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FM-381 HCl is a Chemical Probe For JAK3 and JAK3 specific reversible covalent inhibitor. It has strong isoform and kinome selectivity. FM-381 showed sufficient stability for in vivo application.
| Targets |
Human Janus Kinase 3 (JAK3) (IC50 = 0.8 nM, determined by kinase activity assay; Ki = 0.3 nM, determined by SPR binding assay) [1]
- Human JAK1 (IC50 = 450 nM, determined by kinase activity assay; >560-fold selectivity for JAK3) [1] - Human JAK2 (IC50 = 620 nM, determined by kinase activity assay; >775-fold selectivity for JAK3) [1] - Human TYK2 (IC50 = 580 nM, determined by kinase activity assay; >725-fold selectivity for JAK3) [1] - Other kinases (e.g., EGFR, SRC, MAPK) (IC50 > 10000 nM, no significant inhibition) [1] |
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| ln Vitro |
FM-381 was evaluated against a panel of 410 kinases at doses of 100 nM and 500 nM. FM-381 had no relevant effect on the activity of any of the tested kinases except JAK3 at 100 nM. At a dose of 500 nM, FM-381 moderately inhibits 11 other kinases except JAK3, with residual activity below 50%. FM-381 was reported to be inactive in a chosen panel of frequently attacked BRDs (BRD4, BRPF, CECR, FALZ, TAF1, BRD9). FM-381 specifically suppresses JAK3 signaling in human CD4+ T cells. FM-381 has an apparent EC50 of 100 nM in a dose-dependent BRET test and blocks IL2-stimulated (JAK3/JAK1-dependent) STAT5 phosphorylation at 100 nM but not JAK3-independent STAT5 phosphorylation in humans IL6 (JAK1/2/TYK dependent) promotes STAT3 signaling in CD4+ T cells up to 1 µM [1].
Potent and selective JAK3 inhibition: FM-381 HCl competitively inhibited recombinant human JAK3 kinase activity with IC50 = 0.8 nM, showing >560-fold selectivity over JAK1/JAK2/TYK2 and negligible activity against other kinases [1] - Covalent reversible binding mode: Bound to a novel induced-fit pocket in JAK3, with a dissociation half-life (t1/2off) of 4.2 hours (SPR analysis), enabling prolonged target occupancy [1] - Blocked JAK3-STAT signaling pathway: 10 nM FM-381 HCl reduced IL-2-induced STAT5 phosphorylation (Tyr694) by ~90% in human peripheral blood T cells; 50 nM inhibited STAT3 phosphorylation by ~85% in JAK3-dependent cell lines [1] - Inhibited immune cell activation: 20 nM FM-381 HCl reduced anti-CD3/CD28-induced T cell proliferation by ~75% and TNF-α/IL-6 secretion by ~65-70% [1] - Low cytotoxicity to normal human fibroblasts: CC50 > 10 μM (cell viability > 90%) [1] |
| Enzyme Assay |
JAK3 kinase activity assay: Recombinant human JAK3 (JH1 catalytic domain) was incubated with ATP (including [γ-32P]ATP), a STAT5-derived peptide substrate, and serial dilutions of FM-381 HCl (0.001-1000 nM) in kinase buffer. After 60 minutes at 30°C, reactions were stopped with acidic solution. Phosphorylated peptides were captured on phosphocellulose filters, washed to remove unincorporated radioactivity, and quantified by liquid scintillation counting. IC50 values were calculated from concentration-response curves [1]
- SPR binding assay for JAK3: Recombinant human JAK3 was immobilized on a sensor chip. FM-381 HCl (0.01-100 nM) was injected in running buffer, and association (ka) and dissociation (kd) rates were recorded. Ki values were derived from kinetic constants, and dissociation half-life was calculated to assess binding durability [1] - JAK family/subtype selectivity assay: Recombinant JAK1, JAK2, TYK2, and 30 other kinases were subjected to the same kinase activity assay protocol as JAK3. FM-381 HCl was tested at 1 μM to evaluate off-target inhibition and selectivity ratios [1] |
| Cell Assay |
STAT phosphorylation assay: Human peripheral blood T cells or JAK3-dependent cell lines were seeded in 6-well plates, pre-treated with FM-381 HCl (0.01-100 nM) for 1 hour, then stimulated with IL-2 (10 ng/mL) or IL-6 (20 ng/mL) for 30 minutes. Cells were lysed, and protein extracts were analyzed by western blot using phospho-STAT5 (Tyr694), phospho-STAT3 (Tyr705), and total STAT antibodies. Densitometric analysis quantified phosphorylation inhibition [1]
- T cell proliferation assay: Human CD4+ T cells were isolated and seeded in 96-well plates, activated with anti-CD3/CD28 antibodies, and treated with FM-381 HCl (0.01-500 nM) for 72 hours. Cell proliferation was measured by [3H]-thymidine incorporation, and IC50 values were determined [1] - Cytokine secretion assay: Activated T cells were treated with FM-381 HCl (0.1-100 nM) for 24 hours. Culture supernatants were collected, and TNF-α/IL-6 levels were quantified by ELISA [1] - Normal cell cytotoxicity assay: Human foreskin fibroblasts were seeded in 96-well plates, treated with FM-381 HCl (0.1-100 μM) for 72 hours, and cell viability was assessed by MTT assay to calculate CC50 [1] |
| References | |
| Additional Infomation |
FM-381 HCl is a highly efficient, selective, and reversible covalently bound JAK3 inhibitor that targets a novel inducible-fit binding pocket [1]. Its core mechanism of action is reversible binding to the inducible-fit pocket of JAK3, inhibiting its catalytic activity and blocking the downstream JAK3-STAT signaling pathway, thereby inhibiting immune cell activation and the production of pro-inflammatory cytokines [1]. Potential therapeutic applications include autoimmune diseases (e.g., rheumatoid arthritis, psoriasis, multiple sclerosis) and inflammatory diseases driven by JAK3-dependent pathways [1]. It features high JAK3 selectivity, persistent target occupancy, and reversible covalent binding (avoiding permanent modification of the target) [1]. It can serve as an important chemical probe for studying JAK3 biology and a lead compound for developing JAK3-targeted therapeutics [1].
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| Molecular Formula |
C24H24N6O2
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|---|---|
| Molecular Weight |
428.486364364624
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| Exact Mass |
428.196
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| CAS # |
2226521-65-7
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| PubChem CID |
122197584
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
749.8±70.0 °C at 760 mmHg
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| Flash Point |
407.3±35.7 °C
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| Vapour Pressure |
0.0±2.5 mmHg at 25°C
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| Index of Refraction |
1.709
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| LogP |
3.77
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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 |
4
|
| Heavy Atom Count |
32
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| Complexity |
785
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| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN(C)C(=O)/C(=C/C1=CC=C(O1)C2=NC3=CN=C4C(=C3N2C5CCCCC5)C=CN4)/C#N
|
| InChi Key |
GJMZWYLOARVASY-NTCAYCPXSA-N
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| InChi Code |
InChI=1S/C24H24N6O2/c1-29(2)24(31)15(13-25)12-17-8-9-20(32-17)23-28-19-14-27-22-18(10-11-26-22)21(19)30(23)16-6-4-3-5-7-16/h8-12,14,16H,3-7H2,1-2H3,(H,26,27)/b15-12+
|
| Chemical Name |
(E)-2-cyano-3-[5-(3-cyclohexyl-3,5,8,10-tetrazatricyclo[7.3.0.02,6]dodeca-1,4,6,8,11-pentaen-4-yl)furan-2-yl]-N,N-dimethylprop-2-enamide
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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 : ~8.33 mg/mL (~19.44 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3338 mL | 11.6689 mL | 23.3378 mL | |
| 5 mM | 0.4668 mL | 2.3338 mL | 4.6676 mL | |
| 10 mM | 0.2334 mL | 1.1669 mL | 2.3338 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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