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| Targets |
The primary target of ITK inhibitor 2 is interleukin-2-inducible T-cell kinase (ITK), a member of the Tec family of non-receptor tyrosine kinases. ITK is predominantly expressed in T cells, natural killer (NK) cells, and mast cells, where it functions as a critical signaling molecule downstream of the T-cell receptor (TCR). Following TCR engagement, ITK is recruited to the plasma membrane via its pleckstrin homology (PH) domain and is activated by phosphorylation mediated by Lck and other kinases. Activated ITK then phosphorylates and activates phospholipase C-γ1 (PLC-γ1), leading to calcium mobilization, activation of the NFAT and NF-κB pathways, and ultimately T-cell proliferation and cytokine production (including IL-2, IL-4, IL-5, IL-10, and IFN-γ). By inhibiting ITK with an IC50 of 2 nM, this compound effectively disrupts this signaling cascade. The high potency and selectivity of ITK inhibitor 2 make it an excellent pharmacological tool for dissecting the specific role of ITK in immune cell function and for evaluating the therapeutic potential of ITK inhibition in various disease contexts.
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| ln Vitro |
ITK inhibitor 2 demonstrates potent in vitro activity as a specific antagonist of ITK, with an IC50 of 2 nM. This high affinity indicates that the compound effectively binds to and inhibits the kinase activity of ITK at nanomolar concentrations. In cell-based assays, inhibition of ITK by this compound leads to a reduction in TCR-mediated signaling events, including decreased calcium flux, reduced phosphorylation of PLC-γ1 and downstream effectors, and diminished production of key cytokines such as IL-2 and IFN-γ. The compound's specificity for ITK over other kinases is a crucial attribute, minimizing off-target effects and allowing researchers to attribute observed biological effects specifically to ITK inhibition. Its physicochemical properties, including a molecular weight of 435.56, a LogP of 3.6, and high solubility in DMSO (up to 125 mg/mL), facilitate its use in a wide range of in vitro experimental setups.
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| ln Vivo |
In vivo activity data for ITK inhibitor 2 are not extensively detailed in publicly available sources. However, given its potent in vitro activity and its role as a modulator of T-cell signaling, the compound is expected to exhibit immunomodulatory effects in animal models. ITK inhibition has been shown to attenuate T-cell-mediated immune responses, suggesting that ITK inhibitor 2 could be effective in models of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, or inflammatory bowel disease, as well as in models of allergic airway inflammation. The compound's oral bioavailability and metabolic stability would be key factors determining its in vivo efficacy. As a research tool, ITK inhibitor 2 is primarily used to validate ITK as a therapeutic target and to study the consequences of ITK inhibition in various disease models.
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| Enzyme Assay |
In vitro enzyme assays for ITK inhibitor 2 typically involve measuring its inhibitory activity against recombinant ITK kinase. A common approach is the use of a kinase activity assay, where the compound is incubated with purified ITK enzyme, ATP, and a peptide substrate. The phosphorylation of the substrate is then quantified using methods such as radioactive counting (e.g., ³³P-ATP incorporation), fluorescence polarization, or time-resolved fluorescence resonance energy transfer (TR-FRET). The IC50 value is determined by plotting the percentage of kinase activity remaining against the logarithm of compound concentration. To assess selectivity, the compound is typically screened against a panel of other kinases, including other Tec family members (such as BTK, RLK, and Tec) and unrelated kinases. This profiling helps to confirm the specificity of the inhibitor and identify potential off-target effects.
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| Cell Assay |
In vitro cell-based assays for ITK inhibitor 2 are designed to evaluate its functional effects on T-cell signaling and function. A common assay involves stimulating T cells (e.g., Jurkat T-cell line or primary human T cells) with an anti-CD3 antibody, which activates the TCR, in the presence or absence of varying concentrations of the compound. Readouts include the measurement of intracellular calcium flux using fluorescent indicators, the quantification of cytokine production (e.g., IL-2, IFN-γ) by ELISA or multiplex assays, and the assessment of T-cell proliferation by [³H]-thymidine incorporation or CFSE dilution. Additionally, the phosphorylation status of key signaling proteins, such as PLC-γ1, ERK, and AKT, can be analyzed by western blotting to confirm target engagement and pathway inhibition. These assays provide a comprehensive view of the compound's cellular activity and its impact on immune cell function.
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| Animal Protocol |
In vivo animal studies for ITK inhibitor 2 are typically conducted in murine models of immune-mediated diseases. For example, in a model of allergic airway inflammation, mice sensitized and challenged with an allergen (e.g., ovalbumin) are treated with the compound, and endpoints such as airway hyperresponsiveness, bronchoalveolar lavage fluid cell counts, and cytokine levels are measured. In models of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) or collagen-induced arthritis, the compound is administered prophylactically or therapeutically, and disease severity is monitored by clinical scoring, histopathological analysis, and measurement of inflammatory markers. Pharmacokinetic parameters, including plasma exposure and tissue distribution, are also evaluated to establish a relationship between drug levels and efficacy.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of ITK inhibitor 2 have been characterized to support its use in preclinical studies. The compound has a molecular weight of 435.56 and a LogP of 3.6, indicating moderate lipophilicity that may facilitate cell permeability and oral absorption. It is highly soluble in DMSO (up to 125 mg/mL) and can be formulated for in vivo administration using vehicles such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. For long-term storage, the compound is stable as a powder at -20°C for up to three years or in solution at -80°C for up to six months. Detailed PK parameters, such as half-life, clearance, and oral bioavailability, are not specified in the available sources but would be determined in standard preclinical PK studies.
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| Toxicity/Toxicokinetics |
The toxicological profile of ITK inhibitor 2 is not extensively documented in publicly available sources. As a potent inhibitor of a key immune signaling pathway, its potential toxicity is likely related to its on-target effects on T-cell function. Chronic inhibition of ITK could lead to immunosuppression, which may increase the risk of infections or impair immune surveillance. Off-target effects on other kinases could also contribute to toxicity. In preclinical studies, the compound would be evaluated for general toxicity, genotoxicity, and organ-specific toxicity to establish a safety profile. For laboratory handling, standard safety precautions for research chemicals should be observed, including the use of personal protective equipment (gloves, lab coat, safety goggles) and working in a well-ventilated area. The compound is intended for research use only and is not for human therapeutic or diagnostic use.
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| References | |
| Additional Infomation |
ITK inhibitor 2 is a research-grade compound with the catalog number V33274 and a purity of ≥98%. It is also known by the aliases JTE-051, JTE051, and JTE 051. The compound was first described in patent WO2011065402A1 as compound 4. Its IUPAC name is (2S)-N-[2-(6,6-dimethyl-1,4,5,7-tetrahydroindazol-3-yl)-1H-indol-6-yl]-N-methyl-2-morpholin-4-ylpropanamide. The compound is supplied as a gray to brown solid powder. It is soluble in DMSO at concentrations up to 125 mg/mL (approximately 287 mM). For in vivo studies, it can be formulated in a vehicle of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. ITK inhibitor 2 is a valuable tool for studying T-cell signaling and the role of ITK in immune function and disease.
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| Molecular Formula |
C25H33N5O2
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| Molecular Weight |
435.56182551384
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| Exact Mass |
435.26
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| Elemental Analysis |
C, 68.94; H, 7.64; N, 16.08; O, 7.35
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| CAS # |
1309784-09-5
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| PubChem CID |
135980528
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| Appearance |
Gray to brown solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
681
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C[C@@H](C(=O)N(C)C1=CC2=C(C=C1)C=C(N2)C3=NNC4=C3CCC(C4)(C)C)N5CCOCC5
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| InChi Key |
ZZZXGCPVQQOASC-INIZCTEOSA-N
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| InChi Code |
InChI=1S/C25H33N5O2/c1-16(30-9-11-32-12-10-30)24(31)29(4)18-6-5-17-13-21(26-20(17)14-18)23-19-7-8-25(2,3)15-22(19)27-28-23/h5-6,13-14,16,26H,7-12,15H2,1-4H3,(H,27,28)/t16-/m0/s1
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| Chemical Name |
(2S)-N-[2-(6,6-dimethyl-1,4,5,7-tetrahydroindazol-3-yl)-1H-indol-6-yl]-N-methyl-2-morpholin-4-ylpropanamide
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| Synonyms |
JTE-051; JTE051; JTE 051;
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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 (~286.99 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 | 2.2959 mL | 11.4795 mL | 22.9589 mL | |
| 5 mM | 0.4592 mL | 2.2959 mL | 4.5918 mL | |
| 10 mM | 0.2296 mL | 1.1479 mL | 2.2959 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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