Cholesterol and cholesterol sulfate have been suggested to be natural ligands for RORα (3, 4) and our recent work identified various oxysterols that bind to both RORα and RORγ with high affinity and regulate their activity (5, 6). There is some controversy as to the nature of the constitutive activity of the RORs observed in cell-based assays. Our data indicates that RORs display the constitutive activity in biochemical assays under conditions where the receptor would be expected to have no endogenous ligand present (denatured and refolded receptor) (5), but others have suggested that endogenous oxysterol ligands may copurifiy leading to the observed constitutive activity (7). Although the physiological significance of these natural ligands for the RORs is unclear, the potential utility of synthetic ligands that modulate the activity of these receptors is apparent. For example, loss of RORα in the staggerer mice results in mice resistant to weight gain and hepatic steatosis when placed on a high fat diet (8). RORγ has been shown to be involved in development of Th17 cells that are implicated in autoimmune diseases and loss of RORγ yields animals that are resistant to development of these diseases (9, 10). RORα has been shown to be required for normal bone development and staggerer mice lacking functional RORα are osteopenic (11) suggesting that RORα agonists may have utility in the treatment of osteoporosis.

We recently identified the first synthetic ligand that binds to and modulates the activity of RORα and RORγ, T0901317 (T1317) (Fig. 1A) (12). T1317 was originally identified as a liver X receptor agonist (LXR), an NR that serves as a physiological receptor for oxysterols (13). We later showed that T1317 showed a degree of promiscuity and also activated another NR that serves as a receptor for bile acids, FXR (14). In an NR specificity screen examining the activity of T1317 at all 48 NRs, we noted that T1317 displayed inverse agonist activity on RORα and RORγ (12). Since both RORs and LXRs bind to oxysterols with high affinity it is not unexpected that they may also display cross-reactivity with regard to synthetic ligands; however, our data indicate that T1317 serves as a very efficacious agonist of LXR while performing as an inverse agonist on RORs. Clearly, T1317 does not display the appropriate pharmacological profile to be used as a chemical tool to probe ROR function since it displays significant activity on LXR and FXR. However, we believed that the benzenesulfonamide scaffold to be a suitable initiation point for development of the first selective ROR ligands.

Using the T1317 as an initial scaffold, we synthesized an array of compounds and assessed their activity at RORα, RORγ, FXR, LXRα, and LXRβ. One compound of particular interest was the amide SR1078 (Fig. 1A) that displayed a unique pharmacological profile indicating a high potential to be used as a chemical probe for assessment of ROR function. The synthetic scheme for SR1078 is shown in Fig. 1B (15). This compounds was initially identified based on its ability to inhibit the constitutive activity of RORα/γ. In a biochemical coactivator interaction assay using Alpha screen technology, increasing doses of SR1078 resulted in a concentration dependent reduction in the ability of RORγ to recruit the TRAP220 coactivator LXXLL NR box (Fig. 1B). In a cell-based chimeric receptor Gal4 DNA-binding domain – NR ligand binding domain cotransfection assay, SR1078 significantly inhibited the constitutive transactivation activity of RORα and RORγ, but had no effect on the activity of FXR, LXRα and LXRβ (Fig. 1C). These data clearly demonstrate that we developed a compound that selectively targeted RORα and RORγ and no longer functioned as a LXR/FXR agonist.

In order to examine the activity of SR1078 in more detail, we performed additional cotransfection assays where we transfected cells with full-length RORα or RORγ and luciferase reporter genes driven by promoters derived from known ROR target genes. Two distinct reporter constructs were utilized: one driven by the glucose-6-phosphatase (G6Pase) promoter and one driven by the fibroblast growth factor-21 (FGF21) promoter. Both of these genes have been shown to be direct target genes of ROR and have characterized ROR response elements within their promoters (5, 16, 17). Unexpectedly, we noted that SR1078 functioned as a ROR agonist, not inverse agonist, when used in the context of the full-length receptors. As shown in Fig. 2A, in a RORα cotransfection assay, treatment of cells with SR1078 resulted in a significant increase in transcription. Similarly, in the RORγ cotransfection assay, SR1078 treatment resulted in a stimulation of RORγ-dependent transcription activity (Fig. 2B). In both cases, these effects were clearly mediated by ROR since the effect was lost when the RORE was mutated in the G6Pase promoter. Consistent with the G6Pase data, when the FGF21 promoter was used in the cotransfection assay, SR1078 behaved as a RORα/γ agonist stimulating ROR activity (Fig. 2C). With both RORα and RORγ using either promoter, we noted that the effects of SR1078 were dose-dependent as shown in Figs 3A-3D. Transcription was stimulated at concentrations of 2 to 5 μM and above. The lack of correlation between recruitment of a cofactor peptide and agonist vs. antagonist activity in cell-based assays has been observed previously with REV-ERBα where the natural ligand heme causes displacement of the cofactor peptide but the ligand acts like an agonist in cells (18, 19).

In order to confirm that SR1078 is indeed an agonist in a more “physiological” context, we tested its activity in an assay that detects its effect on the expression of actual target genes in a target cell line expressing endogenous levels of RORα and RORγ. HepG2 cells were treated with SR1078 for 24h followed by assessment of G6Pase and FGF21 gene expression. As shown in Fig. 4A, SR1078 treatment resulted in a significant 3-fold increase in FGF21 mRNA expression. G6Pase mRNA expression was also significantly stimulated ~2-fold by SR1078 treatment (Fig. 4B). These data support the results from the cotransfection data indicating that SR1078 functions as a ROR agonist unlike its precursor T1317 which functions as an inverse agonist is these same assays.

We examined the pharmacokinetic properties of SR1078 in mice and noted significant in vivo exposure. Plasma concentrations reached 3.6 μM 1h after a 10 mg/kg i.p. injection of SR1078 and sustained levels of above 800 nM even 8h after the single injection (Fig. 4C). These levels were sufficient to perform a proof-of-principle experiment to determine if SR1078 treatment would stimulate ROR target gene expression in an animal model. Mice were treated with SR1078 (10 mg/kg i.p.) and 2h after the injection the livers were harvested and mRNA purified for assessment of G6Pase and FGF21 gene expression. As shown in Fig. 4D & 4F the expression of both FGF21 and G6Pase was significantly stimulated by SR1078 treatment vs. vehicle control.

In summary, we report the identification of a synthetic ligand for RORα and RORγ that functions as an agonist in the context of the full-length receptors. Thus, SR1078 represents the first synthetic ligand that is able to function as an ROR agonist. In cotransfection assays, SR1078 activates the transcription driven by ROR target gene promoters in a RORE-dependent manner. Furthermore, treatment of cells that express RORα and RORγ endogenously with SR1078 results in stimulation of expression of ROR target genes. It is worth noting that this compound activates the receptor beyond the level of its constitutive activity that is normally observed. The fact that the initial lead compound, T1317, functions as a RORα/γ inverse agonist and SR1078 functions as a RORα/γ agonist indicates that it is possible to develop synthetic ligands that will either suppress the constitutive activity of the receptors or further activate the receptors. Our work leading to the identification of SR1078 also demonstrates that it is possible to develop ROR selective synthetic ligands that lack activity at related receptors such as LXR and FXR. This degree of promiscuity that is displayed by T1317 limits the ability to utilize this particular compound as a chemical tool to probe the function of the RORs. Additionally, we show that SR1078 displays pharmacokinetic properties that allow it to be used in vivo and as would be expected for a RORα/γ agonist, administration of this compound to mice results in an increase in expression of ROR target genes in the liver. This proof-of-principle study demonstrates that additional experiments are warranted to examine the pharmacological profile of this compound in vivo.