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Kira8 (AMG18; Kira-8; AMG-18) is an effective and allosteric inhibitor of rnase attenuator (kira) that inhibits IRE1α kinase and has antifibrotic properties. With an IC50 of 5.9 nM, it allosterically reduces IRE1α RNase activity. It may encourage the reversal of already-existing fibrosis.
Physicochemical Properties
| Molecular Formula | C31H29CLN6O3S |
| Molecular Weight | 601.118364095688 |
| Exact Mass | 600.17 |
| Elemental Analysis | C, 61.94; H, 4.86; Cl, 5.90; N, 13.98; O, 7.98; S, 5.33 |
| CAS # | 1630086-20-2 |
| Related CAS # | Kira8 Hydrochloride;2250019-92-0 |
| PubChem CID | 118721244 |
| Appearance | Light yellow to yellow solid powder |
| LogP | 5.9 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 42 |
| Complexity | 971 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | CC1=C(C2=C(C=C1)C(=CC=C2)NS(=O)(=O)C3=CC=CC=C3Cl)OC4=C(C=CC=N4)C5=NC(=NC=C5)N[C@H]6CCCNC6 |
| InChi Key | XMWUCMFVDXDRDE-NRFANRHFSA-N |
| InChi Code | InChI=1S/C31H29ClN6O3S/c1-20-13-14-22-23(8-4-11-27(22)38-42(39,40)28-12-3-2-10-25(28)32)29(20)41-30-24(9-6-17-34-30)26-15-18-35-31(37-26)36-21-7-5-16-33-19-21/h2-4,6,8-15,17-18,21,33,38H,5,7,16,19H2,1H3,(H,35,36,37)/t21-/m0/s1 |
| Chemical Name | 2-chloro-N-[6-methyl-5-[3-[2-[[(3S)-piperidin-3-yl]amino]pyrimidin-4-yl]pyridin-2-yl]oxynaphthalen-1-yl]benzenesulfonamide |
| Synonyms | KIRA8; KIRA-8; KIRA 8; AMG-18; AMG 18; AMG18; Amgen IRE1α Inhibitor |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
IRE1α (IC50 = 5.9 nM) Kira8 effectively suppresses IRE1α RNase activity against XBP1 and Ins2 RNAs and prevents IRE1α oligomerization. Kira8 reverses XBP1 splicing that is stimulated by GNF-2 and more effectively than KIRA6 reduces IRE1α-driven apoptosis in INS-1 cells[1]. |
| ln Vitro |
Kira8 effectively suppresses IRE1α RNase activity against XBP1 and Ins2 RNAs and prevents IRE1α oligomerization. Kira8 reverses XBP1 splicing that is stimulated by GNF-2 and more effectively than KIRA6 reduces IRE1α-driven apoptosis in INS-1 cells[1]. KIRA8 blocks IRE1α oligomerization, as shown by DSS crosslinking experiments where it reduced the oligomer/monomer ratio. [1] KIRA8 potently inhibits IRE1α RNase activity against both a XBP1 mini-substrate RNA (IC50 5.9 nM) and a full-length, internally ³²P-labeled mouse Ins2 RNA substrate in vitro. [1] In INS-1 rat insulinoma cells engineered to overexpress IRE1α, treatment with KIRA8 significantly reduced ER stress-induced apoptosis (measured by Annexin V staining) more potently than the earlier inhibitor KIRA6. [1] KIRA8 reverses XBP1 mRNA splicing promoted by the allosteric c-Abl inhibitor GNF-2 in INS-1 cells. [1] |
| ln Vivo |
Male Ins2+/Akita mice receive intraperitoneal injections of KIRA8 (50 mg/kg) every day for 35 days. Over the course of several weeks, a notable decrease in hyperglycemia is observed[1]. Kira8 (50 mg/kg, i.p., once daily) treatment for one week significantly reduces islet XBP1 splicing and TXNIP mRNAs while preserving Ins1/Ins2, BiP, and MANF mRNAs in pre-diabetic NODs mice[1]. In pre-diabetic Non-Obese Diabetic (NOD) mice, daily intraperitoneal (i.p.) administration of KIRA8 (50 mg/kg) for one week starting at 10 weeks of age led to significant reductions in islet XBP1 mRNA splicing and TXNIP mRNA levels, while preserving Ins1/Ins2, BiP, and MANF mRNA levels. [1] In a longer-term prevention study, 10-week-old pre-diabetic NOD mice treated daily with KIRA8 (50 mg/kg, i.p.) for 4 weeks showed a preserved first-phase insulin response. After 6 weeks of treatment, pancreatic insulin-positive area was significantly increased (approximately three-fold) compared to vehicle-treated controls. [1] In a diabetes reversal study, NOD mice with newly established diabetes (blood glucose >250 mg/dL) were treated daily with KIRA8 (50 mg/kg, i.p.) upon disease onset. Random blood glucose levels rapidly decreased in the KIRA8-treated group compared to controls. By four weeks, a statistically significant difference in the percentage of diabetic mice was apparent, with a remarkable >90% diabetes reversal rate in the KIRA8 cohort. [1] In 3-week-old male pre-diabetic Akita (Ins2+/Akita) mice, daily i.p. dosing of KIRA8 (50 mg/kg) led to a significant reduction in hyperglycemia over several weeks. [1] Treatment of C57BL/6 mice with KIRA8 under similar conditions did not show significant effects on T-UPR endpoints, suggesting its effects in NOD mice are specific to the disease context. [1] |
| Enzyme Assay |
For the IRE1α RNase kinetic assay, recombinant IRE1α (the cytosolic kinase and RNase domains) was incubated with varying concentrations of the inhibitor. The reaction was initiated by adding a fluorescently labeled XBP1 mini-substrate (5´-Alex647-CAUGUCCGCAGCGCAUG-IowaBlack-FQ-3´). Fluorescence was monitored in real-time at excitation/emission wavelengths of 650/665 nm. Reaction velocity (V0) was calculated from the linear range of the fluorescence curve to determine percentage activity and IC50 values. [1] For endpoint RNase activity measurement, IRE1α was incubated with a 5´FAM-3´BHQ-labeled XBP1 single stem-loop mini-substrate. After incubation, reaction mixtures were resolved by urea 15% PAGE. [1] Internally ³²P-labeled mouse Insulin2 (Ins2) RNA was also used as a substrate in RNase assays to confirm activity against a physiological mRNA target. [1] To assess inhibition of IRE1α oligomerization, IRE1α was crosslinked with disuccinimidyl suberate (DSS) in the presence or absence of KIRA8. The crosslinked products were then analyzed by immunoblotting to visualize oligomeric and monomeric states. [1] |
| Cell Assay |
Apoptosis was assessed by Annexin V staining and flow cytometry. Cells (e.g., INS-1) were plated, treated with ER stress agents (e.g., tunicamycin) or compounds (e.g., KIRA8, GNF-2) for indicated times. Cells were then trypsinized, washed, resuspended in Annexin V binding buffer with Annexin V FITC, and analyzed on a flow cytometer. [1] For XBP1 mRNA splicing analysis, total RNA was isolated from cells or tissues, reverse transcribed, and then amplified by PCR using primers flanking the unconventional 26-nt intron. The PCR products were digested with PstI (which cuts only the unspliced fragment) and resolved on agarose gels. The ratio of spliced to unspliced product was quantified by densitometry. [1] Gene expression levels were measured by quantitative real-time PCR (qPCR). RNA was isolated, reverse transcribed, and amplified using SYBR Green chemistry and gene-specific primers. Expression was normalized to housekeeping genes like Beta Actin or Hprt1. [1] Protein levels were analyzed by western blot. Cells were lysed, proteins separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies (e.g., against TXNIP, phospho-/total IRE1α, phospho-/total c-Abl, proinsulin). Detection was performed using near-infrared-dye-conjugated secondary antibodies and an imaging scanner. [1] For subcellular localization studies, INS-1 cells stably expressing superfolder GFP-tagged IRE1α (sfGFP-IRE1α) and/or mCherry-tagged c-Abl were treated with compounds (e.g., DTT, imatinib, GNF-2) and imaged live using confocal microscopy. [1] |
| Animal Protocol |
Male Ins2+/Akita mice[1] 50 mg/kg Injected i.p.; daily; for 35 days For pre-diabetic studies in NOD mice, 8- or 10-week-old euglycemic female NOD mice were randomized to treatment or vehicle groups. KIRA8 was dissolved in a vehicle of 3% ethanol, 7% Tween-80, and 90% saline. Mice were injected intraperitoneally (i.p.) with KIRA8 at a dose of 50 mg/kg once daily. Control mice received the vehicle alone. [1] For diabetes reversal studies in NOD mice, treatment with KIRA8 or vehicle commenced immediately at the onset of diabetes (defined as blood glucose >250 mg/dL) and continued daily for 4-6 weeks. Blood glucose and body weight were monitored weekly. [1] For studies in Akita mice, 3-week-old male pre-diabetic Ins2+/Akita mice were injected i.p. daily with KIRA8 (50 mg/kg) or vehicle. [1] Blood glucose was measured from tail snips using a glucose meter. For intraperitoneal glucose tolerance tests (IPGTT) and first-phase insulin response, mice were fasted for 17 hours before i.p. injection of glucose (1.5 g/kg). Blood samples were collected from the tail vein immediately before and 2 minutes after injection for serum insulin measurement by ELISA. [1] For histology, pancreata were harvested, fixed, embedded in paraffin, and sectioned. Sections were stained for insulin by immunofluorescence. The insulin-positive area was quantified as a percentage of total pancreatic area using image analysis software. Insulitis was scored on hematoxylin and eosin-stained sections based on lymphocyte infiltration. [1] Islets were isolated from mice for ex vivo analysis. RNA and protein were extracted from islets for qPCR and western blot analysis of UPR markers. [1] |
| References |
[1]. Targeting ABL-IRE1α Signaling Spares ER-Stressed Pancreatic β Cells to Reverse Autoimmune Diabetes. Cell Metab. 2017 Apr 4;25(4):883-897.e8. |
| Additional Infomation |
KIRA8, also referred to as compound 18 in a prior publication, is described as a mono-selective IRE1α inhibitor that acts as a KIRA (Kinase-inhibitory RNase Attenuator). It allosterically inhibits IRE1α's RNase activity by disrupting oligomer formation. [1] The study proposes that during autoimmune diabetes pathogenesis, endoplasmic reticulum (ER) stress in pancreatic β-cells leads to IRE1α hyperactivation and apoptosis. KIRA8 directly targets this pathway by attenuating IRE1α activity, thereby sparing β-cells, preserving their function, and reversing established diabetes in the NOD mouse model. [1] The efficacy of KIRA8 in reversing diabetes in the complex NOD model, which involves autoimmune attack, suggests that targeting ER stress-induced β-cell degeneration is a promising therapeutic strategy for type 1 diabetes. [1] The chemical synthesis and characterization data (HPLC purity, ¹H-NMR, ESI-MS) for KIRA8 are provided in the Experimental Procedures. [1] |
Solubility Data
| Solubility (In Vitro) |
Ethanol: 76.9~100 mg/mL (128~166.4 mM) DMSO: ~65 mg/mL (~108.1 mM) H2O: ~30 mg/mL (~49.9 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: 4 mg/mL (6.65 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 40.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 2: ≥ 2.31 mg/mL (3.84 mM) (saturation unknown) in 3% ethanol, 7% Tween-80, and 90% normal Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: ≥ 2.17 mg/mL (3.61 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 21.7 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 4: ≥ 2.17 mg/mL (3.61 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 21.7 mg/mL clear DMSO stock solution to 900 μL 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 | 1.6636 mL | 8.3178 mL | 16.6356 mL | |
| 5 mM | 0.3327 mL | 1.6636 mL | 3.3271 mL | |
| 10 mM | 0.1664 mL | 0.8318 mL | 1.6636 mL |