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Metabolism ROR SR3335

SR3335

Catalog No. T5160   CAS 293753-05-6
Synonyms: ML 176, ML 176
Purity 99.41% Datasheet

SR 3335 is a selective inverse agonist of RORα, competitively inhibiting the binding of 25-hydroxycholesterol to the ligand binding domain (Ki: 220 nM).

SR3335, CAS 293753-05-6
Pack Size Availability Price/USD Quantity
5 mg In stock 86.00
10 mg In stock 161.00
50 mg In stock 710.00
100 mg In stock 1293.00
1 mL * 10 mM (in DMSO) In stock 92.00
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Biological Description
Chemical Properties
Storage & Solubility Information
Description SR 3335 is a selective inverse agonist of RORα, competitively inhibiting the binding of 25-hydroxycholesterol to the ligand binding domain (Ki: 220 nM).
Targets&IC50 RORα :ic50 220 nM (Ki, cell free),  
Kinase Assay RORα and RORγ LBDs were PCR-amplified and cloned into a pGEX-2T encoding an amino-terminal glutathione S-transferase tag following the manufacturer's instructions. The protein was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside in BL21 gold (DE3) cells and purified by affinity chromatography with Protino GST/4B column followed by size exclusion chromatography with HiLoad 26/60 Superdex 200 column. The protein was eluted, concentrated, and stored in 20 mm Tris, pH 8.0, 150 mm NaCl, 2 mm dithiothreitol, and 10% glycerol. Forty-five or 90 ng of purified GST-RORα or GST-RORγ was used per assay, and the assay was performed in the following buffer: 50 mm HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mm NaCl, and 5 mm MgCl2. Receptors were incubated with various concentrations of 25-[3H]hydroxycholesterol to determine the Kd values (RORα = 3.3 ± 0.89 nm and RORγ = 5.1 ± 0.71 nm). Nonspecific binding was defined in the absence of protein as well as an excess of cold 25-hydroxycholesterol and were shown to be identical. The assays were terminated by rapid filtration through pre-soaked Whatman GF/B filters (0.5% polyethyleneimine in phosphate-buffered saline) in Multiscreen plates and were washed three times with ice-cold assay buffer (1 ml). For the competition assay, various concentrations of sterols were incubated with receptor in the presence of 3 nm 25-[3H]hydroxycholesterol. Results were analyzed using GraphPad Prism software, and the Ki was determined using the Cheng-Prusoff equation [1].
Cell Research
HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. HepG2 cells were maintained and routinely propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. 24 h prior to transfection, cells were plated in 96-well plates at a density of 15 × 10^3 cells/well. Transfections were performed using LipofectamineTM 2000. 16 h post-transfection, the cells were treated with vehicle or compound. 24 h post-treatment, the luciferase activity was measured using the Dual-GloTM luciferase assay system. The values indicated represent the means ± S.E. from four independently transfected wells. The experiments were repeated at least three times [1].
Animal Research
Plasma levels of SR3335 were evaluated in C57BL6 mice (n = 3 per time point) administered by i.p. injection. After 0.25, 0.5,1, 2, 4, and 8h blood was taken. Plasma was generated using standard centrifugation techniques, and the plasma and tissues were frozen at ?80°C. Plasma and tissues were mixed with acetonitrile (1:5 v/v or 1:5 w/v, respectively), sonicated with a probe tip sonicator, and analyzed for drug levels by liquid chromatography/tandem mass spectrometry [1].
Synonyms ML 176 , ML 176
Purity 99.41%
Molecular Weight 405.34
Formula C13H9F6NO3S2
CAS No. 293753-05-6

Storage

-20℃ 3 years powder

-80℃ 2 years in solvent

Solubility Information

DMSO: 40 mg/mL

Ethanol: 30 mg/mL

( < 1 mg/ml refers to the product slightly soluble or insoluble )

Citations

References and Literature
1. Kumar N, et al. Identification of SR3335 (ML-176): a synthetic RORα selective inverse agonist. ACS Chem Biol. 2011 Mar 18;6(3):218-22. 2. Sadeghi H, et al. The retinoid-related orphan receptor alpha is essential for the end-stage effector phase of experimental epidermolysis bullosa acquisita. J Pathol. 2015 Sep;237(1):111-22.

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