4μ8C (IRE1 Inhibitor III)

IRE1 Rnase (IC50 = 76 nM)
Inositol-requiring enzyme 1 (IRE1, ERN1) (IC50=1.2 μM, inhibiting IRE1 RNase activity; no obvious inhibition on IRE1 kinase activity, Ki>100 μM) [1][2]

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]

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人宫颈癌细胞（HeLa）、肝癌细胞（HepG2）中，4μ8C（1-10 μM）可剂量依赖性抑制IRE1介导的XBP1 mRNA剪接，5 μM浓度时XBP1s（剪接型XBP1）表达降低75%，同时减少内质网应激标志物CHOP、GRP78的mRNA及蛋白表达（分别降低62%和55%）[1]
- 人乳腺癌细胞（MCF-7、MDA-MB-231）中，4μ8C 可抑制细胞增殖，IC50值分别为4.8 μM和6.3 μM，处理48小时后凋亡率较对照组升高3.2倍，伴随caspase-3激活和PARP剪切[2]
- 小鼠胰腺腺泡细胞中，4μ8C（5 μM）可阻断雨蛙素诱导的内质网应激，减少XBP1剪接和IL-6分泌（降低58%），抑制腺泡细胞坏死（坏死率从42%降至15%）[3]
- 人肾小管上皮细胞（HK-2）中，4μ8C（3 μM）可减轻高糖诱导的内质网应激损伤，细胞存活率从52%升至82%，减少ROS生成（降低45%）和α-SMA表达（抑制上皮间质转化）[3]
- 体外酶学实验显示，4μ8C 对IRE1 RNase活性的抑制具有特异性，对其他RNase（如RNase A、RNase T1）无明显抑制作用，靶点选择性良好[1]

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]。

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裸鼠MDA-MB-231乳腺癌异种移植模型中，4μ8C 以10 mg/kg剂量腹腔注射，每日一次，连续21天，肿瘤体积较对照组缩小58%，肿瘤重量减轻55%，肿瘤组织中XBP1s、CHOP表达降低，凋亡细胞比例升高（TUNEL阳性率从9%升至38%）[2]
- 小鼠雨蛙素诱导急性胰腺炎模型中，4μ8C（5 mg/kg，腹腔注射，造模后0、6、12小时各一次）可显著减轻胰腺水肿（胰腺湿重/体重比从0.8%降至0.45%），降低血清淀粉酶、脂肪酶水平（分别降低62%和58%），减少胰腺组织炎症浸润和坏死[3]
- db/db糖尿病小鼠模型中，4μ8C（10 mg/kg，口服，每日一次，连续4周）可改善胰岛素抵抗，空腹血糖从26.3 mmol/L降至15.8 mmol/L，减少肾脏组织内质网应激标志物表达，减轻肾小球系膜增生[3]
- 实验期间，给药动物体重无明显下降（体重变化率≤4%），血清ALT、AST、肌酐水平与对照组无显著差异，主要器官无明显病理损伤[2][3][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。

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IRE1 RNase活性测定：重组人IRE1胞内域蛋白与荧光标记的XBP1 mRNA底物在反应缓冲液中孵育，加入梯度浓度（0.1-20 μM）的4μ8C，37℃反应60分钟后，通过变性聚丙烯酰胺凝胶电泳分离剪接产物，检测荧光强度，计算RNase活性抑制率及IC50值[1]
- IRE1激酶活性测定：免疫沉淀法分离细胞内IRE1复合物，与ATP和特异性底物肽在反应缓冲液中孵育，加入4μ8C（1-100 μM）后30℃反应30分钟，检测底物磷酸化水平，评估激酶活性抑制效果[1]
- 靶点选择性检测：采用相同RNase活性测定体系，分别以RNase A、RNase T1、RNAse L为对照酶，加入10 μM 4μ8C 后检测酶活性，验证对IRE1的特异性抑制[1]

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。

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XBP1剪接检测：HeLa或HepG2细胞经衣霉素（Tunicamycin）诱导内质网应激后，加入梯度浓度（0.5-10 μM）的4μ8C，培养24小时后提取总RNA，RT-PCR扩增XBP1片段，电泳分离剪接型（XBP1s）和未剪接型（XBP1u），定量分析剪接抑制率[1][2]
- 细胞增殖与凋亡检测：肿瘤细胞（MCF-7、MDA-MB-231）接种于96孔板，加入4μ8C（0.1-50 μM），培养72小时后MTT法检测细胞活力并计算IC50；培养48小时后Annexin V/PI双染，流式细胞仪检测凋亡率，Western blot检测caspase-3、PARP剪切体[2]
- 内质网应激标志物检测：细胞经高糖、衣霉素或雨蛙素处理后，加入4μ8C（3-5 μM），培养24小时后提取蛋白和RNA，Western blot检测GRP78、CHOP、IRE1磷酸化水平，RT-PCR检测对应mRNA表达[1][3]
- 胰腺腺泡细胞损伤检测：小鼠原代胰腺腺泡细胞分离后，加入雨蛙素和4μ8C（5 μM），培养12小时后，LDH试剂盒检测细胞毒性，免疫荧光染色观察细胞坏死形态[3]

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]

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Breast cancer xenograft model experiment: 6-8 week-old nude mice were subcutaneously inoculated with MDA-MB-231 cells (5×10^6 cells/mouse) on the right back. Seven days after inoculation, mice were randomly divided into a control group and a treatment group (8 mice per group). 4μ8C was dissolved in 5% DMSO + 95% normal saline. The treatment group was given intraperitoneal injection at 10 mg/kg once daily for 21 consecutive days; the control group was given an equal volume of vehicle. Tumor volume was measured every 3 days. After the experiment, tumors were stripped to detect XBP1s, CHOP expressions, and apoptosis [2]
- Acute pancreatitis model experiment: 8-week-old C57BL/6 mice were randomly grouped. The model group and treatment group were intraperitoneally injected with cerulein (50 μg/kg, once per hour for 7 times) to establish the model. At 0, 6, and 12 hours after modeling, the treatment group was intraperitoneally injected with 4μ8C (5 mg/kg); the control group was given an equal volume of normal saline. Mice were sacrificed 24 hours after modeling, and serum and pancreatic tissues were collected to detect amylase, lipase, and pathological damage [3]
- Diabetic nephropathy model experiment: 12-week-old db/db mice were randomly divided into a control group and a treatment group (10 mice per group). 4μ8C was dissolved in 0.5% sodium carboxymethylcellulose. The treatment group was given oral administration at 10 mg/kg once daily for 4 consecutive weeks; the control group was given an equal volume of vehicle. Fasting blood glucose was monitored weekly. After the experiment, kidney tissues were collected for pathological analysis and ER stress marker detection [3]
- Acute toxicity experiment: ICR mice were randomly divided into 5 groups (6 mice per group). Different doses of 4μ8C (25, 50, 100, 200, 400 mg/kg) were injected intraperitoneally once. The survival status, weight change, and behavioral performance of mice were observed within 14 days, and serum biochemical indicators and organ pathological sections were detected [4]

In vivo pharmacokinetics in mice showed that after intraperitoneal injection of 4μ8C (10 mg/kg), the time to peak plasma drug concentration (Tmax) was 1 hour, the peak concentration (Cmax) was 8.5 μM, and the elimination half-life (t1/2) was 3.2 hours [4]
- The oral bioavailability (20 mg/kg) was 28%, and the drug could distribute to tissues such as tumors, pancreas, and kidneys with a tissue/plasma drug concentration ratio of 1.8-2.5 times, without obvious brain penetration [4]
- The drug was mainly metabolized by cytochrome P450 3A4 in the liver, and metabolites were excreted through urine and feces with an excretion rate of 65% within 24 hours [4]

In acute toxicity experiments, the median lethal dose (LD50) of 4μ8C after a single intraperitoneal injection in mice was 285 mg/kg. No death was observed at doses ≤100 mg/kg, and no obvious toxic symptoms were found [4]
- After long-term administration (10 mg/kg, intraperitoneal injection for 28 consecutive days), there were no significant differences in blood routine, liver and kidney function indicators (ALT, AST, creatinine, urea nitrogen) between mice and the control group, and no abnormalities were found in pathological sections of major organs such as liver, kidney, heart, and lung [2][4]
- In vitro toxicity showed that the IC50 of 4μ8C to normal human fibroblasts (WI-38) was 35 μM, significantly higher than that of tumor cells, with a selectivity index of about 5-7 times [2]
- The plasma protein binding rate was 82%, and it did not inhibit major cytochrome P450 enzyme subtypes (CYP1A2, CYP2C9, CYP2D6, CYP3A4), indicating low risk of drug-drug interactions [4]

[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]

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4μ8C is the first specific IRE1 RNase inhibitor. It binds to the RNase domain of IRE1, blocks IRE1-mediated XBP1 mRNA splicing, and inhibits the IRE1 branch of the unfolded protein response (UPR), thereby alleviating endoplasmic reticulum stress [1][2]
- Its antitumor mechanism is related to blocking the ER stress adaptation of tumor cells and inducing apoptosis. It is more sensitive to tumor cells with high IRE1 expression, and can be used as a potential therapeutic drug for ER stress-related tumors [2]
- In ER stress-related diseases such as acute pancreatitis and diabetic nephropathy, 4μ8C alleviates tissue damage and inflammatory response by inhibiting the IRE1-XBP1 pathway, showing potential for multi-indication applications [3]
- 4μ8C does not inhibit IRE1 kinase activity and only targets RNase function, avoiding off-target effects that may be caused by comprehensive IRE1 blockade, with better safety [1]

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。