Retinoic acid receptor-related orphan receptor γ (RORγ) [1].
IC₅₀: ~1.5 μM (for RORγ in a GAL4 co-transfection assay); ~1 μM (for RORγ in a competitive radioligand binding assay) [1].

In a GAL4-NR chimeric co-transfection assay, SR1555 repressed the transcriptional activity of GAL4-RORγ in a dose-dependent manner with an IC₅₀ of approximately 1.5 μM. It showed no activity on GAL4-LXRα, GAL4-FXR, or GAL4-RORα at the concentrations tested [1].
In a competitive radioligand binding assay, SR1555 displaced [³H]-T0901317 from the ligand-binding domain (LBD) of RORγ with an IC₅₀ of 1 μM. No displacement was observed for RORα [1].
In an IL-17 promoter-driven luciferase reporter assay, SR1555 dose-dependently suppressed RORγ-dependent, but not RORα-dependent, transcriptional activity, confirming its selectivity as a RORγ-specific inverse agonist [1].
In EL4 murine thymoma cells, treatment with 10 μM SR1555 for 24 hours resulted in a greater than 70% inhibition of endogenous Il17a gene expression, as measured by quantitative real-time PCR [1].
In murine splenocytes cultured under Th17-polarizing conditions (TGFβ and IL-6) for three days, 10 μM SR1555 inhibited Il17a mRNA expression. It was less effective at inhibiting Il17f, Il21, and Il22 mRNA expression [1].
In murine splenocytes cultured under Th17-polarizing conditions for four days, 10 μM SR1555 significantly inhibited IL-17A protein expression, as assessed by intracellular cytokine staining and flow cytometry. This inhibition was observed both when SR1555 was present for the entire differentiation period and when it was added only 24 hours prior to analysis [1].
In a cell viability assay using Annexin V and propidium iodide staining, treatment with 10 μM SR1555 did not induce cell death during Th17 cell differentiation compared to the vehicle control [1].
In murine splenocytes cultured under T regulatory cell (iTreg)-polarizing conditions (TGFβ and IL-2) for three days, 10 μM SR1555 increased Foxp3 gene expression while simultaneously suppressing Rorγt gene expression, as measured by real-time PCR [1].
In murine splenocytes cultured under iTreg-polarizing conditions for five days, 10 μM SR1555 increased the frequency of Foxp3⁺ T regulatory cells by approximately two-fold, as determined by intracellular cytokine staining and flow cytometry [1].

Competitive Radioligand Binding Assay: To examine direct binding of SR1555 to RORα and RORγ, a competition radioligand binding assay was performed. The ligand-binding domains (LBDs) of the receptors were incubated with [³H]-T0901317 as the radioligand and increasing concentrations of SR1555. The ability of SR1555 to displace the radioligand was measured. For RORγ, the IC₅₀ was determined to be 1 μM, confirming direct binding. No displacement was observed for RORα [1].

GAL4-NR Chimeric Co-transfection Assay: HEK293 cells were transiently transfected with a GAL4 DNA-binding domain fused to the ligand-binding domain (LBD) of various nuclear receptors (LXRα, RORα, RORγ, FXR) along with a UAS-luciferase reporter gene. Cells were treated with increasing concentrations of SR1555 or vehicle control. After 20 hours, luciferase activity was measured to assess the compound's effect on the transcriptional activity of each receptor [1].
IL-17 Promoter-Driven Luciferase Reporter Assay: HEK293 cells were transiently co-transfected with an IL-17 promoter-driven luciferase reporter construct and either full-length RORα or RORγ. Cells were treated with increasing concentrations of SR1555. Luciferase activity was measured to determine the compound's effect on ROR-dependent activation of the IL-17 promoter [1].
Endogenous Gene Expression Analysis in EL4 Cells: EL4 murine thymoma cells, which endogenously express RORα, RORγt, and IL-17A, were treated with 10 μM SR1555 or DMSO control for 24 hours. Total RNA was extracted, and Il17a gene expression was analyzed by quantitative real-time PCR. Data were normalized to a housekeeping gene (e.g., GAPDH) [1].
Murine Splenocyte Th17 Differentiation Assay: Splenocytes from C57BL/6 mice were cultured under Th17-polarizing conditions. These conditions included stimulation with anti-CD3 and anti-CD28 antibodies in the presence of TGFβ, IL-6, and neutralizing antibodies against IFNγ and IL-4. Cells were treated with 10 μM SR1555 or DMSO for 3-4 days. After the culture period, cells were re-stimulated with PMA and ionomycin. Il17a, Il17f, Il21, and Il22 mRNA expression was analyzed by real-time PCR. For protein analysis, intracellular cytokine staining for IL-17A was performed and analyzed by flow cytometry. Cell viability was assessed by Annexin V and propidium iodide staining [1].
Murine Splenocyte iTreg Differentiation Assay: Splenocytes were cultured under T regulatory cell (iTreg)-polarizing conditions. These conditions included stimulation with anti-CD3 and anti-CD28 antibodies in the presence of TGFβ, IL-2, and neutralizing antibodies against IFNγ and IL-4. Cells were treated with 10 μM SR1555 or DMSO for 3-5 days. Foxp3 and Rorγt mRNA expression was analyzed by real-time PCR. For protein analysis, intracellular staining for Foxp3 was performed and analyzed by flow cytometry [1].

[1]. Identification of a selective RORγ ligand that suppresses T(H)17 cells and stimulates T regulatory cells. ACS Chem Biol. 2012 Sep 21;7(9):1515-9.

SR1555 is a synthetic, selective RORγ inverse agonist that not only inhibits Th17 cell development and function but also increases the frequency of T regulatory cells in vitro [1].
The structure of SR1555 is 1,1,1,3,3,3-hexafluoro-2-(4-(1-(acetyl)piperazine)methyl)-[1,1-biphenyl]-4-yl)propan-2-ol [1].
SR1555 is structurally similar to SR2211, another RORγ-selective ligand; however, SR2211 does not alter T regulatory cell proliferation, suggesting that the effects on Th17 versus Treg cells are separable and may be optimized independently [1].

C22H23CLF6N2O2

496.87

496.135

C, 53.18; H, 4.67; Cl, 7.13; F, 22.94; N, 5.64; O, 6.44

2309312-90-9

1386439-51-5

132285125

Typically exists as solid at room temperature

2

9

4

33

615

0

Cl.FC(C(C(F)(F)F)(C1C=CC(=CC=1)C1C=CC(=CC=1)CN1CCN(C(C)=O)CC1)O)(F)F

OHUOFZJUUXXRGG-UHFFFAOYSA-N

InChI=1S/C22H22F6N2O2.ClH/c1-15(31)30-12-10-29(11-13-30)14-16-2-4-17(5-3-16)18-6-8-19(9-7-18)20(32,21(23,24)25)22(26,27)28;/h2-9,32H,10-14H2,1H3;1H

1-[4-[[4-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]phenyl]methyl]piperazin-1-yl]ethanone;hydrochloride

2309312-90-9; SR1555 HCl; 1-(4-((4'-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1'-biphenyl]-4-yl)methyl)piperazin-1-yl)ethanone,monohydrochloride; 1-[4-[[4-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]phenyl]methyl]piperazin-1-yl]ethanone;hydrochloride; SR 1555 hydrochloride;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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