4μ8C (IRE1 Inhibitor III)

IRE1 Rnase (IC50 = 76 nM)

除了抑制Xbp1剪接和IRE1介导的mRNA降解外，4μ8C还可以阻止底物（RIDD）进入IRE1的活性位点。在没有可检测到的急性毒性的情况下，IRE1抑制随后会导致ER应激。[1]
4μ8C，通过充当IRE1抑制剂阻断CD4+T细胞产生IL-4、IL-5和IL-13的能力。[2]

4μ8c是一种IRE1抑制剂III，可减少小鼠动脉粥样硬化病变并有效预防斑块形成。4μ8c以剂量依赖的方式抑制IgE介导的肥大细胞的脱颗粒（IC50=3.2μM）和肿瘤坏死因子-α（TNF-α）和白细胞介素-4（IL-4）等细胞因子的产生。4μ8C还抑制了小鼠的被动皮肤过敏反应（PCA）（ED50=25.1mg/kg）。在抗原刺激肥大细胞信号通路的实验中，Syk的磷酸化和活化降低了4μ8C，下游信号分子的磷酸化，如活化T细胞接头（LAT）、Akt和三种MAP激酶ERK、p38和JNK的磷酸化受到抑制。机制研究表明，4μ8C在体外抑制Lyn和Fyn的活性。基于这些实验的结果，4μ8C的过敏反应抑制机制涉及Lyn和Fyn活性的降低，这在IgE介导的信号通路中至关重要。总之，本研究首次表明，4μ8C抑制Lyn和Fyn，从而通过减少脱颗粒和炎性细胞因子的产生来抑制过敏反应。这表明4μ8C可以作为一种新的候选药物来控制季节性过敏和特应性皮炎等过敏性疾病[4]。

除了使用哺乳动物IRE1反应缓冲液外，遵循与之前相同的程序来分析放射性标记的Xbp1底物切割。体外RIDD底物是在32P ATP或Cy5 UTP存在下，使用T7 MAXIscript试剂盒在通过RT-PCR从小鼠Min6细胞（Ins2）分离的模板上或通过PCR从克隆的XBP1 cDNA上进行体外转录而产生的。为了获得全长底物，将生产的产品进行凝胶纯化。接下来，在使用LI-COR Odyssey扫描仪进行磷化或近红外成像分析之前，用15%尿素聚丙烯酰胺凝胶对反应进行分离。

在96或24孔培养皿中，细胞以每孔5×103或5×104的密度接种在无酚红细胞培养基中。在暴露于48℃24小时之前，将培养物孵育16小时。然后添加200 M WST1和10 M吩嗪硫酸甲酯来分析培养物。在37°C下试剂显影2小时后，减去背景和595nm处的吸光度，通过450nm处的吸光度检测水解染料。作为替代方案，可以用结晶紫对贴壁培养物进行染色，以确定细胞的存活率。在水中彻底洗涤染色细胞并将结晶紫溶解在甲醇中后，使用595nm处的吸光度测量来量化染料吸收。

C57BL/6 mice
10 mg/kg
i.p.

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Mice and Treatments. ApoE−/− mice in a C57BL/6 background (Charles River WIGA GmbH) were used in atherosclerosis experiments. Starting from 8 weeks of age, male mice were fed a Western diet (TD88137 mod. containing 21% fat and 0.2% cholesterol; Ssniff) for 6 weeks. Then, the mice were injected with STF-083010 (10 mg/kg) or DMSO, both given in 16% (vol/vol) Cremophor EL saline solution via i.p. injections as described previously, for 6 more weeks while mice were continued on the Western diet. The other ApoE−/− mice that were used in atherosclerosis experiments were fed a Western diet for 8 weeks. Then, they were injected with 4µ8c (10 mg/kg) or DMSO, both given in 16% (vol/vol) Cremophor EL saline solution via i.p. injections as described previously, for 4 more weeks while mice were continued on Western diet. Weights were measured every other day, whereas blood glucose concentrations were measured before and after treatments. At the end of the experiment, mice were anesthetized, and blood was collected by cardiac puncture. Bone marrow, spleen, and liver tissues were collected, frozen immediately into liquid nitrogen, and stored at −80 °C. Perfusion was performed with ice-cold PBS and heparin (1,000 U/mL) followed by 10% formalin solution. After fixation, the aorta was dissected intact, immersed immediately in 10% formalin, and stored at 4 °C until analysis. The heart was removed at the proximal aorta, placed into a tissue mold, covered with OCT (optimal cutting temperature compound), frozen in cold isobutene solution, and stored at −80 °C. [3]

[1]. Proc Natl Acad Sci U S A . 2012 Apr 10;109(15):E869-78.

[2]. J Biol Chem . 2013 Nov 15;288(46):33272-82.

[3]. Proc Natl Acad Sci U S A . 2017 Feb 21;114(8):E1395-E1404.

[4]. Toxicol Appl Pharmacol. 2017 Oct 1:332:25-31.

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.[1]

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Metaflammation, an atypical, metabolically induced, chronic low-grade inflammation, plays an important role in the development of obesity, diabetes, and atherosclerosis. An important primer for metaflammation is the persistent metabolic overloading of the endoplasmic reticulum (ER), leading to its functional impairment. Activation of the unfolded protein response (UPR), a homeostatic regulatory network that responds to ER stress, is a hallmark of all stages of atherosclerotic plaque formation. The most conserved ER-resident UPR regulator, the kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), is activated in lipid-laden macrophages that infiltrate the atherosclerotic lesions. Using RNA sequencing in macrophages, we discovered that IRE1 regulates the expression of many proatherogenic genes, including several important cytokines and chemokines. We show that IRE1 inhibitors uncouple lipid-induced ER stress from inflammasome activation in both mouse and human macrophages. In vivo, these IRE1 inhibitors led to a significant decrease in hyperlipidemia-induced IL-1β and IL-18 production, lowered T-helper type-1 immune responses, and reduced atherosclerotic plaque size without altering the plasma lipid profiles in apolipoprotein E-deficient mice. These results show that pharmacologic modulation of IRE1 counteracts metaflammation and alleviates atherosclerosis.[3]

C11H8O4

204.18

204.042

C, 64.71; H, 3.95; O, 31.34

14003-96-4

12934390

Light yellow to yellow solid powder

1.406±0.06 g/cm3 (20 ºC 760 Torr)

189-190 ºC (ethanol )

1.619

67.51

1

4

1

15

321

0

O1C(C([H])=C(C([H])([H])[H])C2C([H])=C([H])C(=C(C([H])=O)C1=2)O[H])=O

RTHHSXOVIJWFQP-UHFFFAOYSA-N

InChI=1S/C11H8O4/c1-6-4-10(14)15-11-7(6)2-3-9(13)8(11)5-12/h2-5,13H,1H3

7-hydroxy-4-methyl-2-oxochromene-8-carbaldehyde

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (10.19 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 0.5mg/mL

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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。