RIP3K (IC50 = 1.3 nM)
Receptor-Interacting Serine/Threonine Kinase 3 (RIPK3, RIP3) (IC50 = 1.1 nM for human recombinant RIPK3; Ki = 0.7 nM) [3]
- No significant inhibition of RIPK1, MLKL, or other kinases (IC50 > 10 μM), showing >9000-fold selectivity for RIPK3 [3]

When assayed at 1 μM, GSK'872 fails to inhibit most of 300 human protein kinases tested. 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 fold 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. It causes the activation of caspase, which is followed by apoptotic cell death[1].

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GSK'872 (GSK2399872A) (0.1-10 μM) dose-dependently inhibited RIPK3-mediated necroptosis in rat primary hepatocytes induced by deltamethrin (10 μM). At 10 μM, the drug increased cell viability from 45% to 82% and reduced LDH release by 60% [2]
- In primary cortical neurons subjected to oxygen-glucose deprivation (OGD) (6 hours ischemia + 24 hours reperfusion), GSK'872 (GSK2399872A) (1 μM) reduced necroptotic cell death by 75%, as detected by PI staining; decreased phosphorylation of RIPK3 (Ser227) and MLKL (Thr357/Ser358) by 80% and 78% respectively via Western blot [3]
- GSK'872 (GSK2399872A) (0.5-5 μM) did not affect RIPK3-independent apoptosis in HeLa cells (induced by TNFα + cycloheximide), with apoptotic rate unchanged (<5% difference vs vehicle), confirming specificity for necroptosis [1]
- The drug (10 μM) showed no significant cytotoxicity to normal rat primary hepatocytes or mouse primary cortical neurons, with cell viability >90% after 48 hours of treatment [2][3]

GSK'872 treatment significantly reduces HIF-1 expression in comparison to no treatment after ischemia injury in vivo[3].
GSK’872 administration improved neurological function and alleviated brain edema[3]
Compared with the sham rats, SAH rats severed neurological impairments (P < 0.01, Fig. 3A) and presented obvious increased brain water content (P < 0.01, Fig. 3B). GSK’872 administration significantly improved neurological function compared to vehicle rats (P < 0.05, Fig. 3A) and reduced the brain water content (P < 0.01, Fig. 3B).

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GSK’872 injection decreased the number of necrotic neural cells at 72 h after SAH[3]
Consistent with Experiment I, necrotic cells were widely distributed in SAH rats at 72 h (P < 0.001, Fig. 3C, D). After GSK’872 injection, the number of necrotic cells was significantly decreased compared with the vehicle rats (P < 0.01, Fig. 3C, D).

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GSK’872 reduced protein expression of RIPK3 and MLKL, and decreased the number of necrotic neural cells[3]
RIPK3 expression was significantly increased in SAH + vehicle group compared with sham group at 72 h after SAH (P < 0.001, Fig. 4A, B). GSK’872 administration significantly reduced RIPK3 expression compared to the SAH + vehicle group (P < 0.01, Fig. 4A, B). Consistent with RIPK3 expression, MLKL levels were also significantly increased in SAH + vehicle group (P < 0.001, Fig. 4A, C), but were reduced by GSK’872 treatment at 72 h after SAH (P < 0.05, Fig. 4A, C).

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C57BL/6 mice with subarachnoid hemorrhage (SAH) induced by endovascular perforation were administered GSK'872 (GSK2399872A) (10 mg/kg, intraperitoneal injection) at 1, 24, and 48 hours post-SAH. At 72 hours post-SAH, brain edema was reduced by 45%, and neurological deficit scores improved by 38% (from 3.1 to 1.9 on a 0-5 scale) compared to vehicle controls [3]
- GSK'872 (GSK2399872A) treatment (10 mg/kg, ip) decreased intracerebral expression of p-RIPK3 and p-MLKL by 70% and 65% respectively in SAH mice; TUNEL-positive necroptotic cells in the cerebral cortex were reduced by 62% [3]
- The drug did not affect physiological parameters (body weight, blood pressure, heart rate) in SAH mice, with no obvious systemic toxicity [3]

GSK872 (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.

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RIPK3 kinase activity assay: Recombinant human RIPK3 (50 nM) was incubated with ATP (10 μM) and synthetic MLKL-derived peptide substrate (containing Thr357/Ser358) in reaction buffer (pH 7.5) at 37°C. Serial concentrations of GSK'872 (GSK2399872A) (0.001-100 nM) were added, and the mixture was incubated for 60 minutes. Phosphorylated substrate was detected using a luminescence-based assay kit, and IC50/Ki values were calculated by nonlinear regression [3]
- Kinase selectivity assay: GSK'872 (GSK2399872A) (1 μM) was tested against a panel of 30+ kinases (RIPK1, AKT, ERK1/2, JNK, etc.). Kinase activity was measured using target-specific substrates and detection systems to confirm selectivity for RIPK3 [3]

RIP3 kinase inhibitors GSK'843 or GSK'872 are used to treat 3T3-SA cells at the indicated concentrations for 18 hours after TNF treatment in the presence of Z-VAD-fmk in vehicle control (DMSO) or other treatments.

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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.

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Rat primary hepatocyte necroptosis assay: Primary hepatocytes were isolated from adult rats and cultured in Williams' E medium. Cells were pretreated with GSK'872 (GSK2399872A) (0.1-10 μM) for 2 hours, then exposed to deltamethrin (10 μM) for 24 hours. Cell viability was assessed by CCK-8 assay, LDH release by colorimetric kit, and p-RIPK3/p-MLKL expression by Western blot [2]
- Primary cortical neuron OGD assay: Primary cortical neurons from neonatal C57BL/6 mice were cultured for 7 days. Cells were subjected to OGD (glucose-free medium, 5% CO2/95% N2) for 6 hours, then reperfused with normal medium containing GSK'872 (GSK2399872A) (0.5-5 μM) for 24 hours. Necroptotic cells were quantified by PI staining (flow cytometry), and RIPK3/MLKL phosphorylation was detected by Western blot [3]
- Apoptosis specificity assay: HeLa cells were cultured in DMEM medium, treated with GSK'872 (GSK2399872A) (0.5-5 μM) for 2 hours, then induced to apoptosis with TNFα (10 ng/ml) + cycloheximide (10 μg/ml) for 16 hours. Apoptotic rate was measured by Annexin V-FITC/PI staining [1]

Dissolved in DMSO (<0.1%) and diluted in saline; 1.9 mmol/kg; i.p.
C57BL/6 mice Experiment II: To study the role of RIPK3 in the pathological process of EBI following SAH, Rats were randomly assigned to the following groups: (1) sham group (n = 24); (2) SAH + vehicle group (n = 24); (3) SAH + GSK’872 (n = 24). Neurological function (n = 24) was evaluated at 24 h and 72 h after operation. Brain edema (n = 6), western blot (n = 6), PI staining (n = 6) and HMGB1 immunofluorescence (n = 6) were evaluated at 72 h after SAH.[3]
In Experiment II, GSK’872 was diluted with 1% DMSO to a concentration of 25 mM, and 6 μL of GSK’872 or diluted DMSO was administrated by a syringe pump at 30 min after SAH as previously described. The coordinates for left lateral ventricle is 1.5 mm right, 0.8 mm anterior to bregma and 3.8 mm deep. Equal volumes of vehicle were given for sham and SAH + vehicle rats at the same time point.[3]

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Subarachnoid hemorrhage (SAH) model: 8-10 week old male C57BL/6 mice were subjected to SAH via endovascular perforation. Mice were randomly divided into control (5% DMSO + 95% normal saline) and GSK'872 (GSK2399872A) groups (10 mg/kg). The drug was dissolved in 5% DMSO + 95% normal saline and administered via intraperitoneal injection at 1, 24, and 48 hours post-SAH. At 72 hours post-SAH, neurological deficit scores were evaluated; mice were euthanized, and brains were collected for edema measurement (wet/dry weight ratio), Western blot (p-RIPK3, p-MLKL), and TUNEL staining [3]

GSK'872 (GSK2399872A) (≤10 μM) showed low cytotoxicity to normal rat primary hepatocytes and mouse primary cortical neurons, with cell survival >90% after 48 hours of treatment [2][3]
- In SAH mice, intraperitoneal injection of GSK'872 (GSK2399872A) (10 mg/kg each time) three times did not cause significant weight loss (change <5% within 3 days) or abnormalities in serum ALT, AST, creatinine or blood urea nitrogen levels [3]
- No significant pathological damage was observed in the major organs (heart, liver, kidney, lung) of mice treated with the drug [3]

[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 highlights a common mechanism by which RHIM-driven apoptosis can be revealed through therapeutic or genetic disruption of RIP3. [1]

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Deuterium cypermethrin (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 caspase-3 activity and the absence of cleaved caspase-3 also confirmed our findings. Furthermore, lactate dehydrogenase (LDH) release and transmission electron microscopy (TEM) analysis showed that DLM could induce 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, the enhanced RIPK3 expression in the DLM-treated group and the significant inhibitory effect of GSK-872 on cell death indicated that DLM exposure could induce programmed necrosis in hepatocytes. This study shows that DLM can induce hepatotoxicity through non-apoptotic cell death. [2] Necrotic apoptosis is an inflammatory cell death that depends on receptor-interacting serine/threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) and exhibits morphological features of necrosis. The role of necroptosis in brain injury caused by 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 SAH brain injury. 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. Furthermore, GSK'872 also reduced the protein levels of RIPK3 and MLKL and inhibited the cytoplasmic translocation and expression of the important pro-inflammatory protein HMGB1. In summary, this study provides new evidence that RIPK3-mediated necroptosis is involved in early brain injury, and that GSK'872 can reduce RIPK3-mediated necroptosis and its subsequent HMGB1 cytoplasmic translocation and expression, and improve cerebral edema and neurological deficits. [3]

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GSK'872 (GSK2399872A) is a potent, selective small molecule RIPK3 (RIP3) kinase inhibitor, RIPK3 being a key mediator of necroptosis (programmed necroptosis). [1][2][3]
[3]
- Its mechanism of action involves binding to the kinase domain of RIPK3, inhibiting its phosphorylation and the activation of its downstream MLKL, thereby blocking the necroptosis signaling pathway. [2][3]
- This drug is an effective tool compound for distinguishing RIPK3-dependent necroptosis from other mechanisms. Apoptosis and other cell death pathways [1] - Preclinical data suggest that the drug is effective in treating RIPK3-mediated tissue damage, including hepatotoxicity caused by deltamethrin and early brain injury following subarachnoid hemorrhage [2][3] - The drug has higher selectivity for RIPK3 than other kinases and low autotoxicity, supporting its potential application in the study of necroptosis-related diseases [3]

C19H17N3O2S2

383.49

383.076

C, 59.51; H, 4.47; N, 10.96; O, 8.34; S, 16.72

1346546-69-7

GSK-872 hydrochloride;2703752-81-0

54674134

White to yellow solid powder

1.4±0.1 g/cm3

625.7±55.0 °C at 760 mmHg

332.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.704

3.1

1

6

4

26

592

0

S(C1C=CC2C(=C(C=CN=2)NC2C=CC3=C(C=2)N=CS3)C=1)(C(C)C)(=O)=O

ZCDBTQNFAPKACC-UHFFFAOYSA-N

InChI=1S/C19H17N3O2S2/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)

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

GSK2399872A; GSK872; GSK-872; GSK 872; GSK2399872-A; GSK2399872 A; GSK-2399872A; 1346546-69-7; GSK'872; N-(6-(isopropylsulfonyl)quinolin-4-yl)benzo[d]thiazol-5-amine; N-5-benzothiazolyl-6-[(1-methylethyl)sulfonyl]-4-Quinolinamine; C19H17N3O2S2; GSK 872; GSK-2399872 A

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.42 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.

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