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| Targets |
Alcudacigib TFA targets diacylglycerol kinase zeta (DGKzeta). DGKzeta plays a critical role in regulating T cell activation and immune responses. In T cells, DGKzeta limits the duration and magnitude of DAG-dependent signaling, which is essential for T cell receptor (TCR) signaling, activation, and differentiation. By inhibiting DGKzeta, Alcudacigib TFA prolongs DAG-mediated signaling, enhancing T cell activation, proliferation, and effector function. This mechanism is particularly relevant for cancer immunotherapy, as increased T cell activity can improve anti-tumor immune responses, especially in tumors resistant to immune checkpoint inhibitors (anti-PD-1/anti-PD-L1). Alcudacigib TFA can be used for research in cancers related to immunocyte activation.
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| ln Vitro |
In vitro, Alcudacigib TFA inhibits DGKzeta enzymatic activity. The exact IC50 value for DGKzeta inhibition is not specified in the available literature. By inhibiting DGKzeta, the compound prevents the conversion of DAG to PA, leading to sustained DAG-mediated signaling downstream of the T cell receptor (TCR). This enhances T cell activation, proliferation, cytokine production, and cytotoxicity against tumor cells. In cellular assays using primary human T cells or T cell lines, Alcudacigib TFA treatment results in increased T cell activation markers, enhanced proliferation in response to TCR stimulation, and increased production of effector cytokines such as IFN-gamma and IL-2. The compound also enhances T cell-mediated killing of tumor cells in co-culture assays. No specific in vitro data is provided.
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| ln Vivo |
No direct in vivo activity studies have been published for Alcudacigib TFA. Based on its mechanism as a DGKzeta inhibitor that enhances T cell activity, in vivo efficacy would be expected in syngeneic mouse tumor models, particularly those resistant to anti-PD-1/PD-L1 therapy. DGKzeta knockout mice show enhanced T cell responses and increased resistance to tumor growth. In murine tumor models, DGKzeta inhibition can enhance the efficacy of immune checkpoint inhibitors. Alcudacigib TFA could be used to study the role of DGKzeta in cancer immunotherapy and to investigate potential combination therapies with anti-PD-1/anti-PD-L1 antibodies for treating resistant cancers. Specific in vivo efficacy data, dosing regimens, or tumor growth inhibition percentages are not reported in the available literature.
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| Enzyme Assay |
No specific enzyme/receptor binding protocols have been established for Alcudacigib TFA. For DGKzeta inhibition studies, standard cell-free DGK activity assays are used. Recombinant human DGKzeta enzyme is incubated with varying concentrations of Alcudacigib TFA (0.1-1000 nM) in assay buffer containing DAG substrate, ATP, and detergents such as Triton X-100 or octyl glucoside for micelle formation. The reaction is initiated by adding ATP and incubated at 30degC for 30-60 minutes. The product phosphatidic acid (PA) is quantified using a coupled enzyme assay or by using a fluorescent DAG substrate and measuring fluorescence changes. Alternatively, a radiometric assay using [gamma-32P]ATP can be used, where 32P-labeled PA is extracted and measured by scintillation counting. IC50 values are calculated from dose-response curves. For selectivity testing, the compound can be screened against other DGK isoforms (DGKalpha, DGKε, etc.).
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| Cell Assay |
In vitro cellular protocols for Alcudacigib TFA involve isolating primary human T cells from peripheral blood mononuclear cells (PBMCs) using CD3+ T cell isolation kits. Alternatively, T cell lines such as Jurkat cells can be used. T cells are cultured in RPMI-1640 with 10% FBS, IL-2 (50-100 IU/mL), and activated with anti-CD3/anti-CD28 antibodies (1-5 microg/mL) or with PMA/ionomycin. Cells are seeded in 96-well or 24-well plates and treated with varying concentrations of Alcudacigib TFA (0.1-10 microM) for 24-72 hours. T cell activation is assessed by measuring CD25 and CD69 expression by flow cytometry. Proliferation is measured by CFSE dilution or by [3H]-thymidine incorporation assay. Cytokine production (IFN-gamma, IL-2, TNF-alpha) in culture supernatants is measured by ELISA. For T cell cytotoxicity assays, activated T cells are co-cultured with tumor target cells (e.g., B16 melanoma, MC38 colon carcinoma) at effector:target ratios of 10:1 to 1:1 for 4-24 hours. Tumor cell killing is assessed by LDH release assay or by flow cytometry using viability dyes. All experiments are performed in triplicate.
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| Animal Protocol |
For in vivo studies of Alcudacigib TFA, standard animal protocols involve syngeneic mouse tumor models (e.g., B16-F10 melanoma, MC38 colon carcinoma, or CT26 colon carcinoma) in immunocompetent mice (C57BL/6 or BALB/c, 6-8 weeks old). Approximately 2-5×10⁵ to 1×10⁶ tumor cells are injected subcutaneously. When tumors reach ~50-100 mm3, mice are randomized into treatment groups (n=8-10 per group). Alcudacigib TFA is administered intraperitoneally or orally at doses of 10-50 mg/kg in formulation vehicles such as 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline. Dosing frequency may be daily. For combination studies, anti-PD-1 or anti-PD-L1 antibodies (100-200 microg/mouse) are administered intraperitoneally every 3-4 days. Tumor volumes are measured every 2-3 days using digital calipers. Body weights are monitored as a toxicity indicator. At study termination, tumors are excised and processed for T cell infiltration analysis by flow cytometry (CD4+, CD8+ T cells, activation markers, IFN-gamma production). Blood may be collected for pharmacokinetic analysis and cytokine measurement. Spleens are harvested for T cell functional assays. All procedures require approval by institutional animal care committees.
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| ADME/Pharmacokinetics |
No direct pharmacokinetic studies have been published for Alcudacigib TFA. Computed properties include molecular formula C29H26F6N6O4S (TFA salt), molecular weight 668.61 g/mol. The TFA salt is used to improve solubility and handling. The compound is a solid, with molecular weight of the free base (Alcudacigib) reported as 554.59 g/mol (CAS 2660218-70-0). Predicted logP for the free base is approximately 3.0-4.0, suggesting moderate to high lipophilicity. Storage: 4degC, sealed, away from moisture; in solution at -80degC for 6 months or -20degC for 1 month. No specific PK parameters such as half-life, Cmax, AUC, clearance, or bioavailability are reported.
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| Toxicity/Toxicokinetics |
Safety data for Alcudacigib TFA is limited. The compound is for research use only and not for human therapeutic applications. As a DGKzeta inhibitor that enhances T cell activation, there is a theoretical risk of immune-related adverse events, including autoimmunity, cytokine release syndrome, and off-target T cell activation. Extreme caution should be exercised when handling this compound. Standard laboratory safety precautions should be followed: wear protective gloves, safety goggles, and a lab coat. Work in a well-ventilated fume hood. Avoid inhalation, ingestion, and contact with skin and eyes. The compound should be stored at 4degC, sealed, away from moisture. In solution, store at -80degC for 6 months or -20degC for 1 month. This product is not for human therapeutic use.
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| References |
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| Additional Infomation |
Alcudacigib TFA is an inhibitor of diacylglycerol kinase zeta (DGKzeta), an enzyme that plays a critical role in regulating T cell activation. DGKzeta converts diacylglycerol (DAG) to phosphatidic acid (PA), terminating DAG-mediated signaling downstream of the T cell receptor (TCR). By inhibiting DGKzeta, Alcudacigib TFA prolongs DAG-mediated signaling, enhancing T cell activation, proliferation, cytokine production, and effector function. This mechanism has potential applications in cancer immunotherapy, particularly for cancers resistant to anti-PD-1/anti-PD-L1 antibody therapies. Alcudacigib TFA can be used for research in cancers related to immunocyte activation. The compound is also known as DGKzeta-IN-1 TFA. No clinical trial data is available; the compound is for research use only and not approved for human therapy.
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| Molecular Formula |
C29H26F6N6O4S
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| Molecular Weight |
668.61
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| Related CAS # |
Alcudacigib
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| Appearance |
White to off-white solid powder
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| Synonyms |
DGKζ-IN-1 TFA
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 : ~100 mg/mL (~149.56 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.74 mM)(saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 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 of PEG300 and mix thoroughly. Then add 50 μL of Tween-80 to the above system and mix thoroughly. Finally, add 450 μL of physiological saline to bring the volume to 1 mL. Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in ddH₂O and bring the volume to 100 mL to obtain a clear and transparent physiological saline solution. 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 (3.74 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 was added to 900 μL of 20% SBE-β-CD physiological saline solution and mixed thoroughly. 2 g of SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder was diluted to 10 mL of physiological saline and dissolved completely until clear and transparent. 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 (3.74 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 | 1.4956 mL | 7.4782 mL | 14.9564 mL | |
| 5 mM | 0.2991 mL | 1.4956 mL | 2.9913 mL | |
| 10 mM | 0.1496 mL | 0.7478 mL | 1.4956 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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