GSK840

RIP3 or RIPK3 (receptor-interacting protein kinase 3) (IC50 = 0.3 nM)
GSK840 targets receptor-interacting serine/threonine-protein kinase 3 (RIP3) (kinase activity IC50 = 1.3 nM; binding Ki = 0.9 nM) [1]

GSK840 (GSK'840)（0.01-3 μM；24 小时）以浓度依赖性方式抑制 TNF 诱导的坏死性凋亡 [1]。 GSK840 比 RIP1 激酶抑制剂具有更广泛的靶点范围，并且在与激酶结构域结合后抑制激酶活性具有良好的特异性。

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]

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1. Radiometric RIP3 kinase activity assay:
- Recombinant human RIP3 kinase domain is diluted in kinase buffer to a final concentration of 50 nM [1]
- GSK840 is serially diluted (0.1 nM to 100 nM) and mixed with RIP3, followed by incubation at room temperature for 30 minutes [1]
- ATP (50 μM, including [γ-32P]ATP) and recombinant MLKL (substrate, 1 μg) are added to initiate the reaction, which is incubated at 30°C for 60 minutes [1]
- The reaction is terminated by adding SDS sample buffer, and proteins are separated by SDS-PAGE [1]
- Phosphorylated MLKL is detected by autoradiography, and IC50 is calculated by quantifying the reduction in radioactive signal [1]
2. Surface Plasmon Resonance (SPR) binding assay:
- RIP3 kinase domain is immobilized on a CM5 sensor chip via amine coupling to a density of ~700 resonance units (RU) [1]
- GSK840 is serially diluted (0.2 nM to 20 nM) in running buffer (PBS with 0.05% Tween-20) and injected over the chip at a flow rate of 25 μl/min [1]
- Association (180 seconds) and dissociation (300 seconds) phases are monitored, and the chip is regenerated with 10 mM glycine-HCl (pH 2.2) [1]
- Binding affinity (Ki) is calculated using a 1:1 Langmuir binding model with reference subtraction [1]

细胞活力测定 [1]
细胞类型：人 HT-29 细胞（TNF 10 ng/ml + zVAD-fmk 20 μM + SMAC007 100 nM）
测试浓度: 0.01-3 μM
孵育时间： 24 小时
实验结果： 阻断 TNF 诱导的坏死细胞凋亡浓度依赖性方式。

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).

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

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]

C21H23N3O3

365.425625085831

365.173

C, 69.02; H, 6.34; N, 11.50; O, 13.13

2361146-30-5

138377545

Light yellow to yellow solid powder

3.1

73.2

1

4

6

27

537

0

O(C(CC1C=CC(=CC=1)N1C=NC2C=C(C(NC)=O)C=CC1=2)=O)C(C)(C)C

UGTLDBJIOSYXRR-UHFFFAOYSA-N

InChI=1S/C21H23N3O3/c1-21(2,3)27-19(25)11-14-5-8-16(9-6-14)24-13-23-17-12-15(20(26)22-4)7-10-18(17)24/h5-10,12-13H,11H2,1-4H3,(H,22,26)

tert-butyl 2-[4-[5-(methylcarbamoyl)benzimidazol-1-yl]phenyl]acetate

GSK840; 2361146-30-5; CHEMBL4439913; tert-butyl 2-(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)phenyl)acetate; tert-butyl 2-[4-[5-(methylcarbamoyl)benzimidazol-1-yl]phenyl]acetate; tert-butyl 2-{4-[5-(methylcarbamoyl)-1H-1,3-benzodiazol-1-yl]phenyl}acetate; SCHEMBL21678866;

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 : ~110 mg/mL (~301.02 mM)

配方 1 中的溶解度: ≥ 2.75 mg/mL (7.53 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 27.5 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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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配方 2 中的溶解度: ≥ 2.75 mg/mL (7.53 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 27.5mg/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 3 中的溶解度: ≥ 2.75 mg/mL (7.53 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 27.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。