Campaign to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR)

The Scripps Research Institute Molecular Screening Center (SRIMSC) implemented a Center-Based Component (CBC) that focuses on the use of a genomic screening platform to support selectivity profiling of high value compounds that emerge from the Molecular Libraries Probe Centers Network (MLPCN) program. One area of focus for the CBC was development of a nuclear receptor (NR) library containing expression vectors for all 48 human NRs in a GAL4 format. In an effort to validate the NR library (PubChem AID-2277), we screened it against a small chemical library containing mostly well characterized NR modulators. Analysis of the validation screen results revealed that the LXR agonist T0901317 (SID-85257301) was capable of repressing the activity of both GAL4-RORα and GAL4-RORγ, but not that of GAL4-RORβ. The activity of T0901317 was confirmed on wildtype receptor on native RORα/γ promoters and direct binding to these receptors was demonstrated. Compounds derived from these initial candidates were purchased as powders or synthesized at the SRIMSC and were tested for their ability to inhibit RORα and RORγ in luciferase-based reporter assays performed at a single concentration of 10 μM or in dose response assays starting at a nominal concentration of 20 μM. Compounds were subsequently counterscreened at a single concentration and/or dose response assays against the liver X receptor (LXR), the farnesoid X receptor (FXR), and glucose-6-phosphatase to determine selectivity. Finally, compounds of interest were tested at a single concentration of 10 μM against VP16 to determine whether they were non-selective or cytotoxic. The above Center-based probe development efforts resulted in the identification of two probes: The first probe, the benzenesulfonamide compound T0901317, ML125 previously identified as a selective agonist of LXR was identified here as a novel RORα/γ inverse agonist probe that decreases the transcriptional activity of both ROR receptors (IC50 values = 2.0 and 1.73 micromolar, respectively). The second probe, ML124 synthesized at the SRIMSC, was found to decrease RORα transcriptional activity (IC50 value = 2.47 micromolar). ML124 represents an improvement over the prior art due to its lack of activity for LXR. ML124 does not have activity against RORγ (IC50 > 20 micromolar). These two probes are useful tools for examining ROR biology.