RIPK3
GSK-843 (3-10 μM; 18 h) induces apoptosis[1].
GSK-843 (0.3-3 μM; 18 h) inhibits virus- and TNF-induced cell necrosis[2].

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Researchers previously identified the RIP3i GSK’843 and GSK’872 by screening conventional small-molecule libraries (Kaiser et al., 2013a) (Figure 1A). These compounds bound RIP3 kinase domain with high affinity (IC50 = 8.6 nM and 1.8 nM, respectively; Figure 1B) and inhibited kinase activity (IC50 = 6.5 nM and 1.3 nM, respectively; Figure 1C). When assayed individually at 1 μM, the three structurally distinct compounds failed to inhibit most of 300 human protein kinases tested, with GSK’840 showing the best profile (Figure S1B; Table S1). All compounds failed to inhibit RIP1 kinase when tested directly (data not shown). Taken together, this demonstrates that GSK’840, GSK’843, and GSK’872 bind to RIP3 kinase domain and inhibit enzyme activity with minimal cross-reactivity[1].

GSK-843 (3-10 μM; 18 h) induces apoptosis[1].
GSK-843 (0.3-3 μM; 18 h) inhibits virus- and TNF-induced cell necrosis[2].

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Researchers previously identified the RIP3i GSK’843 and GSK’872 by screening conventional small-molecule libraries (Kaiser et al., 2013a) (Figure 1A). These compounds bound RIP3 kinase domain with high affinity (IC50 = 8.6 nM and 1.8 nM, respectively; Figure 1B) and inhibited kinase activity (IC50 = 6.5 nM and 1.3 nM, respectively; Figure 1C). When assayed individually at 1 μM, the three structurally distinct compounds failed to inhibit most of 300 human protein kinases tested, with GSK’840 showing the best profile (Figure S1B; Table S1). All compounds failed to inhibit RIP1 kinase when tested directly (data not shown). Taken together, this demonstrates that GSK’840, GSK’843, and GSK’872 bind to RIP3 kinase domain and inhibit enzyme activity with minimal cross-reactivity[1].

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GSK'843 inhibits TNF-induced necroptosis in human HT-29 cells in a concentration-dependent manner, with a significant rightward shift in IC50 (100- to 1000-fold) compared to cell-free biochemical assays [1].
GSK'843 blocks necroptosis in mouse cells, including bone marrow-derived macrophages (BMDM), thioglycolate-elicited peritoneal macrophages (PECs), and 3T3SA fibroblasts, at concentrations ranging from 0.04 to 1 μM [1].
GSK'843 inhibits Toll-like receptor 3 (TLR3)-induced necroptosis triggered by poly(I:C) in the presence of the pan-caspase inhibitor zVAD [1].
At high concentrations (3 μM and 10 μM), GSK'843 triggers caspase-dependent apoptotic cell death in various cell types (e.g., SVEC, L929, 3T3SA, MEF), which is reversed by zVAD [1].
This apoptosis is characterized by caspase-3 cleavage, increased effector caspase activity (DEVDase), membrane blebbing, and apoptotic morphology under electron microscopy [1].
The pro-apoptotic activity of GSK'843 requires the presence of RIP3, as demonstrated by reduced sensitivity in RIP3-low NIH3T3 cells, resistance in Rip3/- MEFs, and restoration of sensitivity in Rip3/- MEFs reconstituted with human RIP3 [1].
The apoptosis induced by GSK'843 is independent of the pro-necrotic machinery (MLKL) but dependent on RHIM (RIP homotypic interaction motif) signaling, as human RIP3 with a mutated RHIM fails to confer sensitivity [1].
Treatment with GSK'843 drives the assembly of a RIP1-FADD-cFLIPL-Casp8 complex (Ripoptosome-like complex) in a concentration-dependent manner, particularly stabilized in the presence of zVAD [1].
This apoptosis requires RIP1, FADD, cFLIPL, and Casp8, but not RIP1 kinase activity, as cells from Rip1K45A/K45A kinase-dead knockin mice remain sensitive [1].
The viral inhibitor of RIP activation (vIRA) from MCMV, which blocks RHIM signaling, suppresses GSK'843-induced apoptosis [1].
GSK'843 shows minimal cross-reactivity, failing to inhibit most of 300 tested human protein kinases at 1 μM and showing no direct inhibition of RIP1 kinase [1].

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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The study describes the generation and characterization of Rip3K51A/K51A kinase-dead knockin mice, which are viable and fertile, to demonstrate that RIP3 kinase activity is dispensable for life and to model the effect of a non-toxic kinase inhibitor [1].

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]

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Candidate small molecule inhibitors were identified targeting the purified, baculovirus-expressed recombinant human RIP3 kinase domain (amino acids 2-328) using binding and kinase inhibition assays [1].
Binding affinity (IC50) of compounds to the RIP3 kinase domain was determined using a fluorescence polarization (FP) assay with an ATP-competitive fluorescent probe [1].
Kinase inhibition (IC50) was assessed using an ADP-Glo assay to measure the suppression of recombinant RIP3 kinase activity [1].
Selectivity profiling was performed by testing compounds at 1 μM against a panel of 300 human protein kinases [1].

Cell viability, caspase activity and, microscopy [1]
Cell viability was measured by indirect ATP detection using Cell Titer-Glo Luminescent Cell Viability Assay kit, lactate dehydrogenase release using Cytotoxicity LDH assay kit, and SYTOX Green uptake by IncuCyte. Effector caspase activity was determined by using the Caspase-Glo 3/7 Activity Assay System or Caspase-Glo-8 Assay System respectively. Transmission electron microscopy (TEM) was performed as described before (Tandon and Mocarski, 2008) and images were obtained at Emory Electron Microscopy Core by JEOL JEM-1400 transmission electron microscope.

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Cell Viability Assays[1]
L929 cells (5000 cells/well), BMDM (30,000 cells/well), NIH3T3 (10,000 cells/well), 3T3-SA (10,000 cells/well), and SVEC4-10 (10,000 cells/well) were seeded into Corning 96-well tissue culture plates (3610). In most experiments, cell viability was assessed by measuring the intracellular levels of ATP using the Cell Titer-Glo luminescent cell viability assay kit according to the manufacturer's instructions, with results graphed relative to control cultures.

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For necroptosis inhibition assays, human HT-29 cells or mouse cells (e.g., L929, 3T3SA, BMDM, PECs) were treated with stimuli (e.g., TNFα, SMAC mimetic, zVAD for HT-29; TNFα+zVAD or poly(I:C)+zVAD for mouse cells) in the presence of increasing concentrations of GSK'843. Cell viability was typically assessed 18-24 hours post-treatment by measuring cellular ATP levels [1].
For apoptosis induction assays, various cell lines were treated with high concentrations (3-10 μM) of GSK'843 in the presence or absence of caspase inhibitors (e.g., zVAD). Cell death was monitored by viability assays (ATP levels), microscopy for membrane permeability (e.g., SYTOX Green uptake), flow cytometry for propidium iodide uptake or cleaved caspase-3, and measurement of caspase-3/7 (DEVDase) or caspase-8 (IETDase) enzymatic activity [1].
Biochemical analysis of complex formation involved immunoprecipitation of FADD or FLAG-tagged RIP3 from cell lysates after treatment with GSK'843 +/- zVAD, followed by immunoblotting for components like RIP3, RIP1, FADD, Casp8, and cFLIP [1].
Gene requirement was validated using genetic knockouts (e.g., Rip3/- , Casp8/- , Fadd/- MEFs), kinase-dead mutants (Rip1K45A/K45A), siRNA/shRNA-mediated knockdown (e.g., RIP3, RIP1, Casp8, MLKL), and reconstitution experiments [1].
The role of viral inhibitors was tested by transducing cells with retroviruses expressing MCMV vIRA (M45) or its mutant form (M45mutRHIM) prior to GSK'843 treatment [1].

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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Generation of a Rip3K51A/K51A kinase inactive knockin mice [1]
The knockin strategy was designed and performed by genOway. The Rip3 gene-targeting vector was constructed from genomic C57BL/6 mouse strain DNA. The K51A point mutation was inserted into Rip3 exon 2 while a neomycin resistance gene cassette was inserted in intron 3 (flanked by FRT sites for further Flp-mediated excision). Exon 2 Including the K51A point mutation was flanked by loxP sites enabling access to constitutive or conditional deletion using Cre-mediated recombination.

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This manuscript does not describe specific animal experiments involving the administration of the compound GSK'843. The in vivo conclusions are based on the phenotypic study of genetically engineered Rip3K51A/K51A knockin mice [1].
The generation of Rip3K51A/K51A mice involved standard gene targeting techniques in embryonic stem cells, followed by breeding to obtain homozygous mutants. Mice were used for viability/fertility assessment, cell isolation (MEFs, BMDMs), and infection models with MCMV [1].
For the MCMV infection model, age-matched mice of different genotypes (WT, Rip3/- , Rip3K51A/K51A) were infected intraperitoneally with MCMV-M45mutRHIM virus. Viral titers in spleen and liver were determined by plaque assay 3 days post-infection [1].
For immune response analysis, mice were primed with MCMV-M45mutRHIM and later challenged with a lacZ-expressing MCMV. Splenocytes were harvested 4 days post-challenge and stimulated with M45-specific peptide to measure IFNγ and TNFα production in CD8+ T cells by intracellular cytokine staining [1].

The main toxicity of GSK'843 observed in this study was its concentration-dependent induction of caspase-8-dependent apoptosis in cell cultures at high concentrations (≥3 μM), a targeting effect associated with RIP3 conformational changes and RHIM signaling pathway activation [1].

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

[2]. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem. 2013 Oct 25;288(43):31268-79.

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 can be revealed through therapeutic or genetic manipulation of RIP3. [1]

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Toll-like receptor (TLR) signaling pathways are triggered by pathogen-associated molecular patterns that mediate known cytokine-driven pathways, activating NF-κB and IRF3/IRF7. In addition, TLR3 drives a caspase 8-regulated programmed cell death pathway, similar to the TNF family death receptor signaling pathway. We found that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 leads to receptor-interacting protein (RIP) 3 kinase-dependent programmed necrosis (CNN), a process that induces the interferon-β (TRIF) or MyD88 signaling pathway via adaptor proteins containing TIR domains. TLR3 or TLR4 directly activates CNN via the RIP isotype-interacting motif-dependent binding of TRIF to RIP3 kinase (also known as RIPK3). In fibroblasts, this pathway is independent of RIP1 or its kinase activity but still depends on mixed lineage kinase domain-like proteins (MLKLs) downstream of RIP3 kinase. This paper describes two small-molecule RIP3 kinase inhibitors and uses them to demonstrate the synergistic role of RIP3 kinase in RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complex-induced CNN. Cell fate determination following the TLR signaling pathway parallels the death receptor signaling pathway and depends on caspase 8 to inhibit RIP3-dependent programmed necrosis, whether the necrosis is directly initiated by the TRIF-RIP3-MLKL pathway or indirectly initiated by TNF activation and the RIP1-RIP3-MLKL necroptosis pathway. [2] GSK'843 is a selective small molecule RIP3 kinase inhibitor that was discovered along with GSK'872 from a routine small molecule compound library screening [1]. This study revealed the dual role of RIP3. The kinase activity of the RIP3 protein drives MLKL-dependent necroptosis, while the RIP3 protein itself can also serve as a scaffold for pro-apoptotic complexes. High concentrations of GSK'843 (and other RIP3 inhibitors) can induce conformational changes in RIP3, promote RHIM-dependent oligomerization, and recruit RIP1, FADD, cFLIPL, and Casp8, ultimately leading to kinase-independent apoptosis [1]. This pro-apoptotic activity poses a potential challenge to the development of RIP3 kinase inhibitors as anti-inflammatory therapies, as it may represent an undesirable target toxicity [1]. This study distinguished various RIP3 kinase inactivating mutants. The D161N mutation spontaneously induced apoptosis (mimicking the effect of high-dose inhibitors), while the K51A mutation did not, unless an inhibitor was present. This suggests that loss of kinase activity itself is not toxic, but specific perturbations (certain mutations or high-concentration inhibitor binding) can convert RIP3 into a pro-apoptotic adaptor protein [1]. The survival rate of Rip3K51A/K51A mice demonstrates that eliminating RIP3 kinase activity in vivo is not fatal or impairs immune function, suggesting that non-toxic RIP3 inhibitors are feasible if they can avoid triggering pro-apoptotic conformational changes [1].

C₁₉H₁₅N₅S₂

377.49

377.076

C, 60.46; H, 4.01; N, 18.55; S, 16.99

1601496-05-2

1601496-05-2

91885439

Light yellow to yellow solid powder

1.5±0.1 g/cm3

640.0±55.0 °C at 760 mmHg

340.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.832

4.9

1

6

2

26

522

0

S1C=C(C2C=CC3=C(C=2)N=CS3)C2C(N)=NC=C(C3=CC(C)=NN3C)C1=2

BPKSNNJTKPIZKR-UHFFFAOYSA-N

InChI=1S/C19H15N5S2/c1-10-5-15(24(2)23-10)12-7-21-19(20)17-13(8-25-18(12)17)11-3-4-16-14(6-11)22-9-26-16/h3-9H,1-2H3,(H2,20,21)

3-(1,3-benzothiazol-5-yl)-7-(2,5-dimethylpyrazol-3-yl)thieno[3,2-c]pyridin-4-amine

GSK843; GSK-843; GSK 843; 1601496-05-2; GSK-843; GSK843; GSK'843; 3-(1,3-benzothiazol-5-yl)-7-(1,3-dimethyl-1H-pyrazol-5-yl)thieno[3,2-c]pyridin-4-amine; CHEMBL4441118; 3-(1,3-BENZOTHIAZOL-5-YL)-7-(2,5-DIMETHYLPYRAZOL-3-YL)THIENO[3,2-C]PYRIDIN-4-AMINE; 3-(benzo[d]thiazol-5-yl)-7-(1,3-dimethyl-1H-pyrazol-5-yl)thieno[3,2-c] pyridin-4-amine; . GSK'843

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)

DMSO: ~50 mg/mL (~132.5 mM)

Solubility in Formulation 1: 4.8 mg/mL (12.72 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 48.0 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: ≥ 2.08 mg/mL (5.51 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.)