IL-6; IL-10; IL-17A
The compound targets the interaction between the T cell receptor (TCR) and the adaptor protein Nck. Specifically, it binds to a non-canonical "DY pocket" in the SH3.1 domain of Nck (Nck1 or Nck2), inhibiting the recruitment of Nck to the proline-rich sequence (PRS) in the cytoplasmic tail of the CD3ε subunit of the TCR [1].

Compared to AX-000 hit, AX-024 hydrochloride has a potency that is >10,000-fold greater in terms of preventing TCR-triggered T cell proliferation. AX-024 hydrochloride has an inhibitory effect at a concentration of 1 pM or less, but its IC50 in this assay is 1 nM. At a concentration of 10 nM, AX-024 hydrochloride significantly outperforms AX-000 as an inhibitor of cytokine release by human peripheral blood mononuclear cells stimulated with anti-CD3 and significantly reduces the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), IL-10, and IL-17A. AX-024 hydrochloride strongly inhibits T cell proliferation in OT1Tg T cells that are WT for the PRS mutation in mice bearing wild-type (WT) CD8+ T cells at a concentration of 0.1 nM. In these cells, coimmunoprecipitation experiments demonstrate that Nck recruitment to the TCR is induced upon stimulation in the absence of drug but is inhibited in the presence of AX-024 hydrochloride in a dose-dependent manner at concentrations beginning at 1 nM[1].

---

Inhibition of T Cell Proliferation: AX-024 HCl selectively inhibits TCR-triggered T cell proliferation. In human blood T cells stimulated with an anti-CD3 antibody, it shows an IC50 (median inhibitory concentration) of approximately 1 nM. It does not inhibit B cell proliferation induced by anti-IgM (BCR stimulation), LPS (TLR4 agonist), or anti-CD40 plus IL-4, even at concentrations up to 10 μM. It also does not inhibit the IL-2-dependent proliferation of T cell lymphoblasts or T cell proliferation induced by PMA plus ionomycin (which bypasses TCR signaling) at concentrations up to 10 μM [1].
Mechanism of Action on TCR Signaling: The compound inhibits early TCR-proximal events. It blocks TCR-triggered actin polymerization in human blood T cells stimulated with anti-CD3, as measured by phalloidin staining. It also inhibits the phosphorylation of ZAP70 on Tyr319 and partially inhibits the phosphorylation of the CD3ζ chain (Tyr142) in Jurkat T cells stimulated with anti-CD3 [1].
Target Specificity Confirmation: In pull-down assays using GST fusion proteins, AX-024 HCl inhibits the binding of CD3ζ from stimulated Jurkat T cell lysates to the GST-Nck(SH3.1) protein in a concentration-dependent manner. It does not inhibit the binding of CD3 to the SH3 domain of Eps8L1 or the binding of c-Cbl to the Nck(SH3.1) domain. In OT1 TCR transgenic mouse T cells, the compound (at 0.1 nM) strongly inhibits antigen-dependent proliferation in wild-type cells but has no additional inhibitory effect on T cells from PRS knock-in mice (KI-PRS), which have a germline mutation preventing Nck recruitment to the TCR. Surface plasmon resonance (SPR) analysis shows that the compound alters the binding kinetics of a CD3ε PRS peptide to the Nck1(SH3.1) domain, increasing the dissociation constant (KD) [1].
Effect on T Cell Differentiation: In vitro differentiation assays using naïve human CD4+ T cells under polarizing conditions, AX-024 HCl (1-100 nM) inhibits differentiation into pro-inflammatory subsets. It reduces the percentage of IFN-γ+ and IL-2+ cells under Th1 conditions, reduces IL-4+ cells under Th2 conditions, and reduces IL-17A+ cells under Th17 conditions. Conversely, it increases the differentiation into FOXP3+ regulatory T cells (Tregs) under Treg-polarizing conditions. Tregs differentiated in the presence of 1 nM AX-024 show enhanced suppressive function, inhibiting the proliferation of naïve CD4+ T cells more effectively than Tregs differentiated without the compound [1].
Gene Expression Analysis: Microarray analysis of purified human blood T cells stimulated with anti-CD3 for 4 hours in the presence of 1 nM AX-024 HCl shows significant changes in gene expression. Pathways related to T helper cell differentiation, IL-15 signaling, and cytokine signaling (e.g., IL-3, Interferon) are predicted to be altered. Upstream regulator analysis predicts inhibition of TCR/CD3 and CD28 signaling pathways [1].

In comparison to the vehicle group, the AX-024 hydrochloride-treated group has fewer scales and lessened skin thickening. Significantly lessening the thickness of both skin layers, AX-024 hydrochloride works best on the dermis, which resembles the skin of mice treated with a control cream devoid of imiquimod (IMQ). In both assays, AX-024 hydrochloride significantly reduces the quantity of inflammatory cells in the airways. In contrast to mice receiving the vehicle, which experience persistent ataxia and loss of the righting reflex, mice receiving AX-024 hydrochloride experience a rapid recovery from neurological impairment and weight loss, becoming symptom-free by day 30[1].

---

Psoriasis Model: In an imiquimod (IMQ)-induced psoriasis mouse model, intraperitoneal administration of AX-024 HCl (50 mg/kg in saline) on days 0, 2, 4, and 6 post-immunization significantly reduces the clinical severity score (ear thickness, scaling, and redness) compared to vehicle-treated mice. It also reduces the cellular infiltrate (CD4+ T cells, neutrophils) and the expression of inflammatory cytokines (IL-17A, IL-22, IFN-γ) in the skin [1].
Allergic Asthma Model: In an ovalbumin (OVA)-induced allergic asthma mouse model, intraperitoneal administration of AX-024 HCl (50 mg/kg in saline) on days 0 and 5 (2 hours before OVA sensitization) inhibits the infiltration of inflammatory cells into the airways. This is shown by a reduced ProSense 680 signal in fluorescence molecular tomography (FMT) imaging 24 hours after OVA challenge. It also significantly reduces the number of Gr-1+CD11b+ inflammatory cells and the levels of cytokines IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid (BALF) 48 hours after OVA challenge [1].
Experimental Autoimmune Encephalomyelitis (EAE) Model - Prophylactic: In a chronic MOG_35-55-induced EAE mouse model, daily oral administration of AX-024 HCl (10 mg/kg) for the first 10 days post-immunization significantly delays the onset and reduces the severity of neurological symptoms (clinical score) and weight loss compared to vehicle-treated mice. Histological analysis shows reduced infiltration of CD4+ T cells and F4/80+ macrophages into the cerebellum and spinal cord [1].
Experimental Autoimmune Encephalomyelitis (EAE) Model - Therapeutic: In a therapeutic setting, daily oral administration of AX-024 HCl (10 mg/kg) starting at the peak of disease (day 13) until day 26 rapidly reduces the clinical score to 0, comparable to fingolimod (0.6 mg/kg). Importantly, after drug withdrawal on day 26, the clinical score in AX-024-treated mice remains at 0, while fingolimod-treated mice experience a rapid and severe relapse [1].
Mechanism in EAE Model: Analysis of draining lymph nodes from MOG-immunized mice treated orally with AX-024 HCl (10 mg/kg daily for 6 days) shows a significant increase in the percentage of FOXP3+ CD4+ regulatory T cells and double-positive IFN-γ+FOXP3+ cells before the onset of neurological symptoms, without significant changes in the percentage of total IFN-γ+ or IL-17+ CD4+ T cells [1].
Viral Infection Model: In mice infected with ectromelia virus (ECTV), daily oral treatment with AX-024 HCl (10 or 40 mg/kg) does not increase susceptibility to infection. All treated mice survive sublethal doses of ECTV at rates similar to vehicle-treated mice. Furthermore, mice that survive the initial infection and are rechallenged 60 days later with a lethal dose of ECTV are protected, regardless of prior AX-024 treatment, indicating that the compound does not prevent the establishment of a protective memory T cell response [1].

Surface Plasmon Resonance (SPR) Assay: The interaction between the compound and the Nck1(SH3.1) domain was characterized using SPR. A recombinant GST-Nck1(SH3.1) fusion protein was immobilized on a sensor chip. A synthetic peptide containing the proline-rich sequence (PRS) of CD3ε was used as the analyte in solution. The association and dissociation rates of the peptide were measured in the continuous presence of increasing concentrations of AX-024 HCl (0, 0.1, 1, 10, and 100 nM). The compound altered the binding kinetics in a concentration-dependent manner. For instance, at 0.1 nM AX-024, the calculated KD increased from 7.5x10⁻⁶ M (no drug) to 5.7x10⁻⁴ M [1].
Nuclear Magnetic Resonance (NMR) Spectroscopy: The binding of AX-024 HCl to the Nck1(SH3.1) domain was confirmed by NMR. Titration of ¹⁵N-labeled Nck1(SH3.1) protein with AX-024 caused significant chemical shift perturbations in the ¹H-¹⁵N HSQC spectrum for specific amino acid residues (e.g., W229, K230, R236, Y253, W254, Y257) that form the "DY pocket" of the SH3.1 domain, confirming direct and specific binding at this site [1].

Cell Trace Violet is used to mark the spleen B cells from C57BL/6 mice. The cells are then incubated for 72 hours with either anti-IgM (10 mg/mL) or anti-CD40 (5 mg/mL), as well as IL-4 (5 ng/mL) or LPS (2.5 mg/mL), in the presence of various concentrations of AX-024 hydrochloride. According to the total number of cell divisions, proliferation is calculated[1].

---

Human T Cell Proliferation Assay: Fresh human peripheral blood lymphocytes (PBLs) were labeled with CFSE and incubated for 4 days at 37°C in 96-well plates coated with an anti-CD3 antibody (OKT3), in the presence of various concentrations of AX-024 HCl. Proliferation of CD4+ T cells was analyzed by flow cytometry based on CFSE dilution, and an IC50 of approximately 1 nM was calculated. As a control for specificity, CFSE-labeled PBLs were stimulated with PMA (10 ng/ml) and ionomycin (1 μM) for 4 days in the presence of the compound, which showed no inhibition of proliferation up to 10 μM [1].
IL-2-Dependent T Cell Proliferation Assay: Human T cell lymphoblasts were generated by stimulating PBLs with phytohemagglutinin, followed by expansion in IL-2-containing medium. These lymphoblasts were then labeled with CFSE and stimulated with IL-2 (100 ng/ml) for 3 days in the presence of various concentrations of AX-024 HCl (or the parental compound AX-000). Proliferation was analyzed by flow cytometry, and no inhibition was observed at concentrations up to 10 μM [1].
Murine OT1 T Cell Proliferation Assay: Lymph node T cells from OT1 TCR transgenic wild-type (WT) or PRS knock-in (KI-PRS) mice were labeled with CellTrace Violet. They were pre-incubated for 1 hour with different concentrations of AX-024 HCl and then stimulated for 3 days with bone marrow-derived dendritic cells loaded with various OVA peptide derivatives: the weak agonist Q4H7 (10 nM) or the strong agonist OVAp (100 pM). Proliferation was analyzed by dye dilution. The compound potently inhibited WT T cell proliferation in response to Q4H7 at picomolar concentrations but required much higher concentrations to inhibit responses to stronger agonists. It showed no additional inhibitory effect on KI-PRS T cells [1].
Western Blot Analysis of TCR Signaling: Enriched human T cells or Jurkat cells were stimulated with soluble anti-CD3 antibody (OKT3, 10 μg/ml) for various times (e.g., 5 min) in the presence or absence of AX-024 HCl (1 nM to 10 μM). Post-nuclear lysates were prepared and analyzed by Western blotting with specific antibodies. The compound inhibited the phosphorylation of ZAP70 on Tyr319 and partially inhibited the phosphorylation of CD3ζ on Tyr142. In pull-down assays, post-nuclear lysates from stimulated or unstimulated Jurkat cells were incubated with GST-Nck(SH3.1), GST-Eps8L1(SH3), or GST alone, along with various concentrations of AX-024. Bound proteins were detected by Western blot, showing that AX-024 specifically inhibited the binding of CD3ζ to Nck(SH3.1) [1].
Human CD4+ T Cell Differentiation Assay: Naïve human CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) for 3 days under various polarizing conditions (Th1, Th2, Th17, Treg) in the presence of 0, 1, or 100 nM AX-024 HCl. After washing, cells were cultured for two additional days without stimulation. On day 5, cells were restimulated with PMA and ionomycin, and the intracellular expression of lineage-specific cytokines and FOXP3 was analyzed by flow cytometry. The results showed a shift from pro-inflammatory subsets towards a Treg phenotype [1].

Acute Toxicity Protocol:** Eight-week-old CD-1 mice were injected intraperitoneally with different amounts of the hydrochloride salt of AX-024 HCl dissolved in 0.5 ml of saline. Animals were observed clinically for adverse reactions twice daily over 14 days. A necropsy was performed on each animal on day 14 to examine abdominal, thoracic, and cranial cavities and associated organs [1].
* **EAE Model Protocol (Prophylactic):** Chronic EAE was induced in female C57BL/6 mice by subcutaneous injection of MOG_35-55 emulsified in Freund's complete adjuvant. Mice received daily intraperitoneal (or oral, as specified) doses of AX-024 HCl (e.g., 10 mg/kg, prepared in PBS or saline) for 10 days starting on the day of immunization. Clinical scores and body weight were monitored daily by an observer blind to the treatment groups [1].
* **EAE Model Protocol (Therapeutic):** EAE was induced as above. Treatment began when more than 50% of the animals reached a clinical score higher than 2 (around day 13). Mice received daily oral doses of AX-024 HCl (10 mg/kg) or fingolimod (0.6 mg/kg) from day 13 to day 26. Clinical scores and body weight were monitored daily [1].
* **Psoriasis Model Protocol (IMQ-induced):** Psoriasis-like skin inflammation was induced in BALB/c mice by daily application of IMQ cream on the shaved back and right ear for 6 consecutive days. AX-024 HCl (50 mg/kg in saline) was administered intraperitoneally on days 0, 2, 4, and 6. Disease severity was scored daily, and skin samples were collected for histological and cytokine analysis at the end of the study [1].
* **Asthma Model Protocol (OVA-induced):** BALB/c mice were sensitized with intraperitoneal injections of OVA in alum on days 0, 5, and 12. AX-024 HCl (50 mg/kg in saline) was injected intraperitoneally on days 0 and 5, 2 hours before OVA administration. On day 12, mice were challenged with an aerosolized OVA solution. Lung inflammation was assessed 24 hours post-challenge by FMT imaging and 48 hours post-challenge by analysis of bronchoalveolar lavage fluid (BALF) [1].
* **Ectromelia Virus Infection Protocol:** C57BL/6 mice were infected intranasally with various doses (10² to 10⁵ PFU) of ECTV. Mice were treated with daily oral doses of AX-024 HCl (10 or 40 mg/kg) or vehicle for up to 10 days post-infection. Survival was monitored. Mice surviving the initial infection were rechallenged 60 days later with a lethal intranasal dose (10⁵ PFU) of ECTV without further treatment to assess memory response [1].

---

Acute Toxicity Protocol: Eight-week-old CD-1 mice were injected intraperitoneally with different amounts of the hydrochloride salt of AX-024 HCl dissolved in 0.5 ml of saline. Animals were observed clinically for adverse reactions twice daily over 14 days. A necropsy was performed on each animal on day 14 to examine abdominal, thoracic, and cranial cavities and associated organs [1].
EAE Model Protocol (Prophylactic): Chronic EAE was induced in female C57BL/6 mice by subcutaneous injection of MOG_35-55 emulsified in Freund's complete adjuvant. Mice received daily intraperitoneal (or oral, as specified) doses of AX-024 HCl (e.g., 10 mg/kg, prepared in PBS or saline) for 10 days starting on the day of immunization. Clinical scores and body weight were monitored daily by an observer blind to the treatment groups [1].
EAE Model Protocol (Therapeutic): EAE was induced as above. Treatment began when more than 50% of the animals reached a clinical score higher than 2 (around day 13). Mice received daily oral doses of AX-024 HCl (10 mg/kg) or fingolimod (0.6 mg/kg) from day 13 to day 26. Clinical scores and body weight were monitored daily [1].
Psoriasis Model Protocol (IMQ-induced): Psoriasis-like skin inflammation was induced in BALB/c mice by daily application of IMQ cream on the shaved back and right ear for 6 consecutive days. AX-024 HCl (50 mg/kg in saline) was administered intraperitoneally on days 0, 2, 4, and 6. Disease severity was scored daily, and skin samples were collected for histological and cytokine analysis at the end of the study [1].
Asthma Model Protocol (OVA-induced): BALB/c mice were sensitized with intraperitoneal injections of OVA in alum on days 0, 5, and 12. AX-024 HCl (50 mg/kg in saline) was injected intraperitoneally on days 0 and 5, 2 hours before OVA administration. On day 12, mice were challenged with an aerosolized OVA solution. Lung inflammation was assessed 24 hours post-challenge by FMT imaging and 48 hours post-challenge by analysis of bronchoalveolar lavage fluid (BALF) [1].
Ectromelia Virus Infection Protocol: C57BL/6 mice were infected intranasally with various doses (10² to 10⁵ PFU) of ECTV. Mice were treated with daily oral doses of AX-024 HCl (10 or 40 mg/kg) or vehicle for up to 10 days post-infection. Survival was monitored. Mice surviving the initial infection were rechallenged 60 days later with a lethal intranasal dose (10⁵ PFU) of ECTV without further treatment to assess memory response [1].

AX-024 HCl is described as an orally available, low-molecular weight inhibitor [1].

Acute Toxicity in Rodents: In acute toxicity studies, single intraperitoneal administration of AX-024 HCl to CD-1 mice at doses as high as 1600 mg/kg did not result in any macroscopically visible adverse reactions or death over a 14-day observation period. Necropsy on day 14 revealed no abnormalities in the abdominal, thoracic, or cranial cavities. The compound is reported to have a low acute toxicity profile [1].
Effect on Thymocyte Differentiation: Administration of AX-024 HCl to 4-week-old mice for 10 days did not have a significant impact on thymocyte subset numbers, suggesting that it does not seriously affect thymic differentiation [1].

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

AX-024 HCl is a first-in-class, orally available small-molecule inhibitor that targets a protein-protein interaction between the TCR and the adaptor Nck, specifically by binding to an SH3 domain. It selectively modulates TCR signaling, inhibiting T cell activation by weak (e.g., self-) antigens but not by strong (e.g., pathogen-derived) antigens. This selectivity allows it to prevent and treat autoimmune diseases (psoriasis, asthma, multiple sclerosis) in animal models without causing generalized immunosuppression or increasing susceptibility to viral infection. The compound promotes the differentiation of T cells towards an anti-inflammatory regulatory T cell (Treg) phenotype. An Investigational Medicinal Product Dossier has been issued and approved, and phase Ia/Ib clinical trials have already been conducted (NCT02243683, NCT02546635) [1].

C21H23CLFNO2

375.864228487015

375.14

C, 67.11; H, 6.17; Cl, 9.43; F, 5.05; N, 3.73; O, 8.51

1704801-24-0

AX-024;1370544-73-2

129909862

White to off-white solid powder

1

4

4

26

480

0

COC1=CC2=C(C=C1)OCC(=C2C3=CC=C(C=C3)F)CN4CCCC4.Cl

DGBCIMPGYRVTPD-UHFFFAOYSA-N

InChI=1S/C21H22FNO2.ClH/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;1H

1-[[4-(4-fluorophenyl)-6-methoxy-2H-chromen-3-yl]methyl]pyrrolidine;hydrochloride

AX-024 hydrochloride; AX-024; AX 024; AX024 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~75 mg/mL (~199.5 mM)
Water: ~4 mg/mL (~10.6 mM)
Ethanol: ~18 mg/mL (~47.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.65 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 (6.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (6.65 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)