IRE1 Rnase (IC50 = 76 nM)

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]

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].

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.

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.

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

204.04

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

189-190 ºC

1.62

67.51

CC1=CC(=O)OC2=C1C=CC(=C2C=O)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.)