Identification of SR3335 (ML-176): a synthetic RORα selective inverse agonist - PubMed (original) (raw)

. 2011 Mar 18;6(3):218-22.

doi: 10.1021/cb1002762. Epub 2010 Dec 6.

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Identification of SR3335 (ML-176): a synthetic RORα selective inverse agonist

Naresh Kumar et al. ACS Chem Biol. 2011.

Abstract

Several nuclear receptors (NRs) are still character-ized as orphan receptors because ligands have not yet been identified for these proteins. The retinoic acid receptor-related receptors (RORs) have no well-defined physiological ligands. Here, we describe the identification of a selective RORα synthetic ligand, SR3335 (ML-176). SR3335 directly binds to RORα, but not other RORs, and functions as a selective partial inverse agonist of RORα in cell-based assays. Furthermore, SR3335 suppresses the expression of endogenous RORα target genes in HepG2 involved in hepatic gluconeogenesis including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Pharmacokinetic studies indicate that SR3335 displays reasonable exposure following an ip injection into mice. We assess the ability of SR3335 to suppress gluconeogenesis in vivo using a diet-induced obesity (DIO) mouse model where the mice where treated with 15 mg/kg b.i.d., ip for 6 days followed by a pyruvate tolerance test. SR3335-treated mice displayed lower plasma glucose levels following the pyruvate challenge consistent with suppression of gluconeogenesis. Thus, we have identified the first selective synthetic RORα inverse agonist, and this compound can be utilized as a chemical tool to probe the function of this receptor both in vitro and in vivo. Additionally, our data suggests that RORα inverse agonists may hold utility for suppression of elevated hepatic glucose production in type 2 diabetics.

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Figures

Figure 1

Figure 1

Identification of a selective RORα synthetic ligand, SR3335. A) Comparison of the chemical structure of T0901317 to SR3335 and SR1078. B) Scheme illustrating the synthesis of SR3335. C) Competition radioligand binding assay illustrating the ability of SR3335 to displace radiolabeled 25-hydroxycholesterol from RORα LBD. D) Competition radioligand binding assay illustrating the inability of SR3335 to displace radiolabeled 25-hydroxycholesterol from RORγ LBD.

Figure 2

Figure 2

SR3335 is a selective RORα partial inverse agonist. Cotransfection of HEK293 cells with RORα, RORγ or LXRα LBD fused to a GAL4 DNA binding domain and a reporter containing 5 copies of the GAL4 UAS upstream of a luciferase reporter. The effect of T1317 is compared to SR3335 in each assay.

Figure 3

Figure 3

SR3335 suppresses the expression of RORα target genes. A) Treatment of HepG2 cells with 5 μM SR3335 results in suppression of transcription in a full-length RORα, G6Pase promoter-luciferase reporter cotransfection assay. G6Pase expression was normalized to cyclophilin. B) Treatment of HepG2 cells with 5 μM SR3335 results in suppression of G6Pase and PEPCK mRNA expression. *, indicates p<0.05.

Figure 4

Figure 4

SR3335 suppresses gluconeogenesis in vivo. A) Pharmcokinetic profile SR3335 following a single injection of 10 mg/kg i.p. B) Pyruvate tolerance test in diet induced obese (DIO) mice (C57Bl/6) following 1 week of b.i.d. dosing (i.p.) 15 mg/kg. C) Gene expression in mice following administration of SR3335 as inidicated in 4B. Gene expression was normalized to cyclophilin. *, indicates p<0.05.

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References

    1. Jetten AM. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl Recept Signal. 2009;7:e003. - PMC - PubMed
    1. Solt LA, Griffin PR, Burris TP. Ligand regulation of retinoic acid receptor-related orphan receptors: implications for development of novel therapeutics. Current Opinion in Lipidology. 21:204–211. - PMC - PubMed
    1. Kallen J, Schlaeppi JM, Bitsch F, Delhon I, Fournier B. Crystal structure of the human ROR alpha ligand binding domain in complex with cholesterol sulfate at 2.2 angstrom. Journal of Biological Chemistry. 2004;279:14033–14038. - PubMed
    1. Kallen JA, Schlaeppi JM, Bitsch F, Geisse S, Geiser M, Delhon I, Fournier B. X-ray structure of the hROR alpha LBD at 1.63 angstrom: Structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of ROR alpha. Structure. 2002;10:1697–1707. - PubMed
    1. Wang Y, Kumar N, Solt LA, Richardson TI, Helvering LM, Crumbley C, Garcia-Ordonez RA, Stayrook KR, Zhang X, Novick S, Chalmers MJ, Griffin PR, Burris TP. Modulation of RORalpha and RORgamma activity by 7-oxygenated sterol ligands. Journal of Biological Chemistry. 2010;285:5013–5025. - PMC - PubMed

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