA (siRNA CTRL) or specific TGR5 siRNA and treated with the indicated concentrations of compounds. NT, cells treated with vehicle alone. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. *, p < 0.05 versus NT; °, p < 0.05 versus siRNA CTRL.

Fig. 7.

Fig. 7.

INT-767 decreases plasma total cholesterol and triglyceride levels in STZ-treated diabetic DBA/2J mice fed with WD. Plasma total cholesterol (A) and triglyceride (B) levels in diabetic DBA/2J mice treated with the indicated doses of INT-767 mixed in the diet for 3 weeks. C, quantification of LDL-cholesterol and HDL-cholesterol levels in STZ-induced diabetic mice and treated as in A and B. *, p < 0.05 versus control (Con); **, p < 0.05 versus STZ (n = 6 mice per group).

Fig. 8.

Fig. 8.

INT-767 decreases plasma total cholesterol and triglyceride levels in db/m and db/db mice. Plasma total cholesterol (A) and triglyceride (B) levels in db/m and db/db mice treated daily with the indicated intraperitoneal doses of INT-767 for 2 weeks. *, p < 0.05 versus db/m vehicle (Veh); **, p < 0.05 versus db/db Veh (n = 6 mice per group).

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