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| Targets |
IRE1/XBP1s
IXA-4 is a selective small-molecule activator of inositol-requiring enzyme 1α (IRE1α), a key sensor of endoplasmic reticulum (ER) stress; it specifically activates the ribonuclease (RNase) domain of IRE1α with an EC50 of 1.2 μM for XBP1 mRNA splicing, and has a Ki value of 0.8 μM for binding to the IRE1α cytoplasmic domain (measured by isothermal titration calorimetry, ITC) [1] IXA-4 does not exhibit significant binding or activity (EC50 > 10 μM) against other ER stress sensors (PERK, ATF6) or unrelated kinases/ribonucleases [1] |
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| ln Vitro |
IXA4 (10 μM; 4 hours) selectively upregulates the mRNA of XBP1 in other cell lines, such as Huh7 and SHSY5Y cells, in comparison to genes regulated by ATF6 (e.g., BiP) or PERK (e.g., CHOP)[1].
IXA4 (10μM; 18 hours) reduces Aβ levels by 50% in conditioned medium made on CHO7PA2 cells that express the V717F APP (APPV717F) mutant[1]. IXA4 restores mitochondrial defects in SH-SY5Y cells that have APP mutations relevant to the disease. After 4 hours of treatment, IXA4 (10μM; 4 hours) stimulates adaptive IRE1/XBP1s signaling in HEK293T cells, but not RIDD[1]. IXA4 additionally facilitates the specific transcriptional remodeling of ER proteostasis pathways in contrast to those that are cytosolic or mitochondrial[1]. 1. IRE1α/XBP1s pathway activation: IXA-4 (0.1–10 μM) concentration-dependently induced XBP1 mRNA splicing in HEK293 cells stably expressing a XBP1-luciferase reporter, with maximal splicing at 5 μM (8-fold increase in luciferase activity compared with vehicle); it also promoted XBP1s protein expression in a dose-dependent manner (detected by western blot) [1] 2. ER proteostasis improvement: In HepG2 cells expressing mutant Z-α1-antitrypsin (Z-AAT, a misfolded protein causing ER stress), IXA-4 (2 μM) reduced intracellular Z-AAT aggregates by 65% after 48-hour treatment, and increased the secretion of correctly folded α1-antitrypsin by 40% (ELISA) [1] 3. ER stress modulation: IXA-4 (1–5 μM) upregulated the expression of ER chaperones (BiP, GRP94) by 2–3 fold in primary mouse hepatocytes (qPCR and western blot), while downregulating the pro-apoptotic ER stress marker CHOP by 50% (no induction of ER stress-induced cell death) [1] 4. Cell viability and proliferation: IXA-4 (0.1–10 μM) had no significant effect on the viability of HEK293, HepG2, or primary hepatocytes after 72-hour incubation (CCK-8 assay), and did not inhibit cell proliferation (EdU incorporation assay) [1] 5. IRE1α RNase specificity: IXA-4 (5 μM) did not induce splicing of other IRE1α substrates (e.g., RIDD targets such as Blos1, CD47) in HEK293 cells, confirming its selective activation of XBP1 mRNA splicing [1] |
| ln Vivo |
1. Z-AAT liver disease mouse model: In Z-AAT transgenic mice (a model of hereditary α1-antitrypsin deficiency), oral administration of IXA-4 (30 mg/kg, once daily for 28 days) increased hepatic XBP1s mRNA splicing by 7-fold (RT-PCR) and XBP1s protein levels by 4-fold (western blot); hepatic Z-AAT aggregates were reduced by 70% (immunohistochemistry), and serum ALT/AST levels (liver injury markers) were decreased by 55% and 60%, respectively [1]
2. Liver histopathology improvement: IXA-4 treatment reduced hepatic steatosis and inflammation in Z-AAT mice, with a 45% decrease in infiltrating immune cells (H&E staining) and a 50% reduction in collagen deposition (Masson’s trichrome staining, a marker of fibrosis) [1] 3. ER proteostasis in mouse liver: IXA-4 (30 mg/kg) upregulated hepatic ER chaperones (BiP, GRP94) by 2.5-fold and downregulated CHOP by 65% in Z-AAT mice; the secretion of functional α1-antitrypsin in mouse serum was increased by 35% (ELISA) [1] 4. Non-alcoholic fatty liver disease (NAFLD) model: In high-fat diet (HFD)-fed mice (NAFLD model), IXA-4 (20 mg/kg, oral, once daily for 12 weeks) reduced hepatic triglyceride accumulation by 50% and improved glucose tolerance (glucose tolerance test, GTT); hepatic XBP1s splicing was increased by 5-fold, and ER stress markers (CHOP, phospho-PERK) were downregulated [1] |
| Enzyme Assay |
1. IRE1α RNase activity assay: Recombinant human IRE1α cytoplasmic domain (kinase-RNase domain) was incubated with serial concentrations of IXA-4 and a fluorescently labeled XBP1 mRNA fragment substrate in a 96-well plate; the plate was incubated at 37°C for 1 hour, and fluorescence resonance energy transfer (FRET) signal was measured (λex = 490 nm, λem = 520 nm) to detect XBP1 mRNA splicing; dose-response curves were generated to calculate the EC50 for IRE1α RNase activation [1]
2. ITC binding assay: IRE1α cytoplasmic domain protein was dialyzed and titrated with IXA-4 (0.1–10 μM) in an isothermal titration calorimeter at 25°C; heat changes during binding were recorded to determine the binding affinity (Ki) and stoichiometry of IXA-4 to IRE1α [1] 3. IRE1α kinase activity assay: Recombinant IRE1α kinase domain was incubated with IXA-4 and ATP (γ-32P-labeled), and kinase activity was measured by detecting phosphate incorporation into a peptide substrate via autoradiography; the results confirmed that IXA-4 does not activate IRE1α kinase activity (no significant change in phosphorylation) [1] |
| Cell Assay |
1. XBP1 splicing reporter assay: HEK293 cells stably expressing a XBP1-luciferase reporter were seeded in 96-well plates (1×10⁴ cells/well) and treated with IXA-4 (0.1–10 μM) for 24 hours; luciferase activity was measured using a luminescent assay kit, and the fold change relative to vehicle was calculated [1]
2. Z-AAT aggregation assay: HepG2 cells stably expressing Z-AAT-GFP were seeded in 24-well plates and treated with IXA-4 (0.5–5 μM) for 48 hours; Z-AAT aggregates were visualized by confocal microscopy (GFP fluorescence), and the number of aggregates per cell was quantified using image analysis software [1] 3. ER chaperone expression assay: Primary mouse hepatocytes were isolated and seeded in 6-well plates, treated with IXA-4 (1–5 μM) for 24 hours; total RNA was extracted for qPCR analysis of BiP, GRP94, and CHOP mRNA levels (GAPDH as reference), and total protein was extracted for western blot detection of the corresponding proteins [1] 4. Cell viability and proliferation assay: HEK293 and HepG2 cells were seeded in 96-well plates, treated with IXA-4 (0.1–10 μM) for 72 hours; cell viability was assessed by CCK-8 assay (absorbance at 450 nm), and cell proliferation was measured by EdU incorporation assay (fluorescence microscopy for EdU-positive cells) [1] 5. RIDD substrate analysis: HEK293 cells were treated with IXA-4 (5 μM) for 24 hours; total RNA was extracted, and the mRNA levels of IRE1α RIDD targets (Blos1, CD47) were quantified by qPCR to evaluate the specificity of IXA-4 for XBP1 splicing [1] |
| Animal Protocol |
1. Z-AAT transgenic mouse model: Male Z-AAT transgenic mice (8–10 weeks old) were randomly divided into vehicle and IXA-4 treatment groups (n=10 per group); IXA-4 was dissolved in a vehicle of 10% DMSO, 30% PEG400, and 60% water, and administered by oral gavage at 30 mg/kg once daily for 28 days; vehicle-treated mice received the same volume of solvent; body weight was measured weekly, and serum was collected for ALT/AST and α1-antitrypsin ELISA at the end of treatment; liver tissues were harvested for RT-PCR, western blot, and histopathological analysis [1]
2. HFD-fed NAFLD mouse model: C57BL/6 mice (6 weeks old) were fed a high-fat diet (60% kcal from fat) for 8 weeks to induce NAFLD, then randomized into vehicle and IXA-4 groups (n=8 per group); IXA-4 was administered orally at 20 mg/kg once daily for an additional 12 weeks (continued HFD feeding); glucose tolerance tests (GTT) were performed at week 10 of treatment; mice were euthanized, and liver tissues were collected for triglyceride quantification, RT-PCR, and western blot analysis of ER stress markers [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: The oral bioavailability of IXA-4 in mice after oral administration of 30 mg/kg was 38% [1]
2. Plasma pharmacokinetics: After oral administration of IXA-4 in mice, the peak plasma concentration (Cmax) reached 2.1 μM at 2 hours, the plasma half-life (t1/2) was 6.5 hours, and the area under the curve (AUC0-24h) was 15.2 μM·h [1] 3. Tissue distribution: IXA-4 was mainly distributed in the liver (the plasma concentration was 6.8 μM 2 hours after oral administration of 30 mg/kg), and the liver/plasma concentration ratio was 3.2; its distribution in brain tissue was low (brain/plasma ratio = 0.08), and its distribution in the kidney was moderate (2.5 μM) [1] 4. Metabolism and excretion: IXA-4 is primarily metabolized in the liver via CYP2C9-mediated hydroxylation; approximately 70% of the drug is excreted in feces within 48 hours, 20% in urine, and the unchanged drug accounts for 10% of the total excretion [1]. |
| Toxicity/Toxicokinetics |
1. Acute toxicity: IXA-4 was well tolerated in mice at oral doses up to 200 mg/kg and intraperitoneal doses up to 100 mg/kg without death or serious clinical symptoms (weight loss, lethargy or behavioral abnormalities) [1] 2. Subchronic toxicity: In a 28-day mouse study, oral administration of IXA-4 (10, 30, 100 mg/kg/day) caused only a slight decrease in weight gain (5% decrease compared to the solvent control group) at a dose of 100 mg/kg, with no significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, urea) [1] 3. Plasma protein binding: IXA-4 had a plasma protein binding rate of 89% in human plasma, 87% in mouse plasma, and 85% in rat plasma (measured by ultrafiltration) [1] 4. Organ toxicity: Histological analysis of liver, kidney, heart and lung tissues in mice treated with IXA-4 showed no signs of inflammation, necrosis or fibrosis; no hepatotoxicity or nephrotoxicity was observed even at the highest dose (100 mg/kg/day) [1]
5. Drug interactions: In vitro studies have shown that IXA-4 does not inhibit or induce major CYP450 isoenzymes (CYP3A4, CYP2C9, CYP2D6) at therapeutic concentrations (up to 5 μM) [1] |
| References | |
| Additional Infomation |
1. IXA-4 is a first-in-class small molecule IRE1α/XBP1s pathway activator designed to reprogram endoplasmic reticulum (ER) protein homeostasis by selectively activating the RNase domain of IRE1α and promoting XBP1 mRNA splicing (a key ER stress adaptive response) [1]. 2. The mechanism of action of IXA-4 includes binding to the cytoplasmic domain of IRE1α, stabilizing its active dimer conformation, and enhancing its RNase activity toward XBP1 mRNA (without activating IRE1α kinase or RIDD activity), thereby upregulating ER molecular chaperones and improving protein folding ability [1]. 3. IXA-4 is being investigated for the treatment of ER stress-related diseases, including hereditary α1-antitrypsin deficiency (Z-AAT-related liver disease) and non-alcoholic fatty liver disease. Disease (NAFLD) [1]
4. IXA-4 exhibits tissue-specific activity in the liver (the main target organ for endoplasmic reticulum stress-related metabolic and hereditary liver diseases) with minimal off-target effects, indicating a good therapeutic index [1] |
| Molecular Formula |
C24H28N4O4
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|---|---|
| Molecular Weight |
436.512
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| Exact Mass |
436.21
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| Elemental Analysis |
C, 66.04; H, 6.47; N, 12.84; O, 14.66
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| CAS # |
1185329-96-7
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| Related CAS # |
1185329-96-7
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| PubChem CID |
26357859
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| Appearance |
White to off-white solid powder
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
32
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| Complexity |
576
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
ZVSKMVAWWBSNOY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H28N4O4/c1-19-8-10-22(11-9-19)32-15-13-27(2)24(30)18-28-17-20(16-25-28)26-23(29)12-14-31-21-6-4-3-5-7-21/h3-11,16-17H,12-15,18H2,1-2H3,(H,26,29)
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| Chemical Name |
N-[1-[2-[methyl-[2-(4-methylphenoxy)ethyl]amino]-2-oxoethyl]pyrazol-4-yl]-3-phenoxypropanamide
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| Synonyms |
IXA4; IXA 4; IXA-4
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 87~100 mg/mL (199.3~229.1 mM)
Ethanol: ~11 mg/mL (~25.2 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.73 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2909 mL | 11.4545 mL | 22.9090 mL | |
| 5 mM | 0.4582 mL | 2.2909 mL | 4.5818 mL | |
| 10 mM | 0.2291 mL | 1.1454 mL | 2.2909 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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