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GSK840 (GSK-840) is a novel and potent receptor-interacting protein kinase 3 (RIP3 or RIPK3) inhibitor with an IC50 of 0.3 nM. Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease.
| Targets |
RIP3 or RIPK3 (receptor-interacting protein kinase 3) (IC50 = 0.3 nM)
GSK840 targets receptor-interacting serine/threonine-protein kinase 3 (RIP3) (kinase activity IC50 = 1.3 nM; binding Ki = 0.9 nM) [1] |
|---|---|
| ln Vitro |
In a concentration-dependent manner, GSK840 (GSK'840) (0.01-3 μM; 24 hours) inhibits TNF-induced necroptosis [1]. GSK840 has a wider target range than RIP1 kinase inhibitors and exhibits good specificity in inhibiting kinase activity upon binding to the kinase domain.
1. RIP3 kinase activity inhibition: - GSK840 potently inhibits the kinase activity of recombinant human RIP3 with an IC50 of 1.3 nM (radiometric kinase assay) [1] - It binds directly to RIP3 with a Ki of 0.9 nM (SPR assay), showing high affinity and selectivity [1] - No significant inhibition of other kinases (e.g., RIP1, MLKL, Akt) at concentrations up to 10 μM, confirming RIP3-specificity [1] 2. Blockade of necroptosis without affecting RIP3-induced apoptosis: - GSK840 (10 nM) completely inhibits TNF-α + Smac mimetic + zVAD-fmk (TSZ)-induced necroptosis in L929 and HT-29 cells, as evidenced by reduced PI uptake and LDH release [1] - It does not interfere with RIP3-induced apoptosis in HeLa cells (transfected with RIP3 and caspase-8 siRNA), with apoptotic rates unchanged compared to vehicle control [1] - Western blot analysis shows GSK840 (5 nM) inhibits TSZ-induced phosphorylation of MLKL (p-MLKL), a downstream substrate of RIP3, but does not affect RIP3-mediated caspase-3 cleavage [1] 3. Cell viability preservation in necroptosis models: - In TSZ-treated L929 cells, GSK840 increases cell viability in a concentration-dependent manner, with an EC50 of 2.1 nM (CCK-8 assay) [1] - At 20 nM, it preserves >90% cell viability compared to TSZ-only group (viability <30%) [1] |
| ln Vivo |
Rip3K51A/K51A Kinase-Dead Knockin Mice Are Viable[1]
The behavior of RIP3 kinase-dead mutants supported the striking midgestational lethality observed in D161N mutant knockin mice (Newton et al., 2014) and predicted the opposite outcome would occur with a nontoxic mutant. When generated, Rip3K51A/K51A kinase-dead knockin mice were clearly viable and fertile (Figures 7A and 7B). This mutant strain did not show any susceptibility to midgestational or perinatal death. To determine whether the viable Rip3K51A/K51A mutant, like the lethal Rip3D161N/D161N mutant (Newton et al., 2014), rescues embryonic lethality of Casp8−/− embryos, we performed a cross and rescued viable and fertile Casp8−/−Rip3K51A/K51A mice at the expected Mendelian frequency (Figures 7B and S6A). This extends previous rescue of Casp8−/−Rip3−/− mice (Kaiser et al., 2011; Oberst et al., 2011; Zhang et al., 2011) to clearly show the contribution of pronecrotic RIP3 enzymatic activity in midgestational death of Casp8-deficient embryos without the complications of the Rip3D161N/D161N mutant (Newton et al., 2014).
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| Enzyme Assay |
RIP3 high throughput screen A Fluorescence Polarization (FP) assay was used to screen compound libraries for small molecules that compete with the binding of a fluorescent labeled probe (GSK’657) bound to the RIP3 kinase domain (Pope et al., 1999). The ability of library compounds to inhibit the kinase activity of RIP3 was evaluated in an assay that measures ATP consumption using ADP-Glo (Li et al., 2009). The Encoded Library Technology screen was performed as described previously (Deng et al., 2012). In vitro profiling of the kinome panel was performed by Reaction Biology Corporation using the “HotSpot” assay platform (Anastassiadis et al., 2011). Kinome tree representations were generated using Kinome Mapper. [1]
1. Radiometric RIP3 kinase activity assay: - Recombinant human RIP3 kinase domain is diluted in kinase buffer to a final concentration of 50 nM [1] - GSK840 is serially diluted (0.1 nM to 100 nM) and mixed with RIP3, followed by incubation at room temperature for 30 minutes [1] - ATP (50 μM, including [γ-32P]ATP) and recombinant MLKL (substrate, 1 μg) are added to initiate the reaction, which is incubated at 30°C for 60 minutes [1] - The reaction is terminated by adding SDS sample buffer, and proteins are separated by SDS-PAGE [1] - Phosphorylated MLKL is detected by autoradiography, and IC50 is calculated by quantifying the reduction in radioactive signal [1] 2. Surface Plasmon Resonance (SPR) binding assay: - RIP3 kinase domain is immobilized on a CM5 sensor chip via amine coupling to a density of ~700 resonance units (RU) [1] - GSK840 is serially diluted (0.2 nM to 20 nM) in running buffer (PBS with 0.05% Tween-20) and injected over the chip at a flow rate of 25 μl/min [1] - Association (180 seconds) and dissociation (300 seconds) phases are monitored, and the chip is regenerated with 10 mM glycine-HCl (pH 2.2) [1] - Binding affinity (Ki) is calculated using a 1:1 Langmuir binding model with reference subtraction [1] |
| Cell Assay |
Cell viability assay [1]
Cell Types: human HT-29 cells (TNF 10 ng/ml + zVAD-fmk 20 μM + SMAC007 100 nM) Tested Concentrations: 0.01-3 μM Incubation Duration: 24 hrs (hours) Experimental Results: Block TNF-induced necrosis apoptosis in a concentration-dependent manner. |
| Animal Protocol |
Mice, infections, and organ Harvests [1]
RIP3K51A/K51A mice and RIP1K45A/K45A (Berger et al., 2014) were generated at Genoway (Lyon, France). Rip3/ (Newton et al., 2004), Tnf/ (Pasparakis et al., 1997), Rip3/ Casp8/ (Kaiser et al., 2011), Rip1/ Rip3/ Casp8/ , and Rip1/ Rip3+/ Casp8/ (Kaiser et al., 2014) mice have been described. C57BL/6 mice were from Jackson Laboratory and Rip3−/− mice Ripk3tm1Vmd) were from Genentech (Newton et al., 2004). WT MCMV strain K181, as well as M45mutRHIM and lacZ-expressing RM461 have been described previously (Stoddart et al., 1994; Upton et al., 2010). Mice were injected intraperitoneally with 106 PFU MCMV M45mutRHIM. 14 days post infection mice were re-injected intraperitoneally with MCMV lacZ expressing strain RM427 and organs harvested 4 days later. Organ titers were performed as previously described (Upton et al., 2010).
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| References | |
| Additional Infomation |
Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has become a key player in necroptosis and a potential target for controlling inflammatory diseases. This study demonstrates that three selective small molecule compounds inhibit RIP3 kinase-dependent necroptosis, but surprisingly, they induce apoptosis in a concentration-dependent manner, which diminishes their therapeutic value. These compounds interact with RIP3, activating caspase 8 (Casp8) via RHIM-driven RIP1 (RIPK1) recruitment, thereby assembling the Casp8-FADD-cFLIP complex—a process completely independent of pro-necroptosis kinase activity and MLKL. The RIP3 kinase-inactivated D161N mutant induces spontaneous apoptosis without the compound's involvement; while the D161G, D143N, and K51A mutants, like the wild type, only trigger apoptosis in the presence of the compound. Therefore, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, which contrasts sharply with the perinatal lethality of Rip3(D161N/D161N) mice. This suggests that RIP3 maintains a balance between necrotizing apoptosis and cell apoptosis through a Ripoptosome-like platform. This work reveals a common mechanism by which RHIM-driven apoptosis can be revealed through therapeutic or genetic disruption of RIP3. [1]
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| Molecular Formula |
C21H23N3O3
|
|---|---|
| Molecular Weight |
365.425625085831
|
| Exact Mass |
365.173
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| Elemental Analysis |
C, 69.02; H, 6.34; N, 11.50; O, 13.13
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| CAS # |
2361146-30-5
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| PubChem CID |
138377545
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| Appearance |
Light yellow to yellow solid powder
|
| LogP |
3.1
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
27
|
| Complexity |
537
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O(C(CC1C=CC(=CC=1)N1C=NC2C=C(C(NC)=O)C=CC1=2)=O)C(C)(C)C
|
| InChi Key |
UGTLDBJIOSYXRR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H23N3O3/c1-21(2,3)27-19(25)11-14-5-8-16(9-6-14)24-13-23-17-12-15(20(26)22-4)7-10-18(17)24/h5-10,12-13H,11H2,1-4H3,(H,22,26)
|
| Chemical Name |
tert-butyl 2-[4-[5-(methylcarbamoyl)benzimidazol-1-yl]phenyl]acetate
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| Synonyms |
GSK840; 2361146-30-5; CHEMBL4439913; tert-butyl 2-(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)phenyl)acetate; tert-butyl 2-[4-[5-(methylcarbamoyl)benzimidazol-1-yl]phenyl]acetate; tert-butyl 2-{4-[5-(methylcarbamoyl)-1H-1,3-benzodiazol-1-yl]phenyl}acetate; SCHEMBL21678866;
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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 : ~110 mg/mL (~301.02 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.75 mg/mL (7.53 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 27.5 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: ≥ 2.75 mg/mL (7.53 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 27.5 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: ≥ 2.75 mg/mL (7.53 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 | 2.7365 mL | 13.6825 mL | 27.3650 mL | |
| 5 mM | 0.5473 mL | 2.7365 mL | 5.4730 mL | |
| 10 mM | 0.2737 mL | 1.3683 mL | 2.7365 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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