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].

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).

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]

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.

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.

[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 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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Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-β (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.[2]

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.)