RIP3K (IC50 = 1.3 nM)

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].

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).

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.

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.

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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Dissolved in DMSO (<0.1%) and diluted in saline; 1.9 mmol/kg; i.p.

C57BL/6 mice

[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 emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.[1]

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Deltamethrin (DLM), a synthetic pyrethroid insecticide, is used all over the world for indoor and field pest management. In the present study, we investigated the elicited pathogenesis of DLM-induced hepatotoxicity in rat primary hepatocytes. DLM-induced cell death was accompanied with increased ROS generation, decreased mitochondrial membrane potential and G2/M arrest. Pre-treatment with N-acetyl cysteine/butylated hydroxyanisole/IM54 could partly rescue hepatocytes suggesting that ROS might play a role in DLM-induced toxicity. Interestingly, DLM treatment resulted in a caspase-independent but non-apoptotic cell death. Pre-treatment with pan-caspase inhibitor (ZVAD-FMK) could not rescue hepatocytes. Unaltered caspase-3 activity and absence of cleaved caspase-3 also corroborated our findings. Further, LDH release and Transmission electron microscopy (TEM) analysis demonstrated that DLM incites membrane disintegrity and necrotic damage. Immunochemical staining revealed an increased expression of inflammatory markers (TNFα, NFκB, iNOS, COX-2) following DLM treatment. Moreover, the enhanced RIPK3 expression in DLM treated groups and prominent rescue from cell death by GSK-872 indicated that DLM exposure could induce programmed necrosis in hepatocytes. The present study demonstrates that DLM could induce hepatotoxicity via non-apoptotic mode of cell death.[2]

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Necroptosis is an inflammatory form of cell death that depends on receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) and displays the morphological characteristics of necrosis. To date, it is unclear to what extent necroptosis contributes to subarachnoid hemorrhage (SAH) induced brain injury. The present study aimed to investigate the RIPK3-mediated necroptosis and the effects of the RIPK3 selective inhibitor GSK'872 in early brain injury following SAH. After SAH, RIPK3 expression increased as early as 6 h and peaked at 72 h. Double immunofluorescence staining revealed that RIPK3 was mainly located in neurons. Most necrotic cells were neurons, which were further confirmed by TEM. Intracerebroventricular injection of GSK'872 (25 mM) could attenuate brain edema and improve neurological function following SAH and reduce the number of necrotic cells. In addition, GSK'872 could also decrease the protein levels of RIPK3 and MLKL, and cytoplasmic translocation and expression of HMGB1, an important pro-inflammatory protein. Taken together, the current study provides the new evidence that RIPK3-mediated necroptosis is involved in early brain injury and GSK'872 decreases the RIPK3-mediated necroptosis and subsequent cytoplasmic translocation and expression of HMGB1, as well as ameliorates brain edema and neurological deficits.[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.)