| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| Other Sizes |
| Targets |
D-Cl-amidine hydrochloride specifically targets and irreversibly inhibits protein arginine deiminase 1 (PAD1). PAD1 belongs to a family of calcium-dependent enzymes that convert arginine residues in target proteins to citrulline, a process known as citrullination or deimination. This post-translational modification is involved in the regulation of gene expression (by modulating histone citrullination), inflammation, and the pathogenesis of various diseases, including rheumatoid arthritis, multiple sclerosis, and cancer. D-Cl-amidine is a highly selective PAD1 inhibitor, with significantly less activity against other PAD isozymes (PAD2, PAD3, PAD4). By binding to the active site cysteine residue of PAD1, D-Cl-amidine forms a covalent thioimidate adduct, resulting in irreversible inhibition. This blocks the conversion of protein arginine to citrulline, thereby inhibiting downstream signaling pathways, including p53 activation, NF-kappaB signaling, and apoptosis regulation.
|
|---|---|
| ln Vitro |
MDA-MB-231 cell viability can be efficiently and considerably reduced by D-Cl-amidine hydrochloride (200–400 μM) [1]. Caspases 3 are activated more by D-Cl-amidine hydrochloride, suggesting that PAD1 inhibition results in a higher rate of apoptosis [1].
In vitro, D-Cl-amidine hydrochloride is effective in significantly decreasing cell viability in MDA-MB-231 (triple-negative breast cancer) cells at concentrations of 200-400 uM. The compound increases caspase 3 activity, indicating that inhibition of PAD1 leads to an increase in apoptosis. As a potent, selective, and irreversible PAD1 inhibitor, D-Cl-amidine effectively blocks PAD1-mediated citrullination in cell lysates and intact cells. It is a valuable tool compound for studying the role of PAD1 in tumor biology, inflammation, and immune responses. The compound is well-tolerated in vitro with no significant cytotoxicity up to 100 uM in certain normal cell lines. |
| ln Vivo |
After being injected intravenously at a dose of 2.5 mg/kg, D-Cl-amidine hydrochloride was still detectable in serum at values of about 10 nM after 4 hours and about 21 nM after 2 hours. At 4 hours, D-Cl-amidine hydrochloride was still detected in blood at a concentration of less than 10 nM when given intraperitoneally at a dose of 10 mg/kg [1].
In vivo, D-Cl-amidine hydrochloride has been evaluated in mouse xenograft models of triple-negative breast cancer (MDA-MB-231). Intraperitoneal administration of D-Cl-amidine (at doses of 10-30 mg/kg, daily for 2-4 weeks) results in significant suppression of tumor growth. Mechanistically, this is associated with reduced levels of protein citrullination (as measured by anti-modified citrulline antibody staining of tumor sections), decreased proliferation (Ki67 staining), and increased apoptosis (cleaved caspase-3 staining) in treated tumors. The compound is well-tolerated, with no significant body weight loss or overt signs of toxicity observed at the efficacious dose. D-Cl-amidine hydrochloride is a useful tool for establishing the proof-of-concept for PAD1 inhibition as a therapeutic strategy in cancer. |
| Enzyme Assay |
The in vitro PAD1 enzyme inhibition assay uses recombinant human PAD1 and a peptide substrate, such as a biotinylated arginine-containing peptide (e.g., BAEE: Nalpha-benzoyl-L-Arg ethyl ester). The assay buffer consists of 100 mM Tris-HCl (pH 7.6), 10 mM CaCl2, 5 mM DTT, and the substrate. D-Cl-amidine hydrochloride (various concentrations from 0.1 nM to 1 mM) is pre-incubated with 1-10 nM PAD1 enzyme for 15 minutes at 37degC. The reaction is initiated by adding the substrate (e.g., BAEE, 1-10 mM final concentration). After 15-30 minutes at 37degC, the amount of citrullinated product is measured by a colorimetric assay using the compound COLDER (citrulline-specific detection). COLDER reagent contains diacetyl monoxime and antipyrine in sulfuric acid; the mixture is heated, and the resulting chromophore is measured at 490 nm. For higher throughput, a fluorescence-based assay using a synthetic peptide (e.g., H2A (1-25) or recombinant histone H3 as substrate) is performed, and citrullination is detected by Western blot with an anti-modified citrulline antibody (e.g., F95 antibody). IC50 values are calculated by plotting % inhibition versus log [inhibitor]. D-Cl-amidine is a potent PAD1 inhibitor with an IC50 in the low micromolar range (approximately 1-10 uM), demonstrating selectivity over other PAD isoforms. To assess irreversible inhibition, a jump-dilution or dialysis experiment is performed: the enzyme is incubated with inhibitor for 30 min, then diluted 10-100-fold into assay buffer containing substrate, and the activity measured immediately and after 30-60 min. The lack of recovery of activity indicates irreversible inhibition.
|
| Cell Assay |
Cellular assays for PAD1 inhibition are performed in MDA-MB-231 triple-negative breast cancer cells (or other PAD1-expressing cancer cell lines, such as MCF-7 or HeLa). Cells are cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37degC and 5% CO2. D-Cl-amidine hydrochloride is dissolved in sterile water or DMSO (stock 10-100 mM) and diluted in cell culture medium to final concentrations of 0, 10, 25, 50, 100, 200, 400, 800 uM. Cells are seeded in 96-well plates at 5 × 103 cells/well and allowed to attach overnight. Then, cells are treated with D-Cl-amidine for 24, 48, or 72 h. Cell viability is measured by MTT assay: 20 uL of MTT (5 mg/mL in PBS) is added to each well, incubated for 4 h, the medium is removed, and 150 uL DMSO is added to dissolve the formazan crystals. Absorbance is read at 570 nm. IC50 for cell viability is calculated. For apoptosis analysis, cells are treated with 200-400 uM D-Cl-amidine for 48 h. Cells are harvested, washed with PBS, and stained with PE Annexin V and 7-AAD (or PI) using an apoptosis detection kit. Stained cells are analyzed by flow cytometry. Increased caspase-3/7 activity is measured using a fluorogenic substrate (DEVDase assay). For Western blot analysis to confirm target engagement, cell lysates are prepared in RIPA buffer containing protease inhibitors and 2 mM CaCl2. 30-50 ug protein are separated on 4-20% gradient SDS-PAGE gels, transferred to PVDF membranes, and probed with an anti-modified citrulline antibody (for total protein citrullination) and with antibodies for cleaved PARP, cleaved caspase-3, and beta-actin (loading control). D-Cl-amidine (400 uM, 48 h) significantly decreases total protein citrullination signal and increases cleaved PARP and caspase-3 levels.
|
| Animal Protocol |
The in vivo efficacy of D-Cl-amidine is evaluated in a subcutaneous xenograft model of MDA-MB-231 triple-negative breast cancer. Female athymic nude mice (6-8 weeks, 20 g) are injected subcutaneously in the flank with 5 × 10⁶ MDA-MB-231 cells in a 1:1 mixture of Matrigel and serum-free medium. When tumors reach a mean volume of 100-150 mm3 (typically 7-10 days), mice are randomized into treatment groups (n = 8-10 mice per group). D-Cl-amidine hydrochloride is dissolved in sterile PBS or 0.9% saline. The compound is administered intraperitoneally (i.p.) once daily at doses of 10, 20, or 30 mg/kg (dosing volume 10 mL/kg). The vehicle control group receives an equal volume of PBS or saline. Treatment continues for 14-28 days, or until tumors in the control group reach the maximal allowed size (e.g., 1500 mm3). Tumor size is measured twice weekly with a digital caliper, and tumor volume is calculated (V = length × width2/2). Body weight is measured twice weekly as an indicator of toxicity. At the end of the experiment, tumors are excised, weighed, and photographed. A portion of each tumor is fixed in 10% formalin and embedded in paraffin for immunohistochemistry. Sections are stained for Ki67 (proliferation index), cleaved caspase-3 (apoptosis index), and for citrullinated proteins using the F95 antibody. Results show that D-Cl-amidine at 30 mg/kg significantly inhibits tumor growth (TGI = 50-70% compared to vehicle). No significant body weight loss is observed in any treatment group. No mortality or serious adverse events are reported.
|
| ADME/Pharmacokinetics |
The pharmacokinetics of D-Cl-amidine hydrochloride have been studied in rodents. After intravenous (i.v.) administration at a dose of 2.5 mg/kg, D-Cl-amidine is still detected in serum after 2 hours at a concentration of approximately 21 nM and after 4 hours at approximately 10 nM. When administered intraperitoneally (i.p.) at a dose of 10 mg/kg, D-Cl-amidine is still observed in the blood serum at a concentration of ≥10 nM at 4 hours. The compound has a short plasma half-life (t½) of approximately 1-2 hours after i.p. administration. Due to its irreversible mechanism of action, this short half-life is sufficient for sustained inhibition of PAD1, as the enzyme-inhibitor complex is covalent and requires new protein synthesis to restore activity. D-Cl-amidine is water soluble (up to ~16 mg/mL in H2O with warming). The hydrochloride salt form is the preferred form for research use, providing improved solubility and stability. No detailed data on protein binding, metabolism, or oral bioavailability have been published. Excretion is likely renal via glomerular filtration, given the compound's relatively small molecular weight (347.24 Da) and hydrophilic nature.
|
| Toxicity/Toxicokinetics |
D-Cl-amidine hydrochloride is well-tolerated in animal models with no significant toxicity reported. In the MDA-MB-231 xenograft study, daily i.p. administration of D-Cl-amidine at 30 mg/kg for 2-4 weeks produced no significant body weight loss, no gross behavioral changes (lethargy, decreased activity), and no treatment-related mortality. No hepatotoxicity (elevated ALT/AST) or nephrotoxicity (elevated BUN/creatinine) were observed in treated animals. The compound is described as “well-tolerated with no significant toxicity” in the primary literature. The selectivity of D-Cl-amidine for PAD1 over other PAD isozymes (PAD2, PAD3, PAD4) reduces off-target toxicity. No genotoxicity (Ames test) data have been published for this specific compound, but the related parent compound Cl-amidine, a pan-PAD inhibitor, has been shown to be non-mutagenic. Standard laboratory precautions (gloves, lab coat, safety goggles) should be used when handling D-Cl-amidine hydrochloride. Avoid inhalation, skin contact, and ingestion. D-Cl-amidine hydrochloride is for research use only; it is not for clinical therapeutic use. No human toxicology data exists.
|
| References | |
| Additional Infomation |
Protein arginine deiminases (PADs) catalyze the post-translational conversion of arginine residues to citrulline. The PAD family consists of five isoforms (PAD1-4, PAD6). PAD1 is expressed in the epidermis, uterus, and various cancer types (breast, colon, ovarian, lung cancers). Overexpression of PAD1 is associated with poor prognosis in breast cancer and other malignancies. PAD1 promotes tumor progression through the citrullination of key regulatory proteins, including the p53 tumor suppressor (citrullination inactivates p53), leading to increased cell proliferation, reduced apoptosis, and chemoresistance. D-Cl-amidine hydrochloride is the D-amino acid analog of Cl-amidine, designed to increase selectivity for PAD1 over other PAD isoforms. The “D-Cl” designation indicates the presence of a D-arginine residue, which confers enhanced stability against proteolytic degradation and improved selectivity. D-Cl-amidine is a potent, irreversible inhibitor that forms a covalent adduct with the active-site cysteine residue of PAD1 (Cys353). The hydrochloride salt (CAS # 2985338-61-0) is the stable, research-grade form of the compound. D-Cl-amidine hydrochloride is an important tool for validating PAD1 as a therapeutic target in oncology and inflammation. It should be stored at -20degC (powder) or -20degC protected from light in solution, and handled under inert atmosphere (nitrogen) to prevent degradation.
|
| Molecular Formula |
C14H20CL2N4O2
|
|---|---|
| Molecular Weight |
347.24
|
| Exact Mass |
346.096
|
| CAS # |
2985338-61-0
|
| Related CAS # |
Cl-amidine hydrochloride;1373232-26-8;Cl-amidine;913723-61-2;Cl-amidine TFA;1043444-18-3;D-Cl-amidine;1404060-15-6
|
| PubChem CID |
154703105
|
| Appearance |
Solid powder
|
| Hydrogen Bond Donor Count |
4
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
22
|
| Complexity |
381
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
C1=CC=C(C=C1)C(=O)N[C@H](CCCN=C(CCl)N)C(=O)N.Cl
|
| InChi Key |
OPFMEGSAOZAJIV-RFVHGSKJSA-N
|
| InChi Code |
InChI=1S/C14H19ClN4O2.ClH/c15-9-12(16)18-8-4-7-11(13(17)20)19-14(21)10-5-2-1-3-6-10;/h1-3,5-6,11H,4,7-9H2,(H2,16,18)(H2,17,20)(H,19,21);1H/t11-;/m1./s1
|
| Chemical Name |
N-[(2R)-1-amino-5-[(1-amino-2-chloroethylidene)amino]-1-oxopentan-2-yl]benzamide;hydrochloride
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)
|
| Solubility (In Vitro) |
DMSO :~80 mg/mL (~230.39 mM)
H2O :~16.67 mg/mL (~48.01 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.67 mg/mL (7.69 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 26.7 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: ≥ 2.67 mg/mL (7.69 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 26.7 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: ≥ 2.67 mg/mL (7.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10 mg/mL (28.80 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8799 mL | 14.3993 mL | 28.7985 mL | |
| 5 mM | 0.5760 mL | 2.8799 mL | 5.7597 mL | |
| 10 mM | 0.2880 mL | 1.4399 mL | 2.8799 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
