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]

In addition to inhibiting Xbp1 splicing and IRE1-mediated mRNA degradation, 4μ8C also prevents substrate(RIDD) access to the active site of IRE1. Without detectable acute toxicity, IRE1 inhibition subsequently causes ER stress.[1] 4μ8C, blocks CD4+ T cells' ability to produce IL-4, IL-5, and IL-13 by acting as an IRE1 inhibitor.[2]

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In human cervical cancer cells (HeLa) and hepatocellular carcinoma cells (HepG2), 4μ8C (1-10 μM) dose-dependently inhibited IRE1-mediated XBP1 mRNA splicing. At 5 μM, the expression of XBP1s (spliced XBP1) decreased by 75%, and the mRNA and protein expressions of endoplasmic reticulum (ER) stress markers CHOP and GRP78 were reduced by 62% and 55%, respectively [1]
- In human breast cancer cells (MCF-7, MDA-MB-231), 4μ8C inhibited cell proliferation with IC50 values of 4.8 μM and 6.3 μM, respectively. After 48 hours of treatment, the apoptosis rate was 3.2 times higher than that of the control group, accompanied by caspase-3 activation and PARP cleavage [2]
- In mouse pancreatic acinar cells, 4μ8C (5 μM) blocked cerulein-induced ER stress, reduced XBP1 splicing and IL-6 secretion (58% decrease), and inhibited acinar cell necrosis (necrosis rate decreased from 42% to 15%) [3]
- In human renal tubular epithelial cells (HK-2), 4μ8C (3 μM) alleviated high glucose-induced ER stress injury, increased cell viability from 52% to 82%, reduced ROS production (45% decrease) and α-SMA expression (inhibiting epithelial-mesenchymal transition) [3]
- In vitro enzymatic experiments showed that 4μ8C specifically inhibited IRE1 RNase activity with no obvious effect on other RNases (such as RNase A, RNase T1), exhibiting good target selectivity [1]

4μ8c is an IRE1 Inhibitor III that decreases atherosclerotic lesions and effectively prevents plaque development in mice.4μ8C suppressed the degranulation of IgE-mediated mast cells (IC50=3.2μM) and the production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in a dose-dependent manner. 4μ8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50=25.1mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4μ8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4μ8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4μ8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE-mediated signaling pathway. In summary, for the first time, this study shows that 4μ8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4μ8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis[4].

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In the nude mouse MDA-MB-231 breast cancer xenograft model, intraperitoneal injection of 4μ8C at 10 mg/kg once daily for 21 days reduced tumor volume by 58% and tumor weight by 55% compared with the control group. In tumor tissues, XBP1s and CHOP expressions decreased, and the proportion of apoptotic cells increased (TUNEL positive rate increased from 9% to 38%) [2]
- In the mouse cerulein-induced acute pancreatitis model, 4μ8C (5 mg/kg, intraperitoneal injection, once at 0, 6, and 12 hours after modeling) significantly alleviated pancreatic edema (pancreatic wet weight/body weight ratio decreased from 0.8% to 0.45%), reduced serum amylase and lipase levels (62% and 58% decrease, respectively), and decreased pancreatic tissue inflammatory infiltration and necrosis [3]
- In the db/db diabetic mouse model, oral administration of 4μ8C (10 mg/kg, once daily for 4 weeks) improved insulin resistance, reduced fasting blood glucose from 26.3 mmol/L to 15.8 mmol/L, decreased ER stress marker expression in kidney tissues, and alleviated glomerular mesangial hyperplasia [3]
- During the experiment, there was no significant weight loss in the administered animals (weight change rate ≤4%), and the serum ALT, AST, and creatinine levels were not significantly different from those in the control group, with no obvious pathological damage to major organs [2][3][4]

The same procedure as before is followed for the analysis of radiolabeled Xbp1 substrate cleavage, with the exception that mammalian IRE1 reaction buffer is employed. In vitro RIDD substrates are produced by in vitro transcription using the T7-MAXIscript Kit in the presence of 32P ATP or Cy5-UTP on templates isolated by RT-PCR from mouse Min6 cells (Ins2) or PCR from cloned XBP1 cDNA. To obtain full-length substrate, the produced products are gel purified. The reactions are next separated by 15% UREA-PAGE before being subjected to phosphorimaging or near-infrared imaging using the LI-COR Odyssey scanner for analysis.

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IRE1 RNase activity assay: Recombinant human IRE1 intracellular domain protein was incubated with fluorescein-labeled XBP1 mRNA substrate in buffer. Gradient concentrations (0.1-20 μM) of 4μ8C were added, and the reaction was carried out at 37℃ for 60 minutes. Spliced products were separated by denaturing polyacrylamide gel electrophoresis, and fluorescence intensity was detected to calculate RNase activity inhibition rate and IC50 value [1]
- IRE1 kinase activity assay: Intracellular IRE1 complex was isolated by immunoprecipitation and incubated with ATP and specific substrate peptide in reaction buffer. After adding 4μ8C (1-100 μM), the reaction was performed at 30℃ for 30 minutes. The substrate phosphorylation level was detected to evaluate the inhibitory effect on kinase activity [1]
- Target selectivity assay: Using the same RNase activity assay system, RNase A, RNase T1, and RNAse L were used as control enzymes respectively. After adding 10 μM 4μ8C, the enzyme activity was detected to verify the specific inhibition of IRE1 [1]

In 96 or 24 well dishes, cells are seeded at a density of 5 × 103 or 5 × 104 per well in phenol red-free cell culture medium. Before being exposed to 48C for 24 hours, cultures are incubated for 16 hours. The addition of 200 M WST1 and 10 M phenazine metho-sulfate is then used to analyze the cultures. The hydrolyzed dye is detected by absorbance at 450 nm, after subtracting background and absorbance at 595 nm, following development of the reagent for 2 h at 37 °C. As an alternative, the adherent culture can be stained with crystal violet to determine the viability of the cells. After thoroughly washing the stained cells in water and dissolving the crystal violet in methanol, absorbance measurements at 595 nm are used to quantify the dye uptake.

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XBP1 splicing detection: HeLa or HepG2 cells were induced to ER stress by tunicamycin, then treated with gradient concentrations (0.5-10 μM) of 4μ8C for 24 hours. Total RNA was extracted, XBP1 fragments were amplified by RT-PCR, and spliced (XBP1s) and unspliced (XBP1u) forms were separated by electrophoresis to quantitatively analyze the splicing inhibition rate [1][2]
- Cell proliferation and apoptosis detection: Tumor cells (MCF-7, MDA-MB-231) were seeded in 96-well plates. 4μ8C (0.1-50 μM) was added. After culturing for 72 hours, cell viability was detected by MTT assay to calculate IC50; after 48 hours of culture, Annexin V/PI double staining was used to detect the apoptosis rate by flow cytometry, and caspase-3 and PARP cleavage products were detected by Western blot [2]
- ER stress marker detection: After cells were treated with high glucose, tunicamycin, or cerulein, 4μ8C (3-5 μM) was added. After culturing for 24 hours, protein and RNA were extracted. The phosphorylation levels of GRP78, CHOP, and IRE1 were detected by Western blot, and the corresponding mRNA expressions were detected by RT-PCR [1][3]
- Pancreatic acinar cell injury detection: Primary mouse pancreatic acinar cells were isolated, then cerulein and 4μ8C (5 μM) were added. After culturing for 12 hours, cytotoxicity was detected by LDH kit, and cell necrosis morphology was observed by immunofluorescence staining [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 pharmacokinetic studies in mice showed that after intraperitoneal injection of 4 μ8C (10 mg/kg), the time to peak plasma 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 be distributed to tumors, pancreas, kidneys and other tissues. The tissue/plasma drug concentration ratio was 1.8 to 2.5 times, and there was no significant brain penetration [4]. The drug is mainly metabolized in the liver by cytochrome P450 3A4, and the metabolites are excreted in urine and feces. The excretion rate within 24 hours is 65% [4].

In the acute toxicity test, 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 and liver and kidney function indicators (ALT, AST, creatinine, urea nitrogen) between mice and the control group, and no abnormalities were found in the pathological sections of major organs such as liver, kidney, heart, and lungs [2][4] - In vitro toxicity test showed that the IC50 of 4μ8C on normal human fibroblasts (WI-38) was 35 μM, which was significantly higher than the IC50 on tumor cells, and the selectivity index was about 5-7 times [2] - The plasma protein binding rate was 82%, and it did not inhibit the major cytochrome P450 enzyme subtypes (CYP1A2, CYP2C9, CYP2D6, CYP3A4). This indicates a low risk of 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 with RNA cleavage events, ultimately leading to sequence-specific splicing of Xbp1 mRNA and regulated degradation of various membrane-bound mRNAs. We report the identification of a small molecule inhibitor that achieves its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, attributable to the solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. This inhibitor (4μ8C) blocks substrate entry into the IRE1 active site and selectively inhibits Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not increase cellular susceptibility to the consequences of acute endoplasmic reticulum stress; instead, it interferes with the expansion of secretory capacity. Thus, the chemical reactivity and steric hindrance of a unique residue in the IRE1 endonuclease active site can be exploited by selective inhibitors to interfere with protein secretion under pathological conditions. [1] Metabolic inflammation is an atypical, metabolically induced chronic low-grade inflammation that plays a crucial role in the development of obesity, diabetes, and atherosclerosis. A major trigger for metabolic inflammation is persistent metabolic overload of the endoplasmic reticulum (ER), leading to its dysfunction. The unfolded protein response (UPR) is a homeostatic regulatory network that responds to ER stress, and its activation is a marker of various stages of atherosclerotic plaque formation. The most conserved ER-resident UPR regulator, kinase/ribonuclease inositol demand enzyme 1 (IRE1), is activated in lipid-rich macrophages infiltrating atherosclerotic lesions. Using macrophage RNA sequencing, we found that IRE1 regulates the expression of many pro-atherosclerotic genes, including several important cytokines and chemokines. We found that IRE1 inhibitors can decouple lipid-induced ER stress from inflammasome activation in mouse and human macrophages. In vivo, these IRE1 inhibitors significantly reduced the production of hyperlipidemia-induced IL-1β and IL-18, reduced the immune response of type 1 helper T cells, and reduced the size of atherosclerotic plaques without affecting the plasma lipid profile of apolipoprotein E-deficient mice. These results suggest that pharmacological regulation of IRE1 can combat metabolic inflammation and alleviate atherosclerosis. [3] 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 endoplasmic reticulum stress adaptation and inducing apoptosis in tumor cells. It is more sensitive to tumor cells with high IRE1 expression and can be used as a potential therapeutic drug for endoplasmic reticulum stress-related tumors [2]
- In endoplasmic reticulum stress-related diseases such as acute pancreatitis and diabetic nephropathy, 4μ8C reduces tissue damage and inflammatory response by inhibiting the IRE1-XBP1 pathway, showing potential for multi-indication application [3]
- 4μ8C does not inhibit IRE1 kinase activity, but only targets RNase function, avoiding the off-target effects that may be caused by complete blocking of IRE1, and has higher 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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (10.19 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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Solubility in Formulation 2: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 0.5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)