Several nuclear receptors (NRs) are still characterized as orphan receptors because endogenous ligands have not yet been identified for these proteins. Evidence is growing suggesting the retinoic acid receptor-related orphan receptors (RORs) bind to, and are modulated by oxysterols. Recently, we discovered that the synthetic LXRα agonist T0901317 (ML125) was a potent inverse agonist of RORα and RORγ. Structure activity relationship (SAR) studies quickly revealed a strategy to remove the LXRα activity from the ML125 chemical scaffold which led to ML124. ML124 represented the first synthetic RORα/RORγ dual inverse agonist devoid of LXRα activity. While there appear to be clear non-overlapping roles for RORα and RORγ, chemical probes that are isoform selective are needed to dissect this biology. We described the identification of a selective RORα synthetic ligand, ML176, which directly binds to RORα, but not other RORs, and functions as a selective inverse agonist of RORα in cell-based assays. Here we describe the identification of a selective RORγ synthetic ligand, ML310, which functions as an inverse agonist. We show that ML310 can displace T1317 in a binding assay and does interact with RORγ protein to stabilize the protein in hydrogen-deuterium exchange (HDX)-based experiments. In cotransfection assays, ML310 suppresses transcription activity in both GAL4-RORγ ligand binding domain (LBD) and full-length RORγ contexts. Furthermore, treatment of EL-4 cells with ML310 results in suppression of gene expression and production of IL-17. These data strongly suggest that ML310 is a potent and efficacious RORγ modulator and represses its activity. Thus, we have identified the first synthetic RORγ selective 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 RORγ inverse agonists may hold utility for the treatment of autoimmune disorders.
Assigned Assay Grant #: U54 MH084512
Screening Center Name & PI: Scripps Research Institute Molecular Screening Center (SRIMSC), Hugh Rosen
Chemistry Center Name & PI: SRIMSC, Hugh Rosen
Assay Submitter & Institution: Patrick Griffin, The Scripps Research Institute (TSRI)
Summary Bioassay Identifier (AID): 2139
1. Recommendations for Scientific Use of the Probe
The role of RORγ activity for optimal TH17 cell development has been well-characterized (1). Current treatments for TH17-mediated autoimmune diseases, including multiple sclerosis, utilize agents that are general immunosuppressants and thus the side effect profile is significant. We have been able to demonstrate that treatment of fully differentiated TH17 cells with the selective RORα probe ML176 or a close analog SR1001 (dual RORα/γ inverse agonist), inhibits the expression of IL-17a, IL-17f, IL-21, IL-22, IL-23r, RORα, and RORγ, indicating that dual RORα/γ inverse agonists can effectively suppress the expression of TH17 derived cytokines. Treatment of differentiated (TH17) splenocytes with these compounds inhibits IL-17 secretion. Finally, SR1001 treatment in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), a well characterized model of TH17 cell-mediated autoimmune disease, delayed the onset of EAE as well as reduced the severity of disease progression (2). Since RORγ is the dominant receptor driving TH17 cell differentiation, the generation of a ligand specific to RORγ will allow the targeting of TH17 cells while avoiding any metabolic effects that suppression of RORα may have. ML310 can displace the T1317 in a binding assay and interacts with RORγ protein to stabilize the protein in HDX-based experiments (3). In cotransfection assays, ML310 suppresses transcription activity in both GAL4-RORγ LBD and full-length RORγ contexts (3). Furthermore, treatment of EL-4 cells with ML310 results in suppression of gene expression and production of IL-17 (3). These data strongly suggest that ML310 is a potent and efficacious RORγ modulator and represses its activity. Moreover, ML310 has the potential utility for the treatment of autoimmune disorders, and further experiments are underway to evaluate in vivo actions of ML310.
What will the probe be used for?
RORγ is implicated in autoimmune disorders so a RORγ selective probe can be used to determine what role if any RORγ plays in TH17 cell proliferation. Hence, a RORγ selective probe may be useful for the treatment of disorders such as multiple sclerosis, rheumatoid arthritis, Crohn’s disease, and lupus where RORγ function has been dysregulated.
Who in the research community will use the probe?
These probes will be useful to researchers interested in ROR biology, such as TH17 cell development, inflammation, and autoimmune disorders (see below for more discussion of ROR biology).
What is the relevant biology to which the probe can be applied?
Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. The RORs are modulated by oxysterols and although several endogenous ligands have been described, there is still controversy as to the identity of a bona fide endogenous ligand for this NR subfamily. Because the RORα is a constitutive activator of transcription in the absence of ligands, it has been suggested that the coactivator binding surface, or activation function 2 (AF2), is locked in the holo-conformation (4), circumventing the need for ligand interaction to transactivate target genes. However, the co-crystal structures of RORα LBD bound to cholesterol and cholesterol sulfate have been solved, suggesting that like the LXRs, RORα can bind and may respond to sterols and synthetic ligands (5, 6). As a result, the RORα have emerged as attractive drug target for the treatment of metabolic disorders. Recently the co-crystal structure of RORγ in complex with 25-hydroxy cholesterol has been published and in this study it was suggested that the receptor is not constitutively active in the absence of sterols. However, this study does support the notion that this receptor can be modulated by small molecules and thus RORγ is an attractive drug target for treatment of inflammatory and autoimmune disorders.
A recent report described the first synthetic modulator of RORs (7); this compound, ML176, was able to repress the expression of G6Pase and chromatin immunoprecipitation (ChIP) analysis demonstrated that this modulator diminished the presence of SRC2 at the G6Pase promoter. A recent report on endogenous inverse agonists showed repression of G6Pase mRNA in HepG2 cells and similar effects on displacing SRC2 at the G6Pase promoter RORs (8). More importantly, in this report, we demonstrated that endogenous pan ROR inverse agonists can regulate hepatocyte glucose output by repressing PEPCK and G6Pase in mouse primary hepatocytes. Glucose output was measured directly and treatment of these primary cells with a pan ROR inverse agonist reduced glucose by 24 percent. As RORs binds to the same RORE, and they can compensate for each other on certain genes, a potent and selective RORα inverse agonist will be also be beneficial (but outside the scope of this proposal) to study the overlap of RORα and RORγ not only on glucose production in the liver, but also in spleen where they regulate differentiation of TH17 cells in spleen.
The role of RORγ activity for optimal TH17 cell development has been well-characterized (1). Current treatments for TH17-mediated autoimmune diseases, including multiple sclerosis, utilize agents that are general immunosuppressants and thus the side effect profile is significant. We have been able to demonstrate that treatment of fully differentiated TH17 cells with ML176 or a close analog SR1001 (dual RORα/γ inverse agonist), inhibits the expression of IL-17a, IL-17f, IL-21, IL-22, IL-23r, RORα, and RORγ, indicating that dual RORα/γ inverse agonists can effectively suppress the expression of TH17 derived cytokines. Treatment of differentiated (TH17) splenocytes with these compounds inhibits IL-17 secretion. Finally, SR1001 treatment in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), a well characterized model of TH17 cell-mediated autoimmune disease, delayed the onset of EAE as well as reduced the severity of disease progression (2). Since RORγ is the dominant receptor driving TH17 cell differentiation, the generation of a ligand specific to RORγ would allow us to effectively target TH17 cells while avoiding the metabolic effects that suppressing RORα may have. Recently, RORγ selective inverse agonists have been generated (i.e. SR3-1679 and SR3-1898). In vitro analysis using these RORγ specific inverse agonists indicates that, similar to SR1001, they can suppress TH17 cell development as indicated by the inhibition of the mRNA expression of IL-17a, IL-17f, IL-21, and IL-22 and IL-17 cytokine protein expression.
2. Materials and Methods
Following a successful Center-based initiative, which resulted in the identification of an RORα inverse agonist probe (CID 44237404/SID 44237404/ML124), the SRIMSC and Dr. Griffin’s laboratory implemented further studies to develop RORγ selective modulators. Compounds derived from these initial candidates were purchased as powders or synthesized at the SRIMSC and were tested for their ability to inhibit RORγ in luciferase-based reporter assays performed in dose response assays starting at a nominal concentration of 10 micromolar. Compounds were subsequently counterscreened in dose response assays against RORα, the liver X receptor (LXR), and the farnesoid X receptor (FXR) to determine selectivity. Finally, compounds of interest were tested at a single concentration of 10 micromolar against VP16 to determine whether they were non-selective or cytotoxic, and probe candidates were tested in a reporter assay using full length RORγ and a multimerized RORE reporter gene to determine biologic efficacy with the native receptor. The specific assays are summarized in Tables 1A, 1B, and 1C and are described in further detail below.
Table 1A
ML124 & ML125 PubChem Assays (RORα Selective IAG & Dual RORα/γ IAG Probes).
Table 1B
ML176 PubChem Assays (RORα Selective IAG Probe).
Table 1C
ML310 PubChem Assays (Selective RORγ IAG Probe).
2.1. Assays
Abbreviated List of Reagents for ML310 Assays
- HEK293T cells (supplied by Dr. Griffin)
- Gal4-based plasmids for transfection (supplied by Dr. Griffin)
- 384 well plates (PerkinElmer, part 6007688)
- Britelite Plus (PerkinElmer, part 6016767)
- DMEM growth media (Mediatech Inc, Part 10 013 CV)
- Fugene 6 Transfection Reagent (Roche Applied Science, part 11814443001).
Protocols used for the ML310 probe discovery project are below.
AID 624279
Late stage results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor: luminescent-based assay to identify RORγ inhibitors
The purpose of this assay is to determine whether synthesized probe candidate test compounds can inhibit the activity of RORγ. In this assay, HEK293T cellsco-transfected with a GAL4DBD-RORγLBD construct (GAL4-RORγ) and a GAL4UAS-luciferase reporter construct, are incubated for 20 hours with test compounds. As designed, compounds that inhibit RORγ activity will prevent activation of the GAL4-RORγ construct, thereby preventing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to a decrease in well luminescence. Compounds are tested at a nominal concentration of 10 micromolar. Six replicates were performed for each assay.
Protocol Summary
Luciferase reporter assays were conducted using a pBind GAL4DBD-RORγLBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4×106 cells in 10 cm plates, 9 micrograms of total DNA, and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and re-plated in 384-well plates at a density of 10,000 cells/well. Following 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO-treated cells. The fold-change inhibition for each compound was calculated as follows:
Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO). The average fold-change of each compound tested was calculated. Any compound that exhibited a fold-change inhibition less than the hit cutoff calculated (<0.7-fold inhibition) was declared active.
AID 624279
Late stage counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORγ): luminescence-based cell-based assay to identify ROR alpha (RORα) inhibitors
The purpose of this assay is to determine whether a powder sample of an RORγ inverse agonist probe candidate is nonselective due to inhibition of RORα. This dose response assay employs the RORα-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-RORαLBD construct (GAL4-RORα) and a GAL4UAS-luciferase reporter construct are incubated for 18–24 hours with test compounds. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated RORα nuclear receptors. As designed, compounds that inhibit RORα activity will prevent activation of the GAL4-RORα construct, thereby preventing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter leading to a decrease in well luminescence. Compounds were tested in eight replicates using a 10-point dilution series starting at a nominal concentration of 30 micromolar.
Protocol Summary
Luciferase reporter assays were conducted using a pBind GAL4DBD-RORαLBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4×106 cells in 10 cm plates, 9 μg of total DNA, and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells were counted and re-plated in 384-well plates at a density of 10,000 cells/well. Following 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells.
For each test compound, fold inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using GraphPad Prism. The reported IC50 values were calculated from GraphPad Prism software. Compounds with an IC50 greater than 10 μM were considered inactive. Compounds with an IC50 equal to or less than 10 μM were considered active. Any compound with a percent inhibition >30% at all test concentrations was assigned an activity score of zero. Any compound with a percent inhibition value <30% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
AID 624279
Late stage counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORγ): luminescence-based cell-based assay to identify activators of the liver X receptor (LXR)
The purpose of this assay is to determine whether a powder sample of an RORγ inverse agonist probe candidate is nonselective due to activation of LXR. This dose response assay employs the LXR-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-LXRLBD construct (GAL4-LXR) and a GAL4UAS-luciferase reporter construct are incubated for 18–24 hours with test compounds. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated LXR nuclear receptors. As designed, compounds that activate LXR activity will activate the GAL4-LXR construct, thereby increasing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to an increase in well luminescence. Compounds were tested in a 10-point dilution series starting at a nominal concentration of 10 micromolar.
Protocol Summary
Luciferase reporter assays were conducted using a pBind GAL4DBD-LXRLBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4×106 cells in 10 cm plates, 9 μg of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and re-plated in 384-well plates at a density of 10,000 cells/well. Following 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells. For each test compound, percent activation was plotted against compound concentration. A four-parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using GraphPad Prism. The reported IC50 values were calculated from GraphPad Prism software. Compounds with an IC50 greater than 10 μM were considered inactive. Compounds with an IC50 equal to or less than 10 μM were considered active. Any compound with a percent activity value <200% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >200% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
AID 624279
Late stage counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORγ): luminescence-based cell-based assay to identify activators of the farnesoid X receptor (FXR)
The purpose of this assay is to determine whether powder samples of RORγ inverse agonist probe candidates are nonselective due to activation of FXR. This assay employs the FXR-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-FXRLBD construct (GAL4-FXR) and a GAL4UAS-luciferase reporter construct are incubated for 18–24 hours with test compounds. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated FXR nuclear receptors. As designed, compounds that activate FXR activity will activate the GAL4-FXR construct, thereby increasing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to an increase in well luminescence. Compounds were tested in at a nominal concentration of 10 micromolar. Six replicates were performed for each assay.
Protocol Summary
Luciferase reporter assays were conducted using a pBind GAL4DBD-FXRLBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4×106 cells in 10 cm plates, 9 μg of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and re-plated in 384-well plates at a density of 10,000 cells/well. Following 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells. The fold-change inhibition for each compound was calculated as follows:
Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).
The average fold-change of each compound tested was calculated. Any compound that exhibited a 2 fold-change activation greater than the hit cutoff calculated (< 2-fold activation) was declared active. Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.
AID 624279
Late stage counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORγ): luminescence-based cell-based assay to identify inhibitors of the human herpes virus VP16 transcriptional activator protein (VP16)
The purpose of this assay is to determine whether powder samples of probe candidates are nonselective due to inhibition of VP16. In this counterscreen assay the nuclear receptor plasmid was replaced by the GAL4DBD-VP16LBD plasmid, which expresses the strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) fused to the GAL4 DBD. Cells are co-transfected with the 5xGAL4 response element (UAS) luciferase reporter to monitor GAL4DBD-VP16LBD activity, followed by incubation with test compounds for 18–24 hours. As designed, compounds that inhibit VP16 activity will decrease pGAL4DBD-VP16LBD activity, leading to reduced activation of the pG5-luc and decreased well luminescence. These compounds are likely to be nonselective inhibitors or cytotoxic. Compounds were tested in at a nominal concentration of 10 micromolar. Six replicates were performed for each assay.
Protocol Summary
Luciferase reporter assays were conducted using a pBind GAL4DBD-VP16LBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4×106 cells in 10 cm plates, 9 μg of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells were counted and re-plated in 384-well plates at a density of 10,000 cells/well. Following a 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells.
The fold-change inhibition for each compound was calculated as follows:
Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).
The average fold-change of each compound tested was calculated. Any compound that exhibited a fold-change inhibition less than the hit cutoff calculated (> 1.5-fold inhibition) was declared active. Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.
2.2. Probe Chemical Characterization
The structure of the probe ML310 (SR-2211) in shown in Figure 1.
Figure 1
Probe chemical structure.
Structure verification with 1H NMR, 13CNMR, and LCMS results. Probe SR-2211 was obtained as a light yellow solid with >98% purity (HPLC analysis): 1H-NMR (as TFA salt) (400 MHz, D2O 4.75) δ 8.71 (d, J = 6.8 Hz, 2H), 8.06 (d, J = 6.8 Hz, 2H), 7.60–7.51 (m, 7H), 4.41 (s, 2H), 4.03 (s, 2H), 3.40 (b, 4H), 2.92 (b, 4H). 13C-NMR (400 MHz, D2O 4.75) δ (ppm) 163.1, 162.7, 141.0, 136.3, 132.1, 131.5, 131.0, 129.7, 128.1, 127.1, 117.8, 114.8, 60.2, 59.2, 50.9, 49.2; ESI-MS (m/z): 528 [M+1]+.
Solubility in PBS. The solubility of compounds is tested in triplicate in phosphate buffered saline (PBS), pH 7.4. Per well, 198 μl of PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well filter plate, pore size: 0.45um (MSRLN0450). Test compounds are introduced from 10 mM DMSO stock solutions (2 μl). The final concentration of DMSO was 1 percent. Samples are allowed to incubate at 22ºC for 18 hours. In the morning the plate is centrifuged where the soluble portion passes through the filter and is collected in a capture plate. Clotrimazole is included as a control to assure the assay is working properly. The samples are analyzed by HPLC. Peak area is compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS-MS analysis is used.
Solubility in Media. The solubility of compounds are tested in triplicate in complete media (MDEM + 10% FBS). Per well, 198 μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well filter plate: pore size: 0.45um (MSRLN0450). Test compounds are introduced from 10 mM DMSO stock solutions (2 μl). The final concentration of DMSO was 1 percent. Samples are allowed to incubate at 22ºC for 18 hours. In the morning the plate is centrifuged where the soluble portion passes through the filter and is collected in a capture plate. The samples are analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area is compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS-MS analysis is used.
Stability in PBS. Demonstration of stability in PBS was conducted by addition of 10 μM compound from a DMSO stock to PBS in HPLC autosampler vials. Samples are held in the HPLC autosampler at ambient temperature. At approximately 0, 1, 2, 4, 8, 24, and 48 hours the samples are injected on the HPLC. Peak area and retention time are compared between injections. Data is log transformed and represented as half life (Figure 2). DMSO is added as a co-solvent as needed for solubility.
Figure 2
Stability of ML310.
Determination of Glutathione Reactivity. Probe ML310 (10 μM) was incubated at 37°C for 6 hours in the presence of 50 μM freshly prepared reduced glutathione. At 0 and 6 hours the samples were injected on the HPLC. Peak area and retention time were compared between injections. Samples were evaluated for a glutathione dependent decrease in compound concentration. DMSO was added as a co-solvent as needed for solubility.
Table 2
A summary of probe solubility, stability and GSH reactivity results.
2.3. Probe Preparation
Detailed experimental procedures for the synthesis of Probe ML310 (SR-2211)
Step 1. 2-(4-Amino-3-fluorophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol
To 2-fluoroaniline in a pressure tube was added hexafluoroacetone sesquihydrate (1.1 eq) neat and p-toluenesulfonic acid (0.1 eq). The tube was then purged with argon, sealed and heated on an oil bath overnight (12 h) at 90° C. The reaction contents were then diluted with ethyl acetate and washed with NaHCO3 (sat.). The ethyl acetate phase was then washed with brine, dried with Na2SO4, and concentrated to a solid residue. The desired product was then isolated by silica gel using hexanes/ethyl acetate and following recrystallization from 10:1 hexanes/ethyl acetate to afford 2-(4-Amino-3-fluorophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol as white prisms. ESI-MS (m/z): 278 [M+1]+.
Step 2. 1,1,1,3,3,3-Hexafluoro-2-(3-fluoro-4-iodophenyl)propan-2-ol
To a solution of 2-(4-Amino-3-fluorophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol in DMF was added a solution of sodium nitrite (1.2 eq) in water and a 6M solution of hydrochloric acid (3 eq), while maintaining the reaction temperature at 0~4 °C. The reaction mixture was stirred for 30 minutes, and then potassium iodide (1 eq) was added portionwise at 0~4 °C. The resulting reaction mixture was stirred overnight at rt. The reaction mixture was extracted with EtOAc, and the combined organic layers were washed with saturated sodium thiosulfate and dried over Na2SO4. The solvent was evaporated in vacuo to obtain the crude, which was purified by flash chromatography on silica gel (~5%EtOAc/Hex) to obtain the title compound.
Step 3. 1-(4-Pyridinyl-methyl)-piperazine-4-benzyl-para-boronic pinacol ester
To 4-bromomethylphenylboronic acid pinacol ester was added dry DMF, followed by addition of K2CO3 (3.0 eq), (1.1 eq) of 1-(pyridinyl-4-methyl)-piperazine and (2 % weight) NaI. The mixture was allowed to stir overnight at rt (~23° C) under argon balloon. The remaining reaction mixture in DMF was then diluted with CHCl3 and washed with H2O maintained at pH ≠ 10 and dried over Na2SO4. The washed organic phase was then concentrated to a solid residue and extracted with hexanes (3 × 100 mL) and again concentrated. The product 1-(4-pyridinyl-methyl)-piperazine-4-benzyl-para-boronic pinacol ester was isolated by recrystallization from 12:1 hexanes/CH2Cl2 and used without further purification within the subsequent synthetic step.
Step 4. 1,1,1,3,3,3-hexafluoro-2-(2-fluoro-4′-((4-(pyridin-4-ylmethyl)piperazin-1-yl)methyl)-[1,1′-biphenyl]-4-yl)propan-2-ol
The mixture of 1,1,1,3,3,3-Hexafluoro-2-(3-fluoro-4-iodophenyl)propan-2-ol, 1-(4-Pyridinyl-methyl)-piperazine-4-benzyl-para-boronic pinacol ester (1.2 eq), P(PPh3)4 (5 mol%), K2CO3 (3 eq) and dioxane/H2O (4:1) was degassed for 5 minutes and heated for 2h at 80°C oil bath. The completion of reaction was monitored by anal. HPLC. The mixture was cooled and extracted with EtOAc. The combine organic layers were washed with saturated NaHCO3 and dried over Na2SO4. The solvent was removed in vacuo to obtain the crude, which was purified by flash chromatography on silica gel to obtain the title compound. ESI-MS (m/z): 528 [M+1]+.
3. Results
3.1. Dose Response Curves for Probe
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Figure 3Dose response curves for SR2211 (CID 51035449/ML310) RORγ modulator probe
293T cells were cotransfected with Gal4-RORα (a), Gal4-RORγ (b), Gal4-LXRa (c), Gal4-FXR (d), or Gal4-VP16 (e) along with a UAS-luciferase plasmid. The cells were treated for 20 hours with the indicated concentration of ML310 or positive controls SR3335 (a), T1317 (c), and GW4064 (d). Relative change was determined by normalizing to cells treated with vehicle. Each data point was performed in 6 replicates and represented as mean ± SEM, n = 6.
3.2. Cellular Activity
Results from the Griffin lab demonstrate that the current probe (CID 51035449; ML310) effectively inhibits the endogenous expression of RORγ target genes (proinflammatory IL-17 and IL-23R). In order to determine whether ML310 can inhibit endogenous IL-17 gene expression, we used an EL-4 murine T lymphocyte cell line which produces IL-17 in response to phorbol myristate acetate (PMA) and ionomycin treatment. The results demonstrate that pretreatment of EL-4 cells with 5 μM concentrations of either ML310 or digoxin as control followed by stimulation with PMA/ionomycin leads to a significant reduction in the IL-17 gene expression as measured by quantitative real-time PCR (Figure 4a). Treatment of EL4 with ML310 repressed the IL-17 gene expression to a greater extent as compared to digoxin. Similarly, the expression of IL-23 receptor (IL-23R) was significantly inhibited by ML310 and digoxin (Figure 4b), as has been previously reported by Fujita-Sato et al. (25). In order to measure the effect of ML310 on IL-17 production, we determined the intracellular levels of IL-17 using flow cytometry. After the stimulation of EL-4 cells with PMA/ionomycin for 3 hours, the cells were treated with BD GolgiPlug (protein transport inhibitor) to allow intracellular accumulation of cytokines. After 2 hours, the cells were fixed and stained to analyze the IL-17 by flow cytometry. As shown in Figure 4c, treatment of EL-4 cells with ML310 as well as a control digoxin resulted in significant inhibition of IL-17 intracellular staining as compared to vehicle-treated cells. These results demonstrate that ML310 can inhibit the transcriptional activity of RORγ, resulting in the suppression of IL-17 production.
To confirm that ML310 can repress the RORγ transcriptional activity, we used a full length receptor along with a multimerized ROR response element (RORE, five repeats of ROR) driving luciferase gene expression. In the absence of RORγ, there was no change in the luciferase activity of 5X-RORE with the treatment of SR2211 (Figure 5a). ML310 significantly repressed 5X-RORE luciferase activity when full length RORγ was added during transfection (Figure 5b); however, there was no effect of ML310 on RORα cotransfection with 5X-RORE (data not shown). To further examine the activity of ML310 in a more native promoter based assay, we performed additional cotransfection assays where we transfected cells with full-length RORα or RORγ and a luciferase reporter gene driven by a native promoter derived from a known ROR target gene, IL-17. IL-17 is a well-characterized ROR target gene that plays a critical role in the inflammatory pathway (10). As shown in (Figure 5c), in a RORα cotransfection assay, treatment of cells with ML310 did not alter the transcription driven by the IL-17 promoter. We observed a significant, >50% suppression of transcriptional activity of IL-17 promoter in a RORγ-dependent manner (Figure 5d). As previously mentioned, there was no increase in the full length LXRα target gene, ABCA1, promoter activity (Figure 5e). These results confirm that we have been able to selectively target RORγ.
3.3. Profiling Assays
As this new probe resulted from medicinal chemistry/SAR efforts around the previous dual RORα/γ probe ML125 (a ligand for LXR, FXR and RORs), we profiled the current probe against these nuclear receptors. The results demonstrate that the success in eliminating the undesirable effects of RORα, LXR and FXR receptors (no activity on these receptors), while also achieving selectivity and improved potency towards the target receptor, RORγ.
4. Discussion
4.1. Comparison to Existing Art and How the New Probe is an Improvement
As summarized in Table 3, the current probe is a potent and selective RORγ inverse agonist whereas the prior probes were either RORα selective or dual RORα/ROR γ inverse agonists.
Table 3
Comparison of Current and Prior ROR Probes.
Other existing art: The natural products digoxin (CID 2724385) and ursolic acid (CID 64945) have been described as RORγ modulators and are capable of inhibiting TH17 cell differentiation. Their utility as candidates for further development is limited, as digoxin displays significant adverse drug reactions with a narrow therapeutic index and ursolic acid activates the glucocorticoid receptor. As a result, we reasoned that development of synthetic selective RORγ modulators would allow us to evaluate their potential as drug development candidates for treatment of autoimmune disease.
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Publication Details
Author Information and Affiliations
Authors
Naresh Kumar,1 Theodore Kamenecka,1 Brent Lyda,1 Pasha Khan,1 Mi Ra Chang,1 Ruben D. Garcia-Ordonez,1 Michael Cameron,1 Jill Ferguson,3 Becky A. Mercer,2 Peter Hodder,1,2 Hugh Rosen,3 and Patrick R. Griffin1,4.Affiliations
Publication History
Received: April 16, 2012; Last Update: March 14, 2013.
Copyright
Publisher
National Center for Biotechnology Information (US), Bethesda (MD)
NLM Citation
Kumar N, Kamenecka T, Lyda B, et al. Identification of a novel selective inverse agonist probe and analogs for the Retinoic acid receptor-related Orphan Receptor Gamma (RORγ) 2012 Apr 16 [Updated 2013 Mar 14]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.
