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Purity: ≥98%
GSK'547 (GSK547; GSK-547) is a novel, potent and highly selective RIP1 (receptor-interacting serine/threonine protein kinase 1) inhibitor with immunomodulatory properties. Compared to GSK'963, it exhibits a 400-fold increase in mouse pharmacokinetic oral exposure. In a STAT1-dependent manner, RIP1 targeting reprogrammed TAMs (tumor-associated macrophages) toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype. Tumor immunity was produced in mice and organotypic models of human PDA by RIP1 inhibition in TAMs, which led to cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype. Immunotherapies based on PD1 and inducible co-stimulators and RIP1 targeting worked well together. RIP1's tumor-promoting effects were not dependent on RIP3 co-association. Our research as a whole characterizes RIP1 as a checkpoint kinase controlling tumor immunity.
| Targets |
RIPK1/receptor-interacting serine/threonine protein kinase 1
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|---|---|
| ln Vitro |
GSK'547 (RIP1i) treatment in vitro directs the programming of bone marrow-derived macrophages (BMDM) toward an immunogenic phenotype, upregulating MHC-II, TNFa, and IFNg, while concomitantly reducing CD206, IL-10, and TGFb expression. Furthermore, STAT1 signaling is upregulated by RIP1i in BMDM, which is linked to M1 programming, but STAT3, STAT5, and STAT6 signaling are downregulated, which is connected to M2-like macrophage differentiation. The ability of macrophages treated with RIP1i to capture antigen is also improved[1].
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| ln Vivo |
GSK'547 (RIP1i) administration in mouse chow results in in vivo steady-state concentrations over the L929 IC90 over a 24-hour period. Over the course of a 6-week treatment regimen, high serum concentrations of RIP1i are maintained. Without obvious pathology, RIP1i therapy is well tolerated. In comparison to mice treated with controls or Nec-1s, those given RIP1i have less tumor burden and longer survival after being exposed to orthotopic PDA (pancreatic ductal adenocarcinoma) tumor cells derived from KPC mice. Aside from new tumors, RIP1i also guards against liver metastases[1].
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| Enzyme Assay |
Fluorescent polarization (FP) binding assay[1]
An FP-based binding assay was used to quantify the interaction between RIP1i and the ATP-binding pocket of RIP1 by competition with a fluorescently labeled ATP-competitive ligand as we previously described (Berger et al., 2015). In brief, purified GST-tagged RIP1 (1-375) was used at a final assay concentration of 200 nM. A fluorescently-labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo [2’,3’]indolizino[8’,7’:5′,6′]pyrano [3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate) was used at a final assay concentration of 5 nM. Samples were read on an Analyst multimode reader and the inhibition was expressed as percent inhibition of internal assay controls. Kinase selectivity and cell-based viability assays[1] Kinase selectivity assays were performed as we previously described (Berger et al., 2015). RIP1i (10 μM) was tested against 371 kinases using a P33-radiolabeled assay according to the manufacturer’s protocol. Reactions were performed in the presence of 10 μM ATP. Data are reported as % enzyme activity relative to DMSO controls. The efficacy of RIP1 inhibitors was tested in vitro using L929 cells. Cell death was induced with recombinant TNFα (100 ng/ml) in the presence of caspase inhibitor QVD-Oph (25 μM; Millipore Sigma). To evaluate the effect of RIP1 inhibition, cells were pretreated with RIP1i at various doses for 30 min. Induced cell death was evaluated 24 hr later by measuring cellular ATP levels using CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer’s protocol. |
| Cell Assay |
For 30 minutes, RIP1i is pretreated with cells in a variety of doses. 24 hours later, cellular ATP levels are used to assess induced cell death.
T cell Proliferation Assays[1] For antibody-based T cell proliferation assays, splenic CD3+ T cells were activated using CD3/CD28 co-ligation in 96-well plates, as we previously described (Daley et al., 2016). In selected wells, TAMs were added in a 1:5 macrophage: T cell ratio. For antigen-restricted T cell stimulation assays, splenic OT-I or OT-II T cells were cultured with macrophages pulsed, respectively, with Ova257-264 or Ova323-339 peptide in a 5:1 ratio. Alternatively, macrophages were loaded with Ovalbumin (1 mg/ml, 60 min). In select wells a neutralizing anti-TNFα mAb (10 μg/ml, MP6-XT22) or isotype control was added. T cell activation was determined at 72 hr by flow cytometry. |
| Animal Protocol |
C57BL/6, OT-I, OT-II, Stat1tm1Dlv, Rag1tm1Mom, and Foxn1nu mice were bred in-house. Ripk3−/− mice were obtained from Genentech. RIP1 KD/KI mice were generated by homologous recombination using a targeting construct that mutated the catalytic lysine residue to alanine (K45A) to eliminate all kinase activity, as we previously described (Kaiser et al., 2014). C57BL/6 mice were used for pharmacokinetic experiments. All mice were housed under pathogen-free conditions. KC mice develop slowly progressive pancreatic neoplasia endogenously by expressing mutant Kras in the progenitor cells of the pancreas (Hingorani et al., 2003). We previously detailed tumor progression and survival in control KC mice (Daley et al., 2016). Pancreatic ductal epithelial cells were harvested from KC mice and cultured in vitro as we previously described (Seifert et al., 2016a). Both male and female mice were used, but animals were age-matched within each experiment. Mice were fed either control chow or RIP1i (~100 mg/kg/day) via food-based dosing. For orthotopic pancreatic tumor challenge, 8-10 week old mice were administered intra-pancreatic injections of FC1242 PDA cells derived from KPC mice, as previously described (Zambirinis et al., 2015). Cells were suspended in PBS with 50% Matrigel and 1x105 tumor cells were injected into the body of the pancreas via laparotomy. Mice were sacrificed 3 weeks later and tumors harvested for analyses.[1]
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| References | |
| Additional Infomation |
GSK547 (GSK'547) is a highly selective and potent inhibitor of receptor-interacting serine/threonine protein kinase 1 (RIPK1), inhibits macrophage-mediated adaptive immune tolerance in pancreatic cancer.
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| Molecular Formula |
C20H18F2N6O
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|---|---|
| Molecular Weight |
396.3933
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| Exact Mass |
396.15
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| Elemental Analysis |
C, 60.60; H, 4.58; F, 9.59; N, 21.20; O, 4.04
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| CAS # |
2226735-55-1
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| Related CAS # |
(Rac)-GSK547
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| PubChem CID |
134521814
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| Appearance |
White to off-white solid powder
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| LogP |
1.9
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
29
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| Complexity |
663
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1CN(CCC1C(=O)N2[C@@H](CC=N2)C3=CC(=CC(=C3)F)F)C4=NC=NC(=C4)C#N
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| InChi Key |
SJVGFKBLUYAEOK-SFHVURJKSA-N
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| InChi Code |
InChI=1S/C20H18F2N6O/c21-15-7-14(8-16(22)9-15)18-1-4-26-28(18)20(29)13-2-5-27(6-3-13)19-10-17(11-23)24-12-25-19/h4,7-10,12-13,18H,1-3,5-6H2/t18-/m0/s1
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| Chemical Name |
6-[4-[(3S)-3-(3,5-difluorophenyl)-3,4-dihydropyrazole-2-carbonyl]piperidin-1-yl]pyrimidine-4-carbonitrile
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| Synonyms |
GSK'547; GSK-547; GSK 547; GSK547; 2226735-55-1; CHEMBL4514271; (S)-6-(4-(5-(3,5-difluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)piperidin-1-yl)pyrimidine-4-carbonitrile; 6-[4-[(3S)-3-(3,5-difluorophenyl)-3,4-dihydropyrazole-2-carbonyl]piperidin-1-yl]pyrimidine-4-carbonitrile; RIP1 inhibitor； RIP1i
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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 (e.g. under nitrogen), 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: 29~250 mg/mL (73.2~630.69 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.31 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.25 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 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.25 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. Solubility in Formulation 4: (saturation unknown) in (add these co-solvents sequentially from left to right, and one by one), |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5228 mL | 12.6138 mL | 25.2277 mL | |
| 5 mM | 0.5046 mL | 2.5228 mL | 5.0455 mL | |
| 10 mM | 0.2523 mL | 1.2614 mL | 2.5228 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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