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AX-024 Sale

目录号 : GC35440

An inhibitor of the CD3ε-Nck interaction

AX-024 Chemical Structure

Cas No.:1370544-73-2

规格 价格 库存 购买数量
10mM (in 1mL DMSO)
¥1,386.00
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2mg
¥743.00
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5mg
¥1,114.00
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10mg
¥1,980.00
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25mg
¥3,935.00
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50mg
¥7,054.00
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100mg
¥12,600.00
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200mg 待询 待询
500mg 待询 待询

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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

Cell experiment:

Spleen B cells from C57BL/6 mice are labeled with Cell Trace Violet and incubated for 72 hours with either anti-IgM (10 mg/mL) or anti-CD40 (5 mg/mL), supplemented with IL-4 (5 ng/mL) or LPS (2.5 mg/mL) in the presence of different concentrations of AX-024. Proliferation is calculated according to the total number of cell divisions[1].

Animal experiment:

Eight-week-old CD-1 mice are injected intraperitoneally with different amounts of the AX-024 dissolved in 0.5 mL of saline. All animals are observed clinically for the appearance of macroscopically visible adverse reactions twice daily over 14 days, as well as immediately after AX-024 administration. A necropsy is carried out on each animal on day 14, and the abdominal, thoracic, and cranial cavities are examined in situ, together with their associated organs[1].

References:

[1]. Borroto A, et al. First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases. Sci Transl Med. 2016 Dec 21;8(370):370ra184.

产品描述

AX-024 is an inhibitor of the CD3ε-Nck interaction.1 It binds to the SH3.1 N-terminal domain of Nck1, preventing its binding to the proline-rich sequence of the CD3ε subunit of T cells. AX-024 inhibits T cell receptor-triggered T cell activation (IC50 = 1 nM for human T cells) and selectively decreases proliferation of T cells over B cells. It prevents CD4+ T cell and macrophage infiltration to the cerebellum and spinal cord and improves neurological impairments in a MOG35-55 mouse model of experimental autoimmune encephalomyelitis (EAE) when administered at a dose of 10 mg/kg per day for ten days starting on the day of immunization. AX-024 (50 mg/kg) prevents the infiltration of eosinophils, macrophages, and neutrophils into the airway, as well as prevents an increase in IL-4 levels in bronchoalveolar lavage fluid (BALF), in an ovalbumin-sensitized mouse model of allergic airway disease. It does not prevent the memory T cell response to an immunodominant B8R poxvirus antigen peptide or to infection by ectromelia virus (mousepox).

1.Borroto, A., Reyes-Garau, D., Jiménez, M.A., et al.First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseasesSci. Transl. Med.8(370)370ra184(2016)

Chemical Properties

Cas No. 1370544-73-2 SDF
Canonical SMILES COC1=CC=C(OC2)C(C(C3=CC=C(F)C=C3)=C2CN4CCCC4)=C1
分子式 C21H22FNO2 分子量 339.4
溶解度 Ethanol: 100 mg/mL (294.64 mM); DMSO: 35 mg/mL (103.12 mM and warming); Water: < 0.1 mg/mL (insoluble) 储存条件 Store at -20°C
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溶解性数据

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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
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Research Update

New Label-Free Biosensing for the Evaluation of the AX-024 Inhibitor: Case Study for the Development of New Drugs in Autoimmune Diseases

Sensors (Basel) 2022 Feb 5;22(3):1218.PMID:35161965DOI:10.3390/s22031218.

We developed a new label-free assay to evaluate the inhibition capacity of AX-024 by means of a new Point-of-Care (PoC) device for application in the development of new drugs in autoimmune diseases. The technology of PoC is based on interferometric optical detection method (IODM). For this purpose, we have optimized and developed an assay protocol whereby a Glutathione S-Transferase modified protein (GST-SH3.1), which contains a functional domain of a protein involved in T-cell activation, together with the AX-024 inhibitor has been studied. The chips used are a sensing surface based on nitrocellulose. We used streptavidin and a biotinylated peptide as links for the immobilization process on the sensing surface. The biotinylated peptide and AX-024 inhibitor compete for the same functional group of the GST-SH3.1 modified protein. When the inhibitor binds its binding site on GST-SH3.1, the biotinylated peptide cannot bind to its pocket on the protein. This competition reduces the total molecular mass of protein fixed onto the biosensor. In order to quantify the inhibition capacity of AX-024, several AX-024:GST-SH3.1 ratios have been studied. We have compared the read-out signal for GST-SH3.1 protein not interfered by the drug, which served as a positive blank, and the response of the GST-SH3.1 modified protein blocked by the inhibitor. The technology has been correlated with confocal fluorescence microscopy.

Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a domain swap in the Nck1-SH3.1 domain

J Biol Chem 2020 Jun 5;295(23):7849-7864.PMID:32317279DOI:10.1074/jbc.RA120.012788.

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.

Lack of Nck1 protein and Nck-CD3 interaction caused the increment of lipid content in Jurkat T cells

BMC Mol Cell Biol 2022 Jul 28;23(1):36.PMID:35902806DOI:10.1186/s12860-022-00436-3.

Background: The non-catalytic region of tyrosine kinase (Nck) is an adaptor protein, which is ubiquitously expressed in many types of cells. In T cells, the Nck1 isoform promotes T cell receptor signalling as well as actin polymerisation. However, the role of Nck1 in the lipid metabolism in T cells is unknown. In the present study, we investigated the effect of the Nck1 protein and Nck-CD3 interaction on lipid metabolism and on the physical and biological properties of Jurkat T cells, using a newly developed holotomographic microscope. Results: Holotomographic microscopy showed that Nck1-knocked-out cells had membrane blebs and were irregular in shape compared to the rounded control cells. The cell size and volume of Nck1-deficient cells were comparable to those of the control cells. Nck1-knocked-out Jurkat T cells had a greater lipid content, lipid mass/cell mass ratio, and lipid metabolite levels than the control cells. Interestingly, treatment with a small molecule, AX-024, which inhibited Nck-CD3 interaction, also caused an increase in the lipid content in wild-type Jurkat T cells, as found in Nck1-deficient cells. Conclusions: Knockout of Nck1 protein and hindrance of the Nck-CD3 interaction cause the elevation of lipid content in Jurkat T cells.

Anti-CD3 Fab Fragments Enhance Tumor Killing by Human γδ T Cells Independent of Nck Recruitment to the γδ T Cell Antigen Receptor

Front Immunol 2018 Jul 9;9:1579.PMID:30038626DOI:10.3389/fimmu.2018.01579.

T lymphocytes expressing the γδ T cell receptor (γδ TCR) can recognize antigens expressed by tumor cells and subsequently kill these cells. γδ T cells are indeed used in cancer immunotherapy clinical trials. The anti-CD3ε antibody UCHT1 enhanced the in vitro tumor killing activity of human γδ T cells by an unknown molecular mechanism. Here, we demonstrate that Fab fragments of UCHT1, which only bind monovalently to the γδ TCR, also enhanced tumor killing by expanded human Vγ9Vδ2 γδ T cells or pan-γδ T cells of the peripheral blood. The Fab fragments induced Nck recruitment to the γδ TCR, suggesting that they stabilized the γδ TCR in an active CD3ε conformation. However, blocking the Nck-CD3ε interaction in γδ T cells using the small molecule inhibitor AX-024 neither reduced the γδ T cells' natural nor the Fab-enhanced tumor killing activity. Likewise, Nck recruitment to CD3ε was not required for intracellular signaling, CD69 and CD25 up-regulation, or cytokine secretion by γδ T cells. Thus, the Nck-CD3ε interaction seems to be dispensable in γδ T cells.