Retinoic acid receptor-related orphan receptor γ (RORγ) [1, 2].
Ki: 105 nM (for RORγ) [1].

IL-17 gene expression was reduced by SR2211 treatment of EL4. In a similar vein, SR2211 markedly reduced the expression of the IL-23 receptor (Il23r). When EL-4 cells were treated with SR2211, IL-17 intracellular staining was dramatically reduced as compared to cells that were treated with vehicle [1].

---

In a radioligand binding assay, SR2211 displaced [³H]-T0901317 from the GST-RORγ-LBD with a calculated Kᵢ value of 105 nM [1].
In a cell-based Gal4 co-transfection assay, SR2211 inhibited the transcriptional activity of Gal4-RORγ LBD with an IC₅₀ of approximately 320 nM. No significant effect was observed on the transcriptional activity of Gal4-RORα, Gal4-LXRα, Gal4-FXR, or the Gal4-VP16 control at 10 μM [1].
In a full-length RORγ reporter assay using a 5X-RORE luciferase construct, SR2211 significantly repressed luciferase activity in a RORγ-dependent manner [1].
In a native promoter assay using an IL-17 luciferase reporter, SR2211 suppressed RORγ-dependent transcriptional activity driven by the IL-17 promoter by more than 50% [1].
In EL-4 murine T lymphocytes, pre-treatment with 5 μM SR2211 followed by stimulation with PMA/ionomycin led to a significant reduction in endogenous IL-17A and IL-23R mRNA expression as measured by quantitative real-time PCR. The repression of IL-17A gene expression by SR2211 was greater than that of digoxin [1].
In EL-4 cells, intracellular cytokine staining by flow cytometry showed that treatment with 5 μM SR2211 significantly inhibited the production of IL-17 protein following PMA/ionomycin stimulation [1].
In a luciferase reporter gene assay (using HEK293 cells transfected with RORγ), SR2211 showed inhibitory activity with an IC₅₀ of 0.11 ± 0.03 μM [2].
In a thermal shift assay (TSA), SR2211 stabilized the RORγ-LBD protein, resulting in a ΔTm of 6.50 °C [2].
In an AlphaScreen assay measuring the disruption of the RORγ-LBD and SRC1 co-activator peptide interaction, SR2211 showed an IC₅₀ of 0.91 ± 0.07 μM [2].

Radioligand Binding Assay (SPA): The assay was performed in a Scintillation Proximity Assay (SPA) format. The reaction mixture contained 0.25 mg of glutathione YSI beads, 1 μg of GST-RORγ-LBD, 5 nM of [³H]-T0901317 as the radioligand, and varying concentrations of SR2211 in assay buffer (50 mM HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol, 1 mM DTT, and a protease inhibitor cocktail). All components were gently mixed and incubated for 20 hours before being read on a TopCount instrument. The binding data were analyzed to calculate the Kᵢ value [1].
Hydrogen/Deuterium Exchange (HDX) Mass Spectrometry: Solution-phase amide HDX was performed using an automated system. 4 μL of a 10 μM RORγ-LBD protein solution was diluted with D₂O-containing HDX buffer and incubated at 25°C for various time points (10 s, 30 s, 60 s, 900 s, 3600 s). Following on-exchange, the protein was denatured by dilution with 0.1% TFA in 3 M urea at 1°C. Samples were passed across an immobilized pepsin column, and the resulting peptides were trapped on a C8 trap cartridge. Peptides were then gradient-eluted across a C18 HPLC column and electrosprayed directly into an Orbitrap mass spectrometer. Data were processed with in-house software to calculate the difference in deuterium uptake between the apo and ligand-bound (SR2211) RORγ-LBD [1].
Thermal Shift Assay (TSA): The thermal stability shift assay was used to detect ligand binding. All reactions were performed in buffer containing 10 mM HEPES (pH 7.5), 150 mM NaCl, and 5% (v/v) glycerol, with a final concentration of 10 μM RORγ-LBD protein and 200 μM of the test compound. The 10 μL reaction mix was added to a 96-well PCR plate. A fluorescence probe was added at a dilution of 1:1000, and the mixture was incubated with compounds on ice for 30 minutes. The plate was heated from 30°C to 80°C at a rate of 0.5°C per minute, and fluorescence readings were recorded at 0.5°C intervals using a real-time PCR system [2].
AlphaScreen Assay: This assay was used to assess the ability of a compound to disrupt the interaction between RORγ-LBD and the SRC1 co-activator peptide. All reactions contained 200 nM RORγ-LBD bound to nickel acceptor beads (5 μg/mL) and 50 nM biotinylated SRC1-4 peptide bound to streptavidin donor beads (5 μg/mL) in the presence of various concentrations of the test compound. The compound concentrations varied from 150 nM to 200 μM. The AlphaScreen assay buffer contained 50 mM MOPS, 50 mM NaF, 0.05 mM CHAPS, and 0.1 mg/mL bovine serum albumin at pH 7.4. The N-terminal biotinylated SRC1-4 peptide sequence was QKPTSPGQTPQAQQKSLIQQLLTE [2].

Gal4 Co-transfection Assay: HEK293 cells were maintained in DMEM with 10% fetal bovine serum. Bulk reverse transfections were performed using 1 × 10⁶ cells in 6 cm plates with 3 μg of total DNA (receptor and reporter in a 1:1 ratio) and a transfection reagent in a 1:3 DNA:lipid ratio. The following day, cells were re-plated in 384-well plates at 10,000 cells/well. After 4 hours, cells were treated with SR2211 or DMSO. Following a 20-hour incubation, luciferase levels were assayed by one-step addition of a detection reagent and read using a plate reader. Data were normalized as fold change over DMSO-treated cells [1].
Full-length RORγ Reporter Assay (5X-RORE): Similar co-transfection assays were performed using a luciferase reporter gene driven by five repeats of a ROR response element (5X-RORE) and either an empty vector or full-length RORγ [1].
Native Promoter Assay (IL-17-Luc): Co-transfection assays were performed using an IL-17 luciferase reporter gene and either full-length RORα or RORγ to assess the effect of SR2211 on a native ROR target promoter [1].
Real-time PCR Analysis in EL-4 Cells: One million EL-4 cells were seeded in 6-well plates and incubated with 5 μM of SR2211, digoxin, or DMSO for 20 hours. Cells were then stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 5 hours. RNA was extracted, and cDNA was synthesized. IL-17A and IL-23R gene expression was quantified by real-time PCR and normalized to the expression of GAPDH [1].
Intracellular Cytokine Staining by Flow Cytometry: EL-4 cells were stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 5 hours. After 3 hours of stimulation, a protein transport inhibitor was added for an additional 2 hours. Cells were then fixed, permeabilized, and stained with an antibody against IL-17A. Cell sorting and analysis were performed using a flow cytometer [1].
Luciferase Reporter Gene Assay (for RORγ inhibition): This assay was performed to test the activity of compounds on RORγ transcription. HEK293T cells were co-transfected with RORγ and a luciferase reporter gene. The cells were then treated with the test compound for 20 hours, and the luciferase activity was measured. For SR2211, an IC₅₀ of 0.11 ± 0.03 μM was reported [2].

[1]. Identification of SR2211: a potent synthetic RORγ-selective modulator. ACS Chem Biol. 2012 Apr 20;7(4):672-7.

[2]. Identification of N-phenyl-2-(N-phenylphenylsulfonamido)acetamides as new RORγ inverse agonists: Virtual screening, structure-based optimization, and biological evaluation. Eur J Med Chem. 2016 Jun 30;116:13-26.

SR2211 is a synthetic, selective RORγ inverse agonist/modulator that potently inhibits the production of IL-17 in cells [1].
The structure of SR2211 is 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. It was derived from SR1001 after several rounds of structure-activity relationship (SAR) studies, retaining the hexafluorophenyl group while modifying the left-hand portion to replace the sulfonamide residue with more lipophilic groups to potentially reduce CNS penetration [1].
SR2211 is used as a positive control in biochemical assays for RORγ inverse agonists [2].

C26H24F7N3O

527.49

527.18

C, 59.20; H, 4.59; F, 25.21; N, 7.97; O, 3.03

1359164-11-6

51035449

Off-white to light brown solid powder

1.4±0.1 g/cm3

552.4±50.0 °C at 760 mmHg

287.9±30.1 °C

0.0±1.6 mmHg at 25°C

1.545

4.79

1

11

6

37

698

0

FC1=C(C2=CC=C(CN3CCN(CC4=CC=NC=C4)CC3)C=C2)C=CC(C(C(F)(F)F)(O)C(F)(F)F)=C1

KVHKWAZUPPBMLL-UHFFFAOYSA-N

InChI=1S/C26H24F7N3O/c27-23-15-21(24(37,25(28,29)30)26(31,32)33)5-6-22(23)20-3-1-18(2-4-20)16-35-11-13-36(14-12-35)17-19-7-9-34-10-8-19/h1-10,15,37H,11-14,16-17H2

1,1,1,3,3,3-hexafluoro-2-[3-fluoro-4-[4-[[4-(pyridin-4-ylmethyl)piperazin-1-yl]methyl]phenyl]phenyl]propan-2-ol

SR2211; SR 2211; SR2211; 1,1,1,3,3,3-hexafluoro-2-[3-fluoro-4-[4-[[4-(pyridin-4-ylmethyl)piperazin-1-yl]methyl]phenyl]phenyl]propan-2-ol; 1,1,1,3,3,3-hexafluoro-2-(3-fluoro-4-(4-((4-(pyridin-4-ylmethyl)piperazin-1-yl)methyl)phenyl)phenyl)propan-2-ol; RefChem:185198; 1359164-11-6; SR-2211

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~189.58 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)