| Size | Price | Stock | Qty |
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| 25mg |
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Purity: ≥98%
GSK583 is a highly potent and selective RIP2 kinase inhibitor with an IC50 value of 5 nM.The innate immune system's RIP2 kinase is essential because it facilitates downstream signaling after the pattern recognition receptors NOD1 and NOD2 are activated, which causes the production of inflammatory cytokines. GSK583 exhibits comparable binding affinity for RIP3 kinase in an in-house FP binding assay configured similarly to the RIP2 FP assay (RIP2 FP IC50 = 5 nM; RIP3 FP IC50 = 16 nM). GSK583 exhibits little to no inhibition of RIP3-dependent necroptotic cell death in a cellular assay up to 10 μM concentration, despite having strong biochemical activity against RIP3 kinase.
| Targets |
RIP2 (IC50 = 5 nM); RIP3 (IC50 = 16 nM)
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| ln Vitro |
GSK583 possesses comparable binding affinity for RIP3 kinase as demonstrated by an in-house FP binding assay configured similarly to the RIP2 FP assay (RIP2 FP IC50 = 5 nM; RIP3 FP IC50 = 16 nM). GSK583 exhibits little to no inhibition of RIP3-dependent necroptotic cell death in a cellular assay up to 10 μM concentration, despite having strong biochemical activity against RIP3 kinase. With an IC50 of 8 nM in primary human monocytes, GSK583 potently and dose-dependently inhibits MDP-stimulated tumor necrosis factor-alpha (TNFα) production. Little pro-inflammatory signaling inhibition is seen after treatment with GSK583 at 1 M upon activation of Toll-like receptors (TLR2, TLR4, TLR7) or cytokine receptors (IL-1R, TNFR), but complete inhibition is seen upon activation of both NOD1 and NOD2 receptors, which signal in a RIP2-dependent manner. Although GSK583 has excellent kinase selectivity, it also inhibits Cyp3A4 and the hERG channel, which prevents it from progressing as a drug candidate[1].
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| ln Vivo |
GSK583 has a low oral bioavailability in rats and mice, a moderate volume of distribution, and a low clearance. Although the oral PK in rats and mice provides sufficient systemic exposure for use as a preclinical in vivo tool molecule in an acute inflammation challenge model[1], GSK583 would not produce a human phamacodynamic response within an acceptable dose range, precluding further development of this molecule as a drug candidate.
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| Enzyme Assay |
A fluorescent polarization based binding assay is developed to quantitate interaction of novel test compounds at the ATP binding pocket of RIP2K by competition with a fluorescently labeled ATP competitive ligand. FLAG in its entirety His tagged RIP2K is isolated from a baculovirus expression system and used in the assay at a concentration that is twice the apparent KD. With a final assay concentration of 5 nM, a fluorescently labeled ligand that is reversible and competitive with the inhibitors is used. The enzyme and the ligand are made in solutions containing 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 1 mM CHAPS. Test substances are prepared in 100 percent DMSO, and 100 nL is dispensed to each well of a multiwell plate. The test compounds are then mixed with 5 L of RIP2K, which is twice as concentrated as the final assay, and left to sit at room temperature for 10 minutes. Each reaction is then given 5 μL of the fluorescently labeled ligand solution, which is added at twice the final assay concentration, and is then given an additional at least 10-minute incubation period at room temperature. An instrument that can measure fluorescent polarization is used to read the samples last. Inhibition by the test compound of internal assay controls is expressed as a percentage (%). The following four parameter logistic equation is used to fit normalized data for concentration response experiments: y = A + ((B-C))/(1+(10x)/(10C)D), where y is the % activity (% inhibition) at a specified compound concentration, A is the minimum % activity, B is the maximum % activity, C = log10(IC50), D = Hill slope, x = log10(compound concentration [M]), and pIC50 = (−C).
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| Cell Assay |
Monocytes are pretreated for 30 min with an inhibitor, then stimulated for 6 h with ligands that selectively agonize NLRs NOD1, NOD2, Toll-like receptors TLR, TLR4, or TLR7, or cytokine receptors IL-1R, or TNFR to measure cellular selectivity. Immunoassay is used to measure the release of pro-inflammatory cytokines, such as TNF (NOD2, TLR2, TLR4, IL1R) or IL-8 (NOD1, TLR7, TNFR). Calculated values include IC50 values or percent inhibition.
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| Animal Protocol |
Mice: Male or female Balb/c or female C57Bl/6 mice (n=10/treatment group) are given oral doses of vehicle or GSK583 (0.1, 1, or 10 mg/kg) 15 min before the MDP challenge. Mice are killed 4 hours after receiving 30 μg of MDP intravenously in order to collect peritoneal fluid for peritoneal neutrophil analysis. A FACS analysis is used to measure peritoneal neutrophils.
Rats: Female Crl:CD(SD) rats (n=8/treatment group) are given oral doses of the drug GSK583 or a vehicle 15 minutes before being challenged with MDP (150 g/rat, IV). Rats are killed 2 hours after the MDP challenge, and terminal serum is made using blood drawn from cardiac punctures. The MSD platform measures the concentrations of serum cytokines (IL-6, IL-8 or KC, IL-1β, and TNFα). |
| References |
| Molecular Formula |
C20H19FN4O2S
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|---|---|---|
| Molecular Weight |
398.45
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| Exact Mass |
398.121
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| Elemental Analysis |
C, 60.29; H, 4.81; F, 4.77; N, 14.06; O, 8.03; S, 8.05
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| CAS # |
1346547-00-9
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| Related CAS # |
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| PubChem CID |
67469084
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
652.9±55.0 °C at 760 mmHg
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| Flash Point |
348.7±31.5 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.679
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| LogP |
3.99
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
662
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1C([H])=C([H])C2C(=C(C([H])=C([H])N=2)N([H])C2C3C([H])=C(C([H])=C([H])C=3N([H])N=2)F)C=1[H])(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(=O)=O
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| InChi Key |
XLOGLWKOHPIJLV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H19FN4O2S/c1-20(2,3)28(26,27)13-5-7-16-14(11-13)17(8-9-22-16)23-19-15-10-12(21)4-6-18(15)24-25-19/h4-11H,1-3H3,(H2,22,23,24,25)
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| Chemical Name |
6-tert-butylsulfonyl-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 25.0 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: ≥ 2.5 mg/mL (6.27 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 25.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5097 mL | 12.5486 mL | 25.0973 mL | |
| 5 mM | 0.5019 mL | 2.5097 mL | 5.0195 mL | |
| 10 mM | 0.2510 mL | 1.2549 mL | 2.5097 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.
J Med Chem.2016 May 26;59(10):4867-80. |
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