The retinoic acid receptor-related orphan receptors ␣ and ␥ (ROR␣ (NR1F1) and ROR␥ (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be ROR␣ ligands, but the physiological significance is unclear. To date, no endogenous ROR␥ ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both ROR␣ and ROR␥ by directly binding to their ligand-binding domains In the late 1980s, as the canonical domain structure and conserved sequence of members of the nuclear hormone receptor (NHR) 3 superfamily became apparent, several laboratories began to isolate additional members of this superfamily that had no identified ligands. Many of these so-called orphan receptors still have no identified ligands. The first member of the ROR subfamily of receptors (ROR␣) was identified in the early 1990s based on sequence similarities to the retinoic acid receptor and the retinoid X receptor, hence the name "retinoic acid receptor-related orphan receptor" (1, 2). The highly similar receptors, ROR␤ and ROR␥, were identified soon after (3, 4).

SummaryThe vitamin D receptor (VDR) functions as an obligate heterodimer with the retinoid X receptor (RXR). These nuclear receptors (NRs) are multidomain proteins and it is unclear how various domains interact with one another within the NR heterodimer. Here we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA binding domains (DBDs), including the coactivator binding surfaces of both coreceptors, and the sequence of the DNA response element can specify the dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that ligand itself may play a role in DNA recognition. These data suggest a mechanism by which NRs can display promoter-specific activity and impart differential effects on various target genes, which provides mechanistic insight for the function of selective NR modulators.

Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we present the first high-affinity synthetic ligand for both ROR␣ and ROR␥. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) -benzenesulfonamide (T0901317) inhibited transactivation activity of ROR␣ and ROR␥ but not ROR␤. T0901317 was found to directly bind to ROR␣ and ROR␥ with high affinity (K i ϭ 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed ROR␣/␥-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by ROR␣ at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine ROR␣/␥ function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently we reported the development of PPARγ antagonist SR1664, designed to block the obesity induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow derived mesenchymal stem cells (MSCs) with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.

Summary Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously we demonstrated that hydrogen/deuterium exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as providing insight into the role of AF-2 dynamics in the action of VDR partial agonists.