RIPK3

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.

GSK'872 treatment significantly reduces HIF-1 expression in comparison to no treatment after ischemia injury in vivo[3].

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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). GSK’872 reduced protein expression of RIPK3 and MLKL, and decreased the number of necrotic neural cells[3]

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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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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.

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.

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

[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]

C19H18CLN3O2S2

419.95

419.052

2703752-81-0

GSK-872;1346546-69-7

155971189

Yellow to brown solid

2

6

4

27

592

0

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.)