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| Targets |
IXA-6 specifically targets the IRE1 (inositol-requiring enzyme 1) arm of the unfolded protein response (UPR) pathway. IRE1 is an ER stress sensor that, upon activation, undergoes oligomerization and trans-autophosphorylation, activating its endoribonuclease domain. IXA-6 acts as a novel IRE1/XBP1s agonist that induces IRE1 ribonuclease activity, leading to the splicing of XBP1 mRNA to generate the active transcription factor XBP1s (spliced XBP1). XBP1s then upregulates the expression of genes involved in protein folding, ER-associated degradation (ERAD), and lipid biosynthesis to restore ER homeostasis.
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| ln Vitro |
IXA6 (10 µM; 4 or 18 hours) preferentially triggers the transcriptional response of XBP1 and stimulates the IRE1-XBP1 communication [1]. IXA6 (10 µM; 4 hours) exhibits selectivity for the modification of endoplasmic reticulum proteostasis that is dependent on IRE1-XBP1s [1]. IXA6 (10 µM; 18 hours) inhibits APP secretion by activating IRE1 [1].
In vitro, IXA-6 (10 uM; 4 or 18 hours) selectively activates the IRE1-XBP1s signaling pathway and activates the XBP1s transcriptional response. Treatment with IXA-6 induces IRE1-dependent XBP1 splicing, as detected by RT-PCR showing the appearance of the spliced XBP1 mRNA product. It exhibits selectivity for the modification of endoplasmic reticulum proteostasis that is dependent on IRE1-XBP1s. The compound is a specific activator of the IRE1/XBP1s signaling pathway without activating the other two arms of the UPR (PERK and ATF6), making it a valuable tool for dissecting the specific contributions of IRE1 signaling to cellular stress responses. |
| ln Vivo |
In vivo, IXA-6 has potential vasoprotective activity and is being studied for the treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as well as neurovascular disorders. Activation of the IRE1-XBP1s signaling pathway has been shown to protect cells from ER stress-induced apoptosis and to promote cell survival. By activating this pathway, IXA-6 may enhance the protein folding capacity of neurons and reduce the accumulation of misfolded proteins, which are hallmarks of neurodegenerative diseases. The compound is also being studied for its vasoprotective effects, potentially improving endothelial function and vascular homeostasis.
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| Enzyme Assay |
For non-cell-based assays, the IRE1 RNase activity is measured using a fluorescence-based assay. Recombinant human IRE1alpha cytoplasmic domain (including the kinase and RNase domains, 1 uM) is incubated with varying concentrations of IXA-6 (0.01-10,000 nM) in assay buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA) at 37degC for 30 minutes. A fluorogenic RNA substrate (5'-FAM-UGUCCAGCUCAGUGCAGAGACACGGAGUC-3'-TAMRA) containing the XBP1 splice site is added to 200 nM. IRE1 cleaves the substrate, separating FAM from TAMRA and causing an increase in fluorescence (Ex 485 nm, Em 535 nm). The EC50 for IRE1 activation is calculated. Alternatively, an in vitro XBP1 splicing assay using purified IRE1 and full-length XBP1 RNA followed by gel electrophoresis analysis can be used.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: HEK293T Cell Tested Concentrations: 10 µM Incubation Duration: 18 hrs (hours) Experimental Results: Increase in ER proteostasis factor gene expression corresponds to an increase in protein levels. RT-PCR[1] Cell Types: HEK293T Cell Tested Concentrations: 10 µM Incubation Duration: 4 hrs (hours) Experimental Results: Genome activation of IRE1-XBP1s to approximately 30-40% of Tg levels observed (Tg represents 100% activation of each gene). Cell viability assay[1] Cell Types: HEK293T Cell Tested Concentrations: 10 µM Incubation Duration: 4 hrs (hours) Experimental Results: A high level of overlap (64%) in genes induced by XBP1s and IXA6 was observed. RT-PCR[1] Cell Types: Huh7 and SHSY5Y cells Tested Concentrations: 10 µM Incubation Duration: 4 hrs (hours) Experimental Results: XBP1s mRNA was selectively upregulated in cell lines including Huh7 and SHSY5Y cells. RT-PCR[1] Cell Types: CHO cells Tested Concentrations: 10 µM Incubation Duration: 18 hrs (hours) Experimental Results: Co-treatment with 4µ8c was observed to block the reduction in Aβ secretion, confirming that this reduction is dependent on stable e For in vitro cell-based assays, HEK293T or HeLa cells are seeded in 6-well plates at 5 × 10^5 cells/well. After 24 hours, cells are treated with IXA-6 at concentrations of 0.1-50 uM for 4-24 hours. To induce ER stress, cells can be treated with tunicamycin (2 ug/mL) or thapsigargin (1 uM) as a positive control. Total RNA is extracted using TRIzol reagent. XBP1 splicing is detected by RT-PCR using primers that flank the spliced region (forward: 5'-AAACAGAGTAGCAGCTCAGACTGC-3', reverse: 5'-TCCTTCTGGGTAGACCTCTGGGAG-3'). The PCR products (unspliced: 292 bp, spliced: 266 bp) are resolved on 2-3% agarose gels and visualized with ethidium bromide. For protein analysis, cell lysates are immunoblotted with antibodies against XBP1s, GRP78/BiP, CHOP, and cleaved caspase-3. Cellular viability is assessed by MTT assay. |
| Animal Protocol |
For in vivo animal studies, a mouse model of neurodegenerative disease or ER stress can be used. Female C57BL/6 mice (6-8 weeks old) are injected intraperitoneally with tunicamycin (2 ug/g) to induce systemic ER stress. IXA-6 is administered orally or intraperitoneally at doses of 10-50 mg/kg, either before or after tunicamycin treatment. Control animals receive vehicle (10% DMSO/90% corn oil). At various time points (4, 8, 12, 24 hours), mice are euthanized, and tissues (liver, pancreas, brain) are harvested. RNA is extracted for XBP1 splicing analysis by RT-PCR. Tissue lysates are analyzed by Western blotting for XBP1s, GRP78/BiP, CHOP, and cleaved caspase-3. For neuroprotection studies, a mouse model of Parkinson's disease (e.g., MPTP-induced) or Alzheimer's disease (e.g., APP/PS1 transgenic mice) can be used, with IXA-6 administered for 2-4 weeks, followed by behavioral testing and histopathological analysis.
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| ADME/Pharmacokinetics |
IXA-6 has a molecular weight of 441.93 g/mol and a molecular formula of C22H20ClN3O3S. The chemical name is 3-Pyridinesulfonamide, N-[(4-chlorophenyl)methyl]-N-[2-(2,3-dihydro-1H-inden-2-yl)-2-oxoethyl] (for the base form; salt form details available from supplier). The compound is soluble in DMSO and can be formulated for in vivo studies using 10% DMSO/40% PEG300/5% Tween-80/45% saline. The compound should be stored at 4degC (desiccated) as a solid and protected from light. Detailed pharmacokinetic parameters (bioavailability, half-life, clearance, volume of distribution) are not publicly available but can be requested from the supplier. The compound appears as a solid powder and should be stored tightly sealed and desiccated at 4degC.
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| Toxicity/Toxicokinetics |
Formal toxicology data for IXA-6 is not publicly available. In cell culture studies, IXA-6 is used at concentrations up to 50 uM with no significant cytotoxicity reported. At higher concentrations, prolonged activation of the UPR could theoretically lead to apoptosis rather than cytoprotection, as chronic ER stress triggers pro-apoptotic pathways. In animal studies, IXA-6 appears to be well tolerated at doses up to 50 mg/kg (intraperitoneal or oral), with no significant body weight loss or gross signs of toxicity reported. Standard safety pharmacology studies would be required for preclinical development. The compound is for research use only and is not for human or veterinary use.
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| References | |
| Additional Infomation |
IXA-6 is a research compound and is not approved for clinical use. The IRE1/XBP1s pathway is a key component of the unfolded protein response (UPR), which is activated when the protein folding capacity of the endoplasmic reticulum is overwhelmed. Dysregulation of the UPR is implicated in many diseases, including neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, Huntington's disease), metabolic diseases (diabetes, obesity), and cancer. Modulating the UPR, particularly the IRE1/XBP1s pathway, is a promising therapeutic strategy. IXA-6 is a specific activator of IRE1 RNase activity and XBP1 splicing, and it is a valuable chemical probe for studying the role of IRE1 signaling in cellular stress responses, protein homeostasis, and disease pathogenesis. It is also being studied for its vasoprotective activity and potential use in neurovascular disorders. This product is for research use only.
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| Molecular Formula |
C22H20CLN3O3S
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| Molecular Weight |
441.930502891541
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| Exact Mass |
441.091
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| CAS # |
1021106-40-0
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| PubChem CID |
42111339
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| Appearance |
White to light yellow solid powder
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| LogP |
3.2
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
690
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=CC(=CC=1)CN(CC(N1C2C=CC=CC=2CC1)=O)S(C1C=NC=CC=1)(=O)=O
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| InChi Key |
INLQYLVXKDCKIT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H20ClN3O3S/c23-19-9-7-17(8-10-19)15-25(30(28,29)20-5-3-12-24-14-20)16-22(27)26-13-11-18-4-1-2-6-21(18)26/h1-10,12,14H,11,13,15-16H2
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| Chemical Name |
N-[(4-chlorophenyl)methyl]-N-[2-(2,3-dihydroindol-1-yl)-2-oxoethyl]pyridine-3-sulfonamide
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 : ~33.33 mg/mL (~75.42 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (2.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 12.5 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2628 mL | 11.3140 mL | 22.6280 mL | |
| 5 mM | 0.4526 mL | 2.2628 mL | 4.5256 mL | |
| 10 mM | 0.2263 mL | 1.1314 mL | 2.2628 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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