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| Targets |
AX-024 targets the interaction between the T cell receptor (TCR) and the Nck adaptor protein. Nck is an adaptor protein that contains SH2 and SH3 domains and plays a critical role in TCR signaling by linking the TCR to downstream signaling pathways. AX-024 specifically targets the SH3 domain of Nck, preventing its interaction with the TCR. By blocking the TCR-Nck interaction, the compound selectively inhibits TCR-triggered T cell activation with an IC50 of approximately 1 nM. This mechanism of action modulates T cell signaling and reduces the production of pro-inflammatory cytokines. AX-024's selectivity for the TCR-Nck interaction makes it a valuable tool for studying T cell signaling and immune regulation.
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| ln Vitro |
When it comes to blocking TCR-triggered T cell proliferation, AX-024 is more than 10,000 times more effective than AX-000. In this assay, AX-024 exhibits inhibitory effects at concentrations of 1 pM or topical, but its IC50 is 1 nM. Furthermore, at a concentration of 10 nM, AX-024 significantly inhibits the production of interleukin-6 (IL-6), tumor cytokine-α (TNF-α), interferon-γ (IFN-γ), IL-10, and IL-17A. It is also a more potent inhibitor of CD3-stimulated cytokine release from human peripheral blood mononuclear cells than AX-000. In CD8+ T cells sliced from wild-type (WT) OT1 TCR fluid (OT1Tg) OT1Tg T cells that were PRS-mutated WT, AX-024 strongly inhibited T cell proliferation at a concentration of 0.1 nM. Co-immunoprecipitation experiments in these cells show that Nck recruitment to the TCR is induced upon stimulation in the absence of the drug but inhibited in a dose-dependent manner when AX-024 is present, starting at 1 nM [1].
AX-024 demonstrates potent in vitro activity as a TCR-Nck interaction inhibitor. The compound selectively inhibits TCR-triggered T cell activation with an IC50 of approximately 1 nM. In blocking TCR-triggered T cell proliferation, AX-024 is more than 10,000 times more potent than AX-000. At a concentration of 10 nM, AX-024 significantly inhibits the production of IL-6, TNF-alpha, IFN-gamma, IL-10, and IL-17A. It is also a more potent inhibitor of CD3-stimulated cytokine release from human peripheral blood mononuclear cells than AX-000. In CD8+ T cells, AX-024 at 0.1 nM strongly inhibits T cell proliferation. Co-immunoprecipitation experiments show that Nck recruitment to the TCR is inhibited by AX-024 in a dose-dependent manner starting from 1 nM. |
| ln Vivo |
The AX-024 treated group showed less skin thickening and fewer scales than the vehicle group. AX-024 markedly decreased the thickening of both skin layers, with the dermal layer being affected more so than the other. This result was comparable to that shown in mice given a control cream that did not include imiquimod (IMQ). In the duplex assay, AX-024 dramatically decreased the quantity of critical airway cells. Unlike mice getting vehicle, which remained later after the ataxic repair and righting crises disappeared, mice receiving AX-024 recovered quickly from neurological impairment and weight loss, with symptoms going away by day 30 [1].
In vivo, AX-024 has demonstrated efficacy in animal models of inflammatory and autoimmune diseases. In a mouse model of imiquimod (IMQ)-induced skin inflammation, AX-024-treated mice showed less skin thickening and less scaling compared to vehicle-treated mice. AX-024 significantly reduced thickening of both skin layers, with the dermis being more affected than other layers. In a dual challenge model, AX-024 significantly reduced the number of key airway cells. In a model of neuroinflammation, AX-024-treated mice rapidly recovered from nerve injury and weight loss, with symptoms disappearing by day 30, whereas vehicle-treated mice did not recover. These in vivo data support the compound's potential for inflammatory disease research. |
| Enzyme Assay |
In vitro binding assays for AX-024 involve measuring the inhibition of the TCR-Nck interaction. Purified Nck SH3 domain or full-length Nck protein is incubated with a peptide corresponding to the Nck binding site on the TCR (or labeled Nck) in the presence of varying concentrations of the test compound. The interaction is measured using surface plasmon resonance (SPR), fluorescence polarization, or ELISA-based binding assays. IC50 values are calculated from dose-response curves. Alternatively, co-immunoprecipitation assays can be performed using T cell lysates to assess the effect of AX-024 on Nck recruitment to the TCR following stimulation. Each concentration is typically tested in duplicate or triplicate with appropriate positive and negative controls.
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| Cell Assay |
In vitro cellular assays for AX-024 are performed using primary human T cells or T cell lines. Cells are stimulated with anti-CD3/anti-CD28 antibodies to activate the TCR, and varying concentrations of AX-024 are added. T cell proliferation is measured by [3H]-thymidine incorporation or CFSE dilution. Cytokine production (IL-6, TNF-alpha, IFN-gamma, IL-10, IL-17A) is measured by ELISA or multiplex bead-based assays. TCR signaling is assessed by measuring phosphorylation of downstream signaling molecules (ZAP-70, LAT, ERK) by Western blot or phospho-flow cytometry. Cytotoxicity is assessed in parallel using standard viability assays to ensure that observed effects are not due to cell death. IC50 values for inhibition of proliferation and cytokine production are calculated from dose-response curves.
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| Animal Protocol |
In vivo animal studies for AX-024 are conducted using mouse models of inflammatory and autoimmune diseases. The IMQ-induced skin inflammation model is used to assess effects on psoriasiform dermatitis. The airway inflammation model is used to assess effects on lung inflammation. The neuroinflammation model is used to assess effects on nerve injury and recovery. AX-024 is administered via oral gavage at various doses and schedules. Disease severity is assessed by clinical scoring, histopathology, and measurement of inflammatory markers. Cytokine levels are measured in serum and tissues. Pharmacokinetic studies assess drug concentrations in plasma. Animals are monitored for clinical signs and body weight. Efficacy is expressed as improvement in disease parameters compared to vehicle-treated controls.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of AX-024 have been characterized in preclinical studies. The compound has a molecular formula of C21H22FNO2 and a molecular weight of 339.40 g/mol. Its chemical name is 1-{[4-(4-fluorophenyl)-6-methoxy-2H-chromen-3-yl]methyl}pyrrolidine. AX-024 is orally bioavailable. The compound is soluble in DMSO and other organic solvents. Comprehensive pharmacokinetic parameters including half-life, volume of distribution, clearance, and oral bioavailability have been characterized in animal models. The compound's pharmacokinetic profile supports its use in preclinical studies of inflammatory and autoimmune diseases. Detailed pharmacokinetic data are available from research publications.
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| Toxicity/Toxicokinetics |
AX-024 is intended for laboratory research use only and has not undergone comprehensive clinical toxicology testing. The compound has low toxicity, high potency, and high selectivity. Standard in vitro cytotoxicity assays in cell lines are typically performed alongside efficacy studies to rule out nonspecific toxicity. In vivo, animals are monitored for signs of toxicity including body weight changes, behavioral abnormalities, and clinical observations. Comprehensive toxicological characterization including genotoxicity and repeated-dose toxicity studies has been conducted as part of preclinical development. The compound is not approved for human use and is strictly intended for research purposes.
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| References | |
| Additional Infomation |
AX-024 is an orally available, first-in-class inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 of approximately 1 nM. It targets the SH3 domain of Nck. AX-024 inhibits IL-6, TNF-alpha, IFN-gamma, IL-10, and IL-17A production. The compound has a molecular formula of C21H22FNO2 and a molecular weight of 339.40 g/mol. AX-024 has not entered clinical trials and is available for research purposes only.
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| Molecular Formula |
C21H22FNO2
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|---|---|
| Molecular Weight |
339.403289318085
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| Exact Mass |
339.163
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| CAS # |
1370544-73-2
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| Related CAS # |
AX-024 hydrochloride;1704801-24-0
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| PubChem CID |
56949412
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.4
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
25
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| Complexity |
480
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1(CC2=C(C3=CC=C(F)C=C3)C3=CC(OC)=CC=C3OC2)CCCC1
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| InChi Key |
VMMKGVRPILDZML-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H22FNO2/c1-24-18-8-9-20-19(12-18)21(15-4-6-17(22)7-5-15)16(14-25-20)13-23-10-2-3-11-23/h4-9,12H,2-3,10-11,13-14H2,1H3
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| Chemical Name |
1-[[4-(4-fluorophenyl)-6-methoxy-2H-chromen-3-yl]methyl]pyrrolidine
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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) |
Ethanol : ~100 mg/mL (~294.64 mM)
DMSO : ~35 mg/mL (~103.12 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.37 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 25.0 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 2: 2.5 mg/mL (7.37 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 ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9464 mL | 14.7319 mL | 29.4638 mL | |
| 5 mM | 0.5893 mL | 2.9464 mL | 5.8928 mL | |
| 10 mM | 0.2946 mL | 1.4732 mL | 2.9464 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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