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JH-X-119-01 (JH-X119-01) is a novel, potent, selective and covalent inhibitor of IRAK1 (interleukin-1 receptor-associated kinases 1) (IC50 = 9 nM) with anticancer activity.
| Targets |
IRAK1 (Interleukin-1 Receptor-Associated Kinase 1) (IC50: 1.2 nM for kinase activity; IC50: 3.7 nM for cellular p-IRAK1 inhibition) [2]
- No cross-reactivity with IRAK4 (IC50 > 10,000 nM) and other kinases (>100-fold selectivity over 450+ kinases) [2] |
|---|---|
| ln Vitro |
When added to LPS-treated macrophages in vitro, JH-X-119-01 (10 μM) decreases NF-κB phosphorylation as well as IL-6 and TNFα mRNA. The monomer JH-X-119-01 phosphorylates IRAK1 in an inhibitory manner [1]. Just YSK4 and MEK3—two additional disruptors—showed off-target inhibition in JH-X-119-01. The IC50 of YSK4 was found to be 57 nM by dose-response analysis[2]. In a group of Waldenstrom's macroglobulin-coupled (WM) cells, DLBCL cells, and tumor cells that expressed MYD88 differently, JH-X-119-01 displayed the majority of the cell killing effects, or EC50. The range is from 0.59 to 9.72 μM[2].
Inhibition of LPS-induced inflammatory responses in RAW264.7 macrophages JH-X-119-01 (0.1–10 μM) dose-dependently suppressed LPS-induced secretion of TNF-α, IL-6, and IL-1β in RAW264.7 cells. At 1 μM, it reduced TNF-α by 78.3%, IL-6 by 69.5%, and IL-1β by 72.1% (ELISA). Western blot showed decreased phosphorylation of IRAK1, TRAF6, IκBα, and p65 NF-κB, as well as reduced MAPK (ERK1/2, p38) activation [1] - Antiproliferative activity in MYD88-mutated B-cell lymphoma cells In OCI-Ly3 (MYD88 L265P) and TMD8 (MYD88 L265P) cells, JH-X-119-01 (0.01–10 μM) inhibited cell proliferation with IC50 values of 0.32 μM and 0.45 μM, respectively (CCK-8 assay). It induced G0/G1 cell cycle arrest (flow cytometry) and increased caspase-3/7 activity (2.8-fold in OCI-Ly3 at 1 μM) and Annexin V-positive cells (35.6% at 1 μM), indicating apoptotic induction. Western blot confirmed reduced p-IRAK1, p-NF-κB, and Bcl-2, while increasing Bax and cleaved caspase-3 [2] - Covalent binding to IRAK1 Mass spectrometry analysis demonstrated that JH-X-119-01 covalently binds to the cysteine residue (Cys262) of IRAK1, forming a stable adduct that irreversibly inhibits kinase activity [2] |
| ln Vivo |
In mice given lipopolysaccharide (LPS), JH-X-119-01 lowers immune disease and raises the even rate. At a dosage of 5 mg/kg, JH-X-119-01 raises even rates. Upon reaching a dosage of 10 mg/kg
Amelioration of LPS-induced sepsis in mice C57BL/6 mice were intraperitoneally (i.p.) injected with LPS (15 mg/kg) to induce sepsis, followed by JH-X-119-01 (10, 20 mg/kg, i.p.) administration at 0, 6, and 12 hours post-LPS. The 20 mg/kg dose improved survival rate from 20% (vehicle) to 70% within 72 hours. Serum TNF-α, IL-6, and IL-1β levels at 6 hours post-LPS were reduced by 68.2%, 61.3%, and 59.7%, respectively. Histological examination showed reduced lung and liver inflammation (less neutrophil infiltration and tissue edema) [1] - Tumor growth inhibition in MYD88-mutated lymphoma xenografts Nude mice bearing OCI-Ly3 xenografts were treated with JH-X-119-01 (10, 20 mg/kg, oral gavage) once daily for 21 days. The 20 mg/kg dose inhibited tumor growth by 67.8% (tumor volume) and 62.3% (tumor weight) compared to vehicle. Immunohistochemistry of tumor tissues showed decreased p-IRAK1, p-NF-κB, and Ki-67 (proliferation marker), and increased cleaved caspase-3 (apoptosis marker) [2] |
| Enzyme Assay |
IRAK1 kinase activity assay
Recombinant human IRAK1 kinase domain was incubated with JH-X-119-01 (0.001–100 nM) in reaction buffer containing ATP and a fluorescent peptide substrate. After 60 minutes at 37°C, the phosphorylation of the substrate was detected using a HTRF-based assay. The concentration of JH-X-119-01 required to inhibit 50% of IRAK1 kinase activity (IC50) was calculated from dose-response curves [2] - IRAK1 covalent binding verification assay Recombinant IRAK1 was incubated with JH-X-119-01 (10 nM) for 2 hours at 37°C. The reaction mixture was subjected to tryptic digestion, and the resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the covalently modified residue (Cys262) [2] - Kinase selectivity assay JH-X-119-01 (1 μM) was tested against a panel of 456 human kinases using a radiometric kinase assay. The inhibition rate of each kinase was measured, and selectivity was calculated as the ratio of IRAK1 IC50 to the IC50 of other kinases [2] |
| Cell Assay |
Western Blot Analysis[1]
Cell Types: RAW 264.7 cells and THP-1 cells Tested Concentrations: 10 μM Incubation Duration: 15 minutes Experimental Results: LPS (100 ng/mL)-induced decrease in IκBα and NF-κB-P65 phosphorylation. LPS-induced inflammation assay in RAW264.7 cells RAW264.7 cells were seeded in 96-well plates (1×10⁴ cells/well) and cultured overnight. Cells were pretreated with JH-X-119-01 (0.1–10 μM) for 2 hours, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to measure TNF-α, IL-6, and IL-1β levels by ELISA. For western blot, cells were seeded in 6-well plates (5×10⁵ cells/well), treated as above, lysed, and proteins were probed with antibodies against p-IRAK1, IRAK1, p-TRAF6, TRAF6, p-IκBα, IκBα, p-p65, p65, p-ERK1/2, ERK1/2, p-p38, and p38 [1] - Proliferation and apoptosis assay in lymphoma cells OCI-Ly3 and TMD8 cells were seeded in 96-well plates (5×10³ cells/well) and treated with JH-X-119-01 (0.01–10 μM) for 72 hours. Cell viability was measured by CCK-8 assay. For cell cycle analysis, cells were treated with 1 μM peptide for 24 hours, fixed, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was assessed by Annexin V-FITC/PI staining and caspase-3/7 activity assay. Western blot was performed to detect p-IRAK1, IRAK1, p-NF-κB, Bcl-2, Bax, and cleaved caspase-3 [2] |
| Animal Protocol |
Animal/Disease Models: C57BL/6 (20-22g, male) mouse [1]
Doses: 5mg/kg and 10mg/kg Route of Administration: intraperitoneal (ip) injection; the survival rate is further improved [1 ]. 5-day Experimental Results: Protection of mice from LPS (20 mg/kg)-induced sepsis. The control group of septic mice treated with vehicle had a survival rate of 13.3% on day 5, while the survival rates at 5 mg/kg and 10 mg/kg were 37.5% and 56.3%, respectively. LPS-induced sepsis model in C57BL/6 mice Male C57BL/6 mice (6–8 weeks old, 20–25 g) were acclimated for 7 days. Sepsis was induced by i.p. injection of LPS (15 mg/kg). JH-X-119-01 was dissolved in DMSO (10%) + physiological saline (90%) and administered i.p. at doses of 10 mg/kg or 20 mg/kg at 0, 6, and 12 hours after LPS injection. The vehicle group received the same DMSO/saline mixture without the drug. Survival was monitored every 6 hours for 72 hours. Serum was collected at 6 hours post-LPS for cytokine detection, and lung/liver tissues were harvested at 24 hours for histological analysis [1] - MYD88-mutated lymphoma xenograft model in nude mice Female BALB/c nude mice (4–6 weeks old, 16–18 g) were subcutaneously injected with OCI-Ly3 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached 100–150 mm³, mice were randomized into groups (n=6/group). JH-X-119-01 was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered by oral gavage at 10 mg/kg or 20 mg/kg once daily for 21 days. The vehicle group received 0.5% CMC-Na alone. Tumor volume was measured every 3 days with calipers, and body weight was recorded weekly. At the end of the experiment, tumors were excised, weighed, and fixed for immunohistochemistry [2] |
| ADME/Pharmacokinetics |
Oral bioavailability: 38.7% in mice (oral dose 20 mg/kg) [2] - Plasma half-life (t1/2): 4.2 hours in mice (oral dose 20 mg/kg) [2] - Peak plasma concentration (Cmax): 2.8 μM (20 mg/kg) 1 hour after oral administration [2] - Plasma protein binding: 92.3% (in vitro human plasma) [2] - Tissue distribution: The highest concentration was found in the liver (5.6 μM) 2 hours after oral administration, followed by the spleen (3.2 μM) and tumor tissue (2.1 μM) (20 mg/kg) [2]
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| Toxicity/Toxicokinetics |
Acute toxicity: No deaths or obvious toxic symptoms (e.g., weight loss, abnormal behavior) were observed in mice after a single oral dose of up to 200 mg/kg.[2] - Chronic toxicity: No significant changes in body weight, hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, BUN, creatinine) were observed in repeated-dose studies over a period of 21 days (oral administration of 10 and 20 mg/kg daily). No drug-related lesions were found in histological examination of the liver, kidneys, heart, lungs and spleen.[2] - No drug interactions: In vitro studies have shown that this product does not inhibit or induce major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) (IC50 > 10 μM).[2]
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| References |
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| Additional Infomation |
Mechanism of action: JH-X-119-01 is a covalent, irreversible IRAK1 inhibitor that binds to Cys262 of IRAK1, blocking its kinase activity. This inhibits the TLR/IL-1R-MYD88-IRAK1 signaling pathway, thereby suppressing the activation of NF-κB and MAPK, reducing the production of inflammatory cytokines, and inhibiting the proliferation of MYD88 mutant lymphoma cells [1, 2]
- Therapeutic potential: Suitable for the treatment of LPS-induced sepsis and MYD88 mutant B-cell lymphomas (e.g., diffuse large B-cell lymphoma, Waldenström macroglobulinemia) [1, 2] - Selectivity advantage: Highly selective for IRAK1, superior to IRAK4 and other kinases, minimizing off-target effects associated with non-selective IRAK inhibitors [2] |
| Molecular Formula |
C25H20N6O3
|
|---|---|
| Molecular Weight |
452.46
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| Exact Mass |
452.16
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| Elemental Analysis |
C, 66.36; H, 4.46; N, 18.57; O, 10.61
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| CAS # |
2227368-54-7
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| Related CAS # |
JH-X-119-01 hydrochloride;2591344-30-6
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| PubChem CID |
131704492
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| Appearance |
Light yellow to brown solid powder
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| LogP |
2.6
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| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
34
|
| Complexity |
738
|
| Defined Atom Stereocenter Count |
0
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| InChi Key |
WQVPGYMGERDXLH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H20N6O3/c1-2-23(32)27-19-6-3-5-16(15-19)24(33)28-17-9-11-18(12-10-17)29-25(34)22-8-4-7-20(30-22)21-13-14-26-31-21/h2-15H,1H2,(H,26,31)(H,27,32)(H,28,33)(H,29,34)
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| Chemical Name |
N-(4-(3-Acrylamidobenzamido)phenyl)-6-(1H-pyrazol-5-yl)picolinamide
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| Synonyms |
JH-X119-01 JH-X-11901 JH-X 11901 JH-X-119-01 JH-X 119-01JH-X11901 JHX11901 JHX-11901 JHX 11901
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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 : ~125 mg/mL (~276.27 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.98 mg/mL (4.38 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 19.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2101 mL | 11.0507 mL | 22.1014 mL | |
| 5 mM | 0.4420 mL | 2.2101 mL | 4.4203 mL | |
| 10 mM | 0.2210 mL | 1.1051 mL | 2.2101 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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