| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg | |||
| 50mg | |||
| 100mg | |||
| 250mg | |||
| Other Sizes |
Purity: ≥98%
| Targets |
RIP1; RIP3
RIP1 (Kd = 12 nM; inhibits kinase activity at 20 nM comparable to 1 μM Nec-1s) [1] RIP3 (Kd = 130 nM; inhibits kinase activity) [1] |
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| ln Vitro |
GSK2593074A (GSK'074; 0.01, 0.1, 1, 10, and 100 nM; 6 hours for MOVAS cells; 3 hours for L929 cells) completely prevents necroptosis in both human and murine cells at an IC50 of ~3 nM. GSK2593074A inhibits necroptosis with an IC50 of ~3 nM in a variety of cell types, including mouse SMCs, fibroblasts (L929), bone marrow-derived macrophages (BMDM), and human colon epithelial cells (HT29)[1].
GSK2593074A inhibits necroptosis with an IC50 of approximately 3 nM in mouse aortic smooth muscle cells (MOVAS), mouse fibroblasts (L929), primary mouse aortic smooth muscle cells, bone marrow-derived macrophages (BMDM), and human colorectal adenocarcinoma HT-29 cells. [1] At 10 nM, GSK074 completely abolished necroptosis in MOVAS cells (7-AAD+ population reduced from 84.36±3.19% to 17.58±2.04%), comparable to 10 μM Nec-1s (15.69±2.45%). [1] In primary mouse aortic SMCs, 10 nM GSK074 reduced 7-AAD+ population from 21.05±1.34% to 8.81±0.41%; in BMDM, cell viability increased from 32.30±3.38% (DMSO) to 87.08±8.76% with 10 nM GSK074. [1] In HT-29 cells, 10 nM GSK074 decreased 7-AAD+ population from 42.90±1.41% to 16.03±4.86%. [1] GSK074 blocks RIP1-RIP3 complex formation as detected by co-immunoprecipitation and proximity ligation assay. [1] GSK074 eliminates RIP3-mediated MLKL serine345 phosphorylation without affecting RIP1/RIP3 protein levels. [1] In L929 cells, 100 nM GSK074 inhibited IFNβ+poly(I:C)+zVAD-induced RIP1-independent necroptosis (7-AAD+ from 63.91±3.21% to 23.90±0.56%). [1] In mouse primary aortic SMCs, 10 nM GSK074 (but not Nec-1s) significantly repressed TNFα (10 ng/ml)-induced IL6 mRNA expression. [1] Unlike GSK843, GSK074 at concentrations up to 20 μM does not induce apoptosis (no increase in Annexin V+/7-AAD- cells) and does not promote formation of the pro-apoptotic ripoptosome complex. [1] In MOVAS and L929 cells, GSK074 did not block apoptosis induced by GSK843 or tunicamycin (ER stress). [1] [1] |
| ln Vivo |
GSK2593074A (GSK’074; 0.93 mg/kg/day; i.p. injection; 14 or 28 days) is administrated to Apoe-/- mice immediately following pump implantation. GSK2593074A-treated mice exhibit significantly lessened aneurysm formation when compared to the DMSO group, as evidenced by a significantly smaller aortic dilatation (DMSO 85.39±15.76% vs GSK2593074A 36.28±5.76%; P<0.05) and a decreased incidence of abdominal aortic aneurysm (AAA) (from 83.3 to 16.7%). GSK2593074A significantly reduces the amount of aortic expansion (DMSO 66.06±9.17% vs GSK2593074A 27.36±8.25%; P<0.05)[1].
In the calcium phosphate-induced AAA model (male C57BL/6J mice), daily intraperitoneal injection of GSK2593074A at 0.93 mg/kg/day significantly attenuated aortic expansion: DMSO 66.06±9.17% vs GSK074 27.36±8.25% (P<0.05). None of the GSK074-treated mice developed aneurysm (0/4), whereas 4/5 DMSO-treated mice reached aneurysm threshold. [1] In the Angiotensin II-induced AAA model (9-month-old female Apoe-/- mice), GSK074 (0.93 mg/kg/day IP) reduced aortic dilatation from 85.39±15.76% (DMSO) to 36.28±5.76% (P<0.05) and decreased AAA incidence from 83.3% to 16.7%. [1] Histologically, GSK074 treatment preserved smooth muscle α-actin expression, reduced propidium iodide-positive necrotic cells, phospho-MLKL staining, TUNEL-positive apoptotic cells, and CD68-positive macrophages in aneurysm-prone aortae. [1] |
| Enzyme Assay |
Competitive binding assay (KINOMEscan™) was performed to determine binding constants (Kd). An 11-point threefold serial dilution of GSK2593074A was prepared in 100% DMSO at 100× final concentration and diluted to 1× in assay buffer. Kds were calculated with a standard dose-response curve using the Hill equation, fitted by nonlinear least square fit with Levenberg-Marquardt algorithm. GSK074 bound to recombinant human RIP3 kinase domain with Kd=130 nM and to RIP1 with Kd=12 nM. No detectable affinity to PKCδ (Kd>30,000 nM). [1]
In vitro kinase activity assay (ADP-Glo) was used to measure RIP1 and RIP3 kinase activities. Test compounds at indicated concentrations in assay buffer (50 mM Hepes pH7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.02% CHAPS, 0.5 mg/mL BSA) were combined with 25 nM kinase and substrate in 384-well plates. After 1 h incubation, 150 μM ATP was added for additional 5 h. Reaction was terminated by sequential addition of ADP-Glo Reagent and ADP-Glo Detection solution. Luminescence was measured and normalized to no kinase control. GSK074 inhibited both RIP1 and RIP3 kinase activities; 20 nM GSK074 inhibited RIP1 to the same extent as 1 μM Nec-1s. [1] Molecular docking: Homology models of RIP1 and RIP3 were built based on human B-Raf structures (PDB:4FK3 for DFG-in, PDB:3IDP for DFG-out) using MOE and minimized with AMBER10 force field. Ligand docking was performed using HYBRID (v3.2.0) with 1000 conformations per ligand and MMFF charge model. GSK074 bound preferentially to DFG-out homology models for both RIP1 and RIP3, suggesting type II inhibition. [1] |
| Cell Assay |
Cell lines used: MOVAS (mouse aortic smooth muscle), L929 (mouse fibroblast), HT-29 (human colorectal adenocarcinoma), primary mouse aortic SMCs, and bone marrow-derived macrophages (BMDM). Necroptosis was induced with TNFα (30 ng/mL for MOVAS, 20 ng/mL for L929, 100 ng/mL for primary SMCs) plus zVAD (60 μM for MOVAS, 40 μM for L929, 60 μM for primary SMCs). For HT-29: 20 ng/mL TNFα + 200 nM Smac mimetic + 20 μM zVAD. For RIP1-independent necroptosis: L929 primed with IFNβ (50 units/mL) for 24 h then treated with poly(I:C) (10 μg/mL) + zVAD (40 μM). Cell viability was measured using CellTiter-Glo. For flow cytometry, cells were stained with 7-AAD or Annexin V-PE/7-AAD. For in situ PLA, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, incubated with primary antibody pairs, then with PLA probes, ligation and amplification performed, and detected with fluorescent oligonucleotides. For co-immunoprecipitation, cells were lysed in Pierce IP Lysis Buffer, protein A magnetic beads incubated with anti-RIP3 antibody or isotype control for 30 min, then incubated with cell lysate for 1 h, washed, eluted in Laemmli buffer, and subjected to immunoblotting. For immunoblotting, cells were lysed in RIPA buffer with protease/phosphatase inhibitors, separated by SDS-PAGE, transferred to PVDF, blocked, incubated with primary antibodies overnight, then HRP-labeled secondary antibodies, visualized by ECL. For RT-PCR, total RNA extracted with Trizol, cDNA synthesized, real-time PCR performed with SYBR Green, relative mRNA levels calculated by 2-ΔΔCT method with β-actin as control. [1]
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| Animal Protocol |
Animals: Male C57BL/6J mice (8-10 weeks old, 3-month-old) and female Apoe-/- mice (9-10 months old). Calcium phosphate-induced AAA: Under midline incision, infraneral aorta isolated, gauze soaked in 0.5 M CaCl2 applied perivascularly for 10 min followed by PBS-soaked gauze for 5 min. Control received 0.5 M NaCl. External aortic diameter measured with digital caliper before and at harvest (day 14). Aneurysm defined as ≥50% increase. Angiotensin II-induced AAA: Female Apoe-/- mice under regular diet, Ang II (1000 ng/kg/min) administered subcutaneously via Alzet osmotic minipump (model 2004) for 28 days. GSK2593074A was dissolved in 8% DMSO and administered daily via intraperitoneal (IP) injection at 0.93 mg/kg/day (2 μmol/kg/day) immediately after aneurysm induction. For calcium phosphate model, treatment continued for 14 days; for Ang II model, for 28 days. In vivo propidium iodide staining: PI (15 mg/kg body weight) injected IP 2 h before euthanization; cryosections (6 μm) mounted with DAPI and visualized by fluorescence microscopy. [1]
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| ADME/Pharmacokinetics |
The half-life of GSK2593074A in liver microsome was approximately 1 hour, similar to that of Nec-1s. [1]
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| References | |
| Additional Infomation |
GSK2593074A is a representative of a new class of necroptosis inhibitors with dual RIP1/RIP3 targeting as type II kinase inhibitors. It was identified from a screen of 1141 kinase inhibitors using Nec-1s as baseline. Selectivity profiling (KINOMEscan at 100 nM) against 468 human kinases showed high selectivity for RIP1 (0% of control) with selectivity score S(1)=0.012 (5 out of 403 non-mutant kinases at ≤1% control). Other bound kinases (KIT, MEK5, CSF1R, EPHB6) have no established roles in cell death and negligible expression in SMCs except MEK5. Unlike the RIP3 inhibitor GSK843 which induces apoptosis at >3 μM via ripoptosome formation, GSK074 lacks pro-apoptotic properties even at 20 μM. GSK074 is likely the first necroptosis inhibitor capable of binding to RIP3 as a type II inhibitor. [1]
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| Molecular Formula |
C27H23N5OS
|
|---|---|
| Molecular Weight |
465.569423913956
|
| Exact Mass |
465.162
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| CAS # |
1337531-06-2
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| Related CAS # |
1337531-06-2
|
| PubChem CID |
53466951
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| Appearance |
Light yellow to khaki solid
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| LogP |
4.1
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
34
|
| Complexity |
732
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN1C=C(C=N1)C2=CN=C(C3=C2SC=C3C4=CC5=C(C=C4)N(CC5)C(=O)CC6=CC=CC=C6)N
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| InChi Key |
LIGGMBSSOOVGAE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H23N5OS/c1-31-15-20(13-30-31)21-14-29-27(28)25-22(16-34-26(21)25)18-7-8-23-19(12-18)9-10-32(23)24(33)11-17-5-3-2-4-6-17/h2-8,12-16H,9-11H2,1H3,(H2,28,29)
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| Chemical Name |
1-[5-[4-amino-7-(1-methylpyrazol-4-yl)thieno[3,2-c]pyridin-3-yl]-2,3-dihydroindol-1-yl]-2-phenylethanone
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| Synonyms |
GSK2593074A; GSK'074; GSK-2593074A; GSK 074; GSK 2593074A; GSK-074
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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 |
| 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: 41.7 mg/mL (~89.5 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1479 mL | 10.7395 mL | 21.4790 mL | |
| 5 mM | 0.4296 mL | 2.1479 mL | 4.2958 mL | |
| 10 mM | 0.2148 mL | 1.0740 mL | 2.1479 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.
 Identification of new necroptosis inhibitors.Cell Death Dis.2019 Mar 6;10(3):226. |
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 GSK’074 inhibits necroptosis in various cell types.Cell Death Dis.2019 Mar 6;10(3):226. |
 GSK’074 inhibits aneurysm formation in mouse models of aneurysms.Cell Death Dis.2019 Mar 6;10(3):226. |
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