| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
EC50: 9.5 μM (PAD2); 0.4 μM (H3 citrullination)[1]
PAD2 (Protein Arginine Deiminase 2). |
|---|---|
| ln Vitro |
Compound 30a, or AFM-30a hydrochloride, exhibits an EC50 of 9.5 μM and high efficacy to penetrate HEK293T/PAD2 cells and covalently alter PAD2[1]. With an EC50 of 0.4 μM, AFM-30a hydrochloride exhibits good capacity to block H3 citrullination[1].
AFM-30a hydrochloride binds to PAD2 with an EC50 of 9.5 microM and penetrates HEK293T/PAD2 cells efficiently to covalently alter the enzyme. It also potently inhibits H3 citrullination (EC50: 0.4 microM), demonstrating superior intracellular activity. |
| ln Vivo |
In PAD2-/-transgenic mice, AFM-30a hydrochloride reduces airway remodeling and inhibits NLRP3 signaling[2].
In vivo data is limited. PAD2 inhibitors like AFM-30a are used to study cancer and autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis. Efficacy is measured by reduction of citrullinated proteins in animal models. |
| Enzyme Assay |
A standard in vitro PAD2 inhibition assay is conducted. Recombinant human PAD2 is incubated with a synthetic substrate (e.g., N-benzoyl-L-arginine ethyl ester) and Ca2+ in Tris buffer. AFM-30a is added to the reaction. The citrulline product is quantified via a colorimetric reaction with diacetyl monoxime and antipyrine.
|
| Cell Assay |
Cell Viability Assay[1]
Cell Types: HEK293T/PAD2 cells Tested Concentrations: Various concentrations Incubation Duration: 24 h Experimental Results: demonstrated low cytotoxicity for cells. HEK293T/PAD2 cells are seeded in 6-well plates and treated with varying concentrations of AFM-30a (1-25 microM) for 6 hours. Cells are lysed in SDS buffer, and proteins are resolved by SDS-PAGE. Western blotting is performed using an anti-citrulline (modified) detection antibody to assess target engagement and inhibition of H3 citrullination. |
| Animal Protocol |
A mouse model of rheumatoid arthritis (Collagen-Induced Arthritis, CIA) is used. Mice are immunized with type II collagen. Once arthritis develops, AFM-30a is administered (e.g., 10-50 mg/kg, IP, daily). Clinical arthritis scores (swelling/redness) are monitored, and paw tissue is collected for histological analysis.
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| ADME/Pharmacokinetics |
PK data for AFM-30a is not published. It is a small molecule with a molecular weight of 502.97, suggesting potential for oral bioavailability. Its mechanism of action involves covalent modification of PAD2, leading to sustained target inhibition even after the compound is cleared from circulation.
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| Toxicity/Toxicokinetics |
No specific toxicity data is available. As a covalent inhibitor of PAD2, potential off-target toxicity could arise from inhibiting other PAD isozymes (PAD1, PAD3, PAD4), though AFM-30a is reported to be highly selective. It is for research use only.
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| References | |
| Additional Infomation |
PAD2-mediated citrullination of histones and other nuclear proteins regulates gene expression. Dysregulation of PAD2 is linked to multiple sclerosis (MS), ulcerative colitis, and rheumatoid arthritis. AFM-30a is a selective chemical probe to dissect the role of PAD2 in these pathologies and is not a clinical drug.
|
| Molecular Formula |
C24H28CLFN6O3
|
|---|---|
| Molecular Weight |
502.97
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| Related CAS # |
AFM-30a;2095107-57-4
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| Appearance |
White to off-white solid powder
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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) |
H2O :~50 mg/mL (~99.41 mM)
DMSO :~50 mg/mL (~99.41 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (9.94 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 50.0 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: ≥ 5 mg/mL (9.94 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 50.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9882 mL | 9.9410 mL | 19.8819 mL | |
| 5 mM | 0.3976 mL | 1.9882 mL | 3.9764 mL | |
| 10 mM | 0.1988 mL | 0.9941 mL | 1.9882 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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