RIPK3
GSK-872 hydrochloride targets receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, a form of programmed cell death. RIPK3 is activated downstream of RIPK1 in response to death receptor stimulation and phosphorylates MLKL, leading to plasma membrane rupture and inflammatory cell death. By binding to the RIP3 kinase domain with high affinity (IC50 = 1.8 nM) and inhibiting its kinase activity (IC50 = 1.3 nM), GSK-872 suppresses downstream MLKL phosphorylation.

GSK-872 fails to inhibit the majority of the 300 human protein kinases tested when measured at 1 μM.Direct tests show that it is unable to inhibit RIP1 kinase. In HT-29 cells, GSK-872 inhibits TNF-induced necroptosis in a concentration-dependent manner. In comparison to cell-free biochemical assays, the IC50 is 100–1000 times higher in cell-based assays. In primary human neutrophils isolated from whole blood, GSK-872 also inhibits necroptosis. GSK-872 blocks two RIP1-independent pathways of necroptosis, TLR3- or DAI-induced death. Caspase activation is brought on by it, and apoptotic cell death follows.
In vitro, GSK-872 hydrochloride binds to the RIP3 kinase domain with an IC50 of 1.8 nM and inhibits kinase activity with an IC50 of 1.3 nM. It is a potent and selective inhibitor of RIPK3 with low-nanomolar activity. GSK-872 inhibits RIPK3-mediated necroptosis and blocks subsequent cytoplasmic translocation and expression of HMGB1. The compound is used to dissect necroptotic signaling and distinguish it from apoptosis-mediated pathways.

GSK-872 treatment significantly decreases HIF-1α expression compared with no treatment after ischemia injury in vivo

GSK-872 (also known as GSK2399872A, GSK872, or GSK-872) is a potent and selective RIPK3 (receptor interacting protein kinase-3) inhibitor. It has a high binding affinity to the RIP3 kinase domain with IC50 value of 1.8 nM, and it inhibits the kinase activity with an IC50 of 1.3 nM.
For in vitro kinase assays, recombinant RIPK3 protein is incubated with a peptide substrate and ATP in kinase assay buffer. The test compound is added at various concentrations (0.001-1000 nM). Kinase activity is measured using radioactive ATP incorporation or luminescent ADP detection assays. IC50 values are calculated by fitting dose-response curves. Binding affinity to the RIP3 kinase domain is assessed using surface plasmon resonance or other biophysical methods.

Viability of 3T3-SA cells at 18 h after treatment with TNF in the presence of Z-VAD-fmk in vehicle control (DMSO) or treated with the indicated concentrations of RIP3 kinase inhibitors, GSK-843 or GSK-872 are assayed.

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Cell viability assay[3]
Cell viability was estimated by Trypan blue exclusion and3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Treatment of inhibitors [N-acetyl Cysteine (NAC), butylated hydroxyanisole (BHA), IM54, Bay11-7082, Z-VAD-FMK, caspase-8 inhibitor, GSK-872 and necrostatin-1 (Nec-1)] was given for 4 h before DLM treatment.
For cell-based necroptosis assays, cells (such as L929 fibroblasts or HT-29 cells) are treated with TNF-α, SMAC mimetic, and z-VAD-FMK (TSZ) to induce necroptosis. GSK-872 hydrochloride is added at concentrations ranging from 0.01-10 µM. Cell viability is assessed using MTT or CellTiter-Glo assays. MLKL phosphorylation is measured by Western blotting to confirm inhibition of necroptotic signaling. HMGB1 release and cytoplasmic translocation can also be assessed.

Eight weeks old Sprague-Dawley male rats with 300-320 g body weight (rat SAH model)[3]
25 mM/6 μL
Syringe pump (intracerebroventricular) at 30 min after SAH
For in vivo studies, GSK-872 hydrochloride can be administered to mice via intraperitoneal or intravenous injection. In early brain injury models, the compound is administered after injury induction, and neurological deficits, brain edema, and HMGB1 levels are assessed. For other disease models (stroke, inflammatory diseases), appropriate animal models are used, and endpoints include tissue damage assessment, inflammatory marker measurement, and survival analysis.

GSK-872 hydrochloride is soluble in DMSO. Storage is recommended at -20°C for long-term stability. The compound is for research use only and is not for human or veterinary use. Further detailed PK parameters including half-life, oral bioavailability, and tissue distribution would be required for in vivo studies. The compound's molecular weight of 419.95 and physicochemical properties suggest it may have drug-like characteristics pending formulation development.

Toxicological data for GSK-872 hydrochloride have not been extensively reported in the available literature. In cell-based assays, the compound is used at concentrations up to 10 µM without significant cytotoxicity. The compound is for research use only and is not intended for human or veterinary use. Standard safety precautions should be followed when handling this compound. Comprehensive toxicology studies would be required before any clinical development.

[1]. RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol Cell. 2014 Nov 20;56(4):481-95.

[2]. Deltamethrin induced RIPK3-mediated caspase-independent non-apoptotic cell death in rat primary hepatocytes. Biochem Biophys Res Commun. 2016 Oct 14;479(2):217-223.

[3]. Inhibiting of RIPK3 attenuates early brain injury following subarachnoid hemorrhage: Possibly through alleviating necroptosis. Biomed Pharmacother. 2018;107:563-570.

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, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 maintains a balance between necrotizing apoptosis and cell death through a Ripoptosome-like platform. This work reveals a common mechanism by which RHIM-driven apoptosis is revealed through therapeutic or genetic disruption of RIP3. [1]

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Deltamethrin (DLM) is a synthetic pyrethroid insecticide widely used worldwide for indoor and field pest control. In this study, we investigated the pathogenesis of DLM-induced hepatotoxicity in rat primary hepatocytes. DLM-induced cell death was accompanied by increased reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, and G2/M phase arrest. Pretreatment with N-acetylcysteine/butylated hydroxyanisole/IM54 partially rescued hepatocytes, suggesting that reactive oxygen species (ROS) may play a role in DLM-induced toxicity. Interestingly, DLM treatment led to a caspase-independent but non-apoptotic cell death. Pretreatment with the pan-caspase inhibitor (ZVAD-FMK) failed to rescue hepatocytes. The absence of altered caspase-3 activity and the absence of detected cleaved caspase-3 further confirmed our findings. Furthermore, lactate dehydrogenase (LDH) release and transmission electron microscopy (TEM) analysis indicated that DLM induced disruption of cell membrane integrity and necrotizing damage. Immunochemical staining showed increased expression of inflammatory markers (TNFα, NFκB, iNOS, COX-2) after DLM treatment. In addition, enhanced RIPK3 expression in the DLM-treated group and the significant inhibitory effect of GSK-872 on cell death suggest that DLM exposure can induce programmed necrosis in hepatocytes. This study demonstrates that DLM can induce hepatotoxicity through non-apoptotic cell death. [2]

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Necrotization is an inflammatory cell death process that depends on receptor-interacting serine/threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) and exhibits necrotic morphological features. The extent of the role of necroptosis in brain injury following subarachnoid hemorrhage (SAH) remains unclear to date. This study aimed to investigate RIPK3-mediated necroptosis and the role of the RIPK3 selective inhibitor GSK'872 in early brain injury following SAH. Following SAH, RIPK3 expression began to increase as early as 6 hours and peaked at 72 hours. Dual immunofluorescence staining showed that RIPK3 was primarily localized in neurons. Most necrotic cells were neurons, further confirmed by transmission electron microscopy (TEM). Intraventricular injection of GSK'872 (25 mM) reduced cerebral edema, improved neurological function, and decreased the number of necrotic cells after SAH. In addition, GSK'872 can reduce the protein levels of RIPK3 and MLKL, and inhibit the cytoplasmic translocation and expression of the important pro-inflammatory protein HMGB1. In summary, this study provides new evidence that RIPK3-mediated necroptosis is associated with early brain injury, and that GSK'872 can reduce RIPK3-mediated necroptosis and subsequent HMGB1 cytoplasmic translocation and expression, and improve cerebral edema and neurological deficits. [3]
GSK-872 hydrochloride is a potent, selective RIPK3 inhibitor that binds the RIP3 kinase domain with IC50 of 1.8 nM and inhibits kinase activity with IC50 of 1.3 nM. It decreases RIPK3-mediated necroptosis and HMGB1 expression, and ameliorates brain edema in early brain injury. The compound is used to probe necroptosis and cell death signaling. GSK-872 hydrochloride is a research tool and is not approved for clinical use.

C19H18CLN3O2S2

419.95

419.052

2703752-81-0

GSK-872;1346546-69-7

155971189

Yellow to brown solid

2

6

4

27

592

0

CC(C)S(=O)(=O)C1=CC2=C(C=CN=C2C=C1)NC3=CC4=C(C=C3)SC=N4.Cl

VKCVPZFWFZKUJY-UHFFFAOYSA-N

InChI=1S/C19H17N3O2S2.ClH/c1-12(2)26(23,24)14-4-5-16-15(10-14)17(7-8-20-16)22-13-3-6-19-18(9-13)21-11-25-19;/h3-12H,1-2H3,(H,20,22);1H

N-(6-propan-2-ylsulfonylquinolin-4-yl)-1,3-benzothiazol-5-amine;hydrochloride

GSK-872 HYDROCHLORIDE; 2703752-81-0; GSK-872 (hydrochloride); GSK2399872A; AKOS040758258; HY-101872A; N-(6-propan-2-ylsulfonylquinolin-4-yl)-1,3-benzothiazol-5-amine;hydrochloride; DA-73928; GSK-872 hydrochloride

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~10 mg/mL (~23.8 mM)
H2O: ~2.5 mg/mL (~6.0 mM)

Solubility in Formulation 1: ≥ 0.21 mg/mL (0.50 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 2.1 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)