| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
CRAC channel 402 nM (IC50) Orai1/Stim1 channels 25 nM (IC50) IL-2
RO2959 monohydrochloride targets the calcium release-activated calcium (CRAC) channel, which is composed of Orai1 (the pore-forming subunit) and STIM1 (the calcium sensor in the endoplasmic reticulum). It is a potent and specific CRAC channel inhibitor with an IC50 of 402 nM. RO2959 mono HCl is also a potent blocker of store-operated calcium entry (SOCE) mediated by Orai1/Stim1 channels with an IC50 of 25 nM. It has an IC50 of 265 nM for SOCE in activated CD4+ T lymphocytes and is a potent inhibitor of human IL-2 production. |
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| ln Vitro |
RO2959 has an IC50 of 25 nM for Orai1 and 530 nM for Orai3 inhibition. With an IC50 value of 265 nM, RO2959 inhibits store operated calcium entry (SOCE) in activated CD4+T lymphocytes[1]. Strong SOCE inhibitor RO2959 inhibits both IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells that exhibit stable human Orai1 and Stim1 expression, as well as SOCE in human primary CD4+ T cells stimulated by TCR activation or thapsigargin therapy. When TCR stimulation or mixed lymphocyte reaction (MLR) is used to stimulate T cells, RO2959 totally blocks both cytokine generation and T cell proliferation[1].
In vitro, RO2959 monohydrochloride is a potent and selective CRAC channel inhibitor (IC50 = 402 nM). It is a potent blocker of store-operated calcium entry (SOCE) mediated by Orai1/Stim1 channels (IC50 = 25 nM). RO2959 also has an IC50 of 265 nM for SOCE in activated CD4+ T lymphocytes. It is a potent inhibitor of human IL-2 production, effectively blocking T cell receptor (TCR)-triggered gene expression and T cell function pathways. RO2959 has an IC50 of 25 nM for Orai1 and 530 nM for Orai3 inhibition. It also strongly inhibits both IP3-dependent current in CRAC-expressing RBL-2H3 cells and SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. |
| ln Vivo |
Specific in vivo activity data for RO2959 monohydrochloride has not been published. However, as a potent CRAC channel inhibitor that blocks T cell activation and IL-2 production, it is hypothesized to be effective in animal models of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis. T cell activation plays a critical role in these diseases, and inhibition of SOCE with RO2959 could reduce T cell-mediated inflammation. The compound also has the potential to be used in transplantation research to prevent graft rejection.
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| Enzyme Assay |
The specific protocol for assessing CRAC channel inhibition uses a thapsigargin-induced SOCE assay in human primary CD4+ T cells or Jurkat T cells. Cells are loaded with a fluorescent calcium indicator (e.g., Fura-2 AM or Fluo-4 AM) in the presence of 2 uM thapsigargin (to deplete ER calcium stores) in Ca2+-free buffer for 30 minutes at 37degC. Cells are then transferred to a fluorescence plate reader or imaging system. Baseline fluorescence is recorded for 60 seconds. RO2959 monohydrochloride (0.1-1000 nM) is added and incubated for 2 minutes. CaCl2 (2 mM) is then added to initiate Ca2+ entry via SOCE. The increase in intracellular calcium (measured as deltaF/F0) is recorded. The IC50 for SOCE inhibition (25 nM) is calculated from the concentration-response curve.
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| Cell Assay |
For in vitro cellular assays, human peripheral blood mononuclear cells (PBMCs) are isolated from healthy donor blood. PBMCs are seeded in 96-well plates at 2×10⁵ cells/well. Cells are stimulated with anti-CD3/CD28 antibodies (1 ug/mL each) to activate T cell receptor (TCR) signaling. RO2959 monohydrochloride is added at concentrations of 0.1-1000 nM at the time of stimulation. After 72 hours, cell culture supernatants are collected. Human IL-2, IFN-gamma, and TNF-alpha levels are quantified by ELISA. T cell proliferation is measured by adding [3H]-thymidine (1 uCi/well) for the final 18 hours of culture, followed by scintillation counting. The IC50 for inhibition of IL-2 production is calculated. In a mixed lymphocyte reaction (MLR), PBMCs from two donors are co-cultured, and RO2959 is added at the start of the culture. After 5-7 days, T cell proliferation is assessed by [3H]-thymidine incorporation or by CFSE dilution flow cytometry.
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| Animal Protocol |
An in vivo protocol for RO2959 would involve a mouse model of T cell-mediated autoimmunity, such as experimental autoimmune encephalomyelitis (EAE, a model of multiple sclerosis) or collagen-induced arthritis (CIA). For the EAE model, female C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG35-55) peptide in complete Freund's adjuvant, followed by pertussis toxin injections. RO2959 monohydrochloride is dissolved in a suitable vehicle (e.g., 10% DMSO/40% PEG300/5% Tween-80/45% saline) and administered orally at doses of 1, 3, or 10 mg/kg once or twice daily starting from day 0 (prophylactic) or from the onset of clinical signs (therapeutic). Disease severity is scored daily (0-5 scale). At the end of the study (day 21-28), spinal cords and brains are collected for histopathological assessment of inflammatory infiltrates. Splenocytes are harvested for ex vivo restimulation with MOG peptide to measure cytokine production by ELISA or ELISpot.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic data for RO2959 monohydrochloride is not publicly available. As a small molecule CRAC channel inhibitor, a standard PK study in C57BL/6 mice would involve IV (1 mg/kg) and PO (5-10 mg/kg) administration. Serial blood samples would be collected at multiple time points (0-24 h). RO2959 monohydrochloride concentrations in plasma would be quantified by LC-MS/MS. Key parameters including T1/2, Cmax, AUC0-∞, clearance, and oral bioavailability would be calculated. The compound is expected to have moderate oral bioavailability and a half-life suitable for once- or twice-daily dosing.
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| Toxicity/Toxicokinetics |
Specific toxicology data for RO2959 monohydrochloride is not available. As a CRAC channel inhibitor that blocks T cell activation, the primary safety concern would be immunosuppression, potentially leading to an increased risk of infections and malignancies. Off-target inhibition of Orai3 (IC50 = 530 nM) could have additional effects on other cell types. Standard safety assessment would include in vitro hERG channel inhibition testing, CYP450 enzyme inhibition screening, and a 14-day repeat-dose oral toxicity study in rats to determine the maximum tolerated dose (MTD) and evaluate effects on immune function (e.g., T cell-dependent antibody response).
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| References | |
| Additional Infomation |
RO2959 monohydrochloride is a research-grade chemical and is not approved for clinical use. Its molecular formula is C21H20ClF2N5OS with a molecular weight of 463.93. It is a potent and selective CRAC channel inhibitor (IC50 = 402 nM) and a potent blocker of store-operated calcium entry (SOCE) mediated by Orai1/Stim1 channels (IC50 = 25 nM). RO2959 monohydrochloride potently inhibits human T cell activation, IL-2 production, and T cell proliferation, making it a valuable tool for studying autoimmune and inflammatory diseases. It is stored at -20degC and is soluble in DMSO.
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| Molecular Formula |
C21H20CLF2N5OS
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|---|---|
| Molecular Weight |
463.931208610535
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| Exact Mass |
463.104
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| CAS # |
2309172-44-7
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| Related CAS # |
RO2959 hydrochloride;1219927-22-6
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| PubChem CID |
45274292
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| Appearance |
Light brown to brown solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
31
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| Complexity |
655
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| Defined Atom Stereocenter Count |
0
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| SMILES |
Cl.S1C(C)=CN=C1N1CC(C2C=NC(=CN=2)NC(C2C(=CC=CC=2F)F)=O)=C(C)CC1
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| InChi Key |
GMLSDODALBRLPE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H19F2N5OS.ClH/c1-12-6-7-28(21-26-8-13(2)30-21)11-14(12)17-9-25-18(10-24-17)27-20(29)19-15(22)4-3-5-16(19)23;/h3-5,8-10H,6-7,11H2,1-2H3,(H,25,27,29);1H
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| Chemical Name |
2,6-difluoro-N-[5-[4-methyl-1-(5-methyl-1,3-thiazol-2-yl)-3,6-dihydro-2H-pyridin-5-yl]pyrazin-2-yl]benzamide;hydrochloride
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 25 mg/mL (53.89 mM)
H2O: < 0.1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.48 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1555 mL | 10.7775 mL | 21.5550 mL | |
| 5 mM | 0.4311 mL | 2.1555 mL | 4.3110 mL | |
| 10 mM | 0.2155 mL | 1.0777 mL | 2.1555 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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