GSK-843

RIPK3

GSK-843（3-10 μM；18 小时）诱导细胞凋亡[1]。 GSK-843（0.3-3 μM；18 小时）抑制病毒和 TNF 诱导的细胞坏死[2]。

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研究人员之前通过筛选常规小分子文库确定了RIP3i GSK ' 843和GSK ' 872 (Kaiser et al., 2013a)（图1A）。这些化合物以高亲和力结合RIP3激酶结构域（IC50分别为8.6 nM和1.8 nM）；图1B)和抑制的激酶活性(IC50分别为6.5 nM和1.3 nM；图1 c)。当在1 μM下单独检测时，这三种结构不同的化合物对300种人蛋白激酶的大部分都没有抑制作用，其中GSK 840表现出最好的抑制效果(图S1B；表S1)。当直接测试时，所有化合物都不能抑制RIP1激酶（数据未显示）。综上所述，这表明GSK ' 840、GSK ' 843和GSK ' 872结合到RIP3激酶结构域，并以最小的交叉反应性抑制酶活性[1]。

Rip3K51A/K51A激酶死亡敲入小鼠存活[1]
RIP3激酶死亡突变体的行为支持在D161N突变敲入小鼠中观察到的惊人的妊娠中期致命性（Newton等人，2014），并预测无毒突变体将出现相反的结果。当产生Rip3K51A/K51A激酶死亡敲入小鼠时，小鼠明显具有活力和生育能力（图7A和7B）。该突变株对妊娠中期或围产期死亡没有表现出任何易感性。为了确定存活的Rip3K51A/K51A突变体是否像致死性Rip3D161N/D161N突变体（Newton et al., 2014）一样，挽救了Casp8−/−胚胎的胚胎致死性，我们进行了杂交，并以预期的孟德尔频率挽救了存活和可育的Casp8−/−Rip3K51A/K51A小鼠（图7B和S6A）。这扩展了先前对Casp8−/−Rip3−/−小鼠的拯救(Kaiser et al., 2011；Oberst et al., 2011；Zhang et al., 2011)清楚地表明，在没有Rip3D161N/D161N突变并发症的casp8缺陷胚胎妊娠中期死亡中，早衰RIP3酶活性的贡献（Newton et al., 2014）。

RIP3高通量筛选荧光极化（FP）试验用于筛选与RIP3激酶结构域荧光标记探针（GSK ' 657）结合竞争的小分子化合物文库（Pope et al., 1999）。文库化合物抑制RIP3激酶活性的能力是通过使用ADP-Glo测量ATP消耗的实验来评估的（Li et al., 2009）。编码库技术筛选按照前面描述进行（Deng et al., 2012）。kinome面板的体外分析由Reaction Biology Corporation使用“HotSpot”分析平台进行（Anastassiadis等，2011）。Kinome树表示是使用Kinome Mapper生成的。[1]

细胞活力、caspase活性和显微观察[1]
使用Cell Titer-Glo发光细胞活力测定试剂盒间接检测ATP，使用细胞毒性LDH测定试剂盒检测乳酸脱氢酶释放，使用IncuCyte对SYTOX Green的摄取。利用caspase - glo 3/7活性测定系统和caspase - glo 8活性测定系统分别测定效应物caspase活性。如前所述（Tandon和Mocarski， 2008）进行透射电子显微镜（TEM），并在Emory electron microscopy Core使用JEOL JEM-1400透射电子显微镜获得图像。

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细胞活力测定[1]
将L929细胞（5000细胞/孔）、BMDM细胞（30000细胞/孔）、NIH3T3细胞（10000细胞/孔）、3T3-SA细胞（10000细胞/孔）、SVEC4-10细胞（10000细胞/孔）分别接种到康宁96孔组织培养板（3610）中。在大多数实验中，根据制造商的说明，使用cell Titer-Glo发光细胞活力测定试剂盒通过测量细胞内ATP水平来评估细胞活力，并将结果与对照培养物相比较。

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

126

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)

配方 1 中的溶解度: 4.8 mg/mL (12.72 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 48.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.51 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。