Cl-amidine
(Synonyms: N-[(1S)-1-(氨基羰基)-4-[(2-氯-1-亚氨基乙基氨基]丁基]-苯甲酰胺) 目录号 : GC35706
Cl-amidine is an irreversible pan-peptidylarginine deiminase (PAD) inhibitor with IC50 values of 5.9 ± 0.3 μM, 0.8 ± 0.3 μM, 6.2 ± 1.0 μM for PAD4, PAD1 and PAD3, respectively. Cl-amidine induces apoptosis.
Cas No.:913723-61-2
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >98.00%
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- Datasheet
| Cell experiment [1]: | |
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Cell lines |
HCT 116 wild-type (WT) colon cancer cells, HCT 116 p53−/− colon carcinoma cells, LS-180 colon cancer cells |
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Preparation Method |
HCT 116 WT and p53−/− cells were cultured in McCoy's medium supplemented with 10% Newborn Calf Serum, 2 mM glutamine, penicillin (10 U/ml) and streptomycin (10 μg/ml). LS-180 colon cancer cells were cultured in DMEM Medium supplemented with 10% Newborn Calf Serum, 2 mM glutamine, penicillin (10 U/mL) and streptomycin (10 μg/mL). |
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Reaction Conditions |
1×106 were incubated in 6 well plates in McCoy's 5A medium 1 day before treatment and then treated with 50 µg/mL Cl-amidine. The cells were harvested and fixed with ice cold ethanol. After washing with 1x PBS containing 0.5% BSA twice, the cells were incubated with DNA staining solution consisting of Propidium Iodide (PI; 10 µg/mL) and RNase (0.1 mg/mL) for 30 min at room temperature in the dark. |
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Applications |
Cl-amidine could lead to a cell cycle arrest at the G1 phase through a p53-mediated mechanism. Cl-amidine could cause miRNA-16 induction in a p53-dependent manner. |
| Animal experiment [2]: | |
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Animal models |
Male C57BL/6J mice (18–26 g) |
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Preparation Method |
Mice were injected with intra-peritoneal Cl-amidine dissolved in DMSO, or vehicle DMSO 1 h after cecal ligation and puncture (CLP). DMSO was also injected into mice that were not subjected to CLP (n = 12/group). Mortality was monitored for 10 days after surgeries. |
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Dosage form |
40 mg/kg |
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Applications |
Cl-amidine protects mice from sepsis-induced lethality. The inhibition of PAD by Cl-amidine significantly suppresses the CitH3 production, while the CitH3 neutralization markedly improves the survival in septic mice. |
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References: [1]. Cui X, et al. The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest. PLoS One. 2013;8(1):e53791. [2]. Ting Z, et al. Protective effect of Cl-amidine against CLP-induced lethal septic shock in mice. Sci Rep. 2016; 6: 36696. |
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Cl-amidine is an irreversible pan-peptidylarginine deiminase (PAD) inhibitor with IC50 values of 5.9 ± 0.3 μM, 0.8 ± 0.3 μM, 6.2 ± 1.0 μM for PAD4, PAD1 and PAD3, respectively. Cl-amidine induces apoptosis.
Cl-amidine antagonizes the PAD4-mediated enhancement of the the p300GBD-GRIP1 interaction in a dose-dependent manner. The inhibitory effect of this compound is not a nonspecific one but is targeted at the active PAD4 enzyme[1]. Cl-amidine increases p53 expression in CD45 positive immune cells. It triggers the differentiation and apoptosis of multiple cancer cell lines that are p53+/+ and p53?/? (e.g., HL60, HT29, TK6, and U2-OS cells). Cl-amidine induces the expression of p53 and several downstream target genes including the cyclin dependent kinase inhibitor p21, GADD45, and the proapoptotic protein PUMA in U2-OS osteosarcoma cells[2].
Cl-amidine treatment inhibits NZM(New Zealand mixed 2328) NET(neutrophil extracellular trap) formation in vivo and significantly alters circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increases the differentiation capacity of bone marrow endothelial progenitor cells, improves endothelium-dependent vasorelaxation, and markedly delays time to arterial thrombosis induced by photochemical injury. Cl-amidine delays thrombosis development in NZM mice. It inhibits PADs in mice without significant toxicity and improves disease phenotypes in animal models of inflammatory arthritis and inflammatory bowel disease[3]. And It is shown to reduce disease severity in mouse models of ulcerative colitis and RA[2].
[1] Luo Y, et al. Biochemistry. 2006, 45(39):11727-36. [2] Knuckley B, et al. Biochemistry. 2010, 49(23):4852-63. [3] Knight JS, et al. J Clin Invest. 2013, 123(7):2981-93.
| Cas No. | 913723-61-2 | SDF | |
| 别名 | N-[(1S)-1-(氨基羰基)-4-[(2-氯-1-亚氨基乙基氨基]丁基]-苯甲酰胺 | ||
| Canonical SMILES | O=C(N[C@H](C(N)=O)CCCNC(CCl)=N)C1=CC=CC=C1 | ||
| 分子式 | C14H19ClN4O2 | 分子量 | 310.78 |
| 溶解度 | Soluble in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.2177 mL | 16.0886 mL | 32.1771 mL |
| 5 mM | 0.6435 mL | 3.2177 mL | 6.4354 mL |
| 10 mM | 0.3218 mL | 1.6089 mL | 3.2177 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Controlled Delivery of Pan-PAD-Inhibitor Cl-amidine Using Poly(3-Hydroxybutyrate) Microspheres
Int J Mol Sci 2021 Nov 27;22(23):12852.PMID:34884657DOI:10.3390/ijms222312852.
This study deals with the process of optimization and synthesis of Poly(3-hydroxybutyrate) microspheres with encapsulated Cl-amidine. Cl-amidine is an inhibitor of peptidylarginine deiminases (PADs), a group of calcium-dependent enzymes, which play critical roles in a number of pathologies, including autoimmune and neurodegenerative diseases, as well as cancer. While Cl-amidine application has been assessed in a number of in vitro and in vivo models; methods of controlled release delivery remain to be investigated. P(3HB) microspheres have proven to be an effective delivery system for several compounds applied in antimicrobial, wound healing, cancer, and cardiovascular and regenerative disease models. In the current study, P(3HB) microspheres with encapsulated Cl-amidine were produced in a size ranging from ~4-5 µm and characterized for surface morphology, porosity, hydrophobicity and protein adsorption, in comparison with empty P(3HB) microspheres. Cl-amidine encapsulation in P(3HB) microspheres was optimized, and these were found to be less hydrophobic, compared with the empty microspheres, and subsequently adsorbed a lower amount of protein on their surface. The release kinetics of Cl-amidine from the microspheres were assessed in vitro and expressed as a function of encapsulation efficiency. There was a burst release of ~50% Cl-amidine in the first 24 h and a zero order release from that point up to 16 days, at which time point ~93% of the drug had been released. As Cl-amidine has been associated with anti-cancer effects, the Cl-amidine encapsulated microspheres were assessed for the inhibition of vascular endothelial growth factor (VEGF) expression in the mammalian breast cancer cell line SK-BR-3, including in the presence of the anti-proliferative drug rapamycin. The cytotoxicity of the combinatorial effect of rapamycin with Cl-amidine encapsulated P(3HB) microspheres was found to be 3.5% more effective within a 24 h period. The cells treated with Cl-amidine encapsulated microspheres alone, were found to have 36.5% reduction in VEGF expression when compared with untreated SK-BR-3 cells. This indicates that controlled release of Cl-amidine from P(3HB) microspheres may be effective in anti-cancer treatment, including in synergy with chemotherapeutic agents. Using controlled drug-delivery of Cl-amidine encapsulated in Poly(3-hydroxybutyrate) microspheres may be a promising novel strategy for application in PAD-associated pathologies.
Cl-amidine Improves Survival and Attenuates Kidney Injury in a Rabbit Model of Endotoxic Shock
Surg Infect (Larchmt) 2021 May;22(4):421-426.PMID:32833601DOI:10.1089/sur.2020.189.
Objective: Sepsis causes millions of deaths on a global scale annually. Activation of peptidylarginine deiminase (PAD) enzymes in sepsis causes citrullination of histones, which results in neutrophil extracellular trap formation and sepsis progression. This study evaluates pan-PAD inhibitor, Cl-amidine, in a model of lipopolysaccharide (LPS)-induced endotoxic shock in rabbits. We hypothesized that Cl-amidine would improve survival and attenuate kidney injury. Methods: In the survival model, rabbits were injected injected intravenously with 1 mg/kg of LPS, and then randomly assigned either to receive dimethyl sulfoxide (DMSO; 1 mcL/g) or Cl-amidine (10 mg/kg diluted in 1 mcL/g DMSO). They were then monitored for 14 days to evaluate survival. In the non-survival experiment, the same insult and treatment were administered, however; the animals were euthanized 12 hours after LPS injection for kidney harvest. Acute kidney injury (AKI) scoring was performed by a histopathologist who was blinded to the group assignment. Serial blood samples were also collected and compared. Results: Rabbits that received Cl-amidine had a higher survival (72%) compared with the rabbits that received DMSO (14%; p < 0.05). Cl-amidine-treated rabbits had lower (p < 0.05) histopathologic AKI scores, as well as plasma creatinine and blood urea nitrogen (BUN) levels 12 hours after insult. Conclusions: Pan-PAD inhibitor Cl-amidine improves survival and attenuates kidney injury in LPS-induced endotoxic shock in rabbits.
Cl-amidine attenuates lipopolysaccharide-induced mouse mastitis by inhibiting NF-κB, MAPK, NLRP3 signaling pathway and neutrophils extracellular traps release
Microb Pathog 2020 Dec;149:104530.PMID:32980473DOI:10.1016/j.micpath.2020.104530.
Cl-amidine, a peptidylarginine deiminase inhibitor, has been shown to ameliorate the disease course and clinical manifestation in variety of disease models. Due to the beneficial effects of Cl-amidine, it has been becoming the hottest compound for the study in inflammatory diseases. However, the anti-inflammatory activity of Cl-amidine in lipopolysaccharide (LPS)-induced mouse mastitis remains unclear. In this study, we investigated the effects of Cl-amidine on LPS-induced mastitis mouse model. The mouse mastitis model was established by injection of LPS through the canals of the mammary gland. Cl-amidine was administered intraperitoneally 1 h before LPS treatment. The results showed that Cl-amidine significantly attenuated the damage of the mammary gland, which suppressed the activity of myeloperoxidase (MPO). The real-time PCR results indicated that Cl-amidine inhibited the production of TNF-α, IL-1β and IL-6 in LPS-induced mouse mastitis. Moreover, the western blot results indicated that Cl-amidine decreased the phosphorylation of IκB, p65, p38, ERK and the expression of NLRP3 in LPS-induced mouse mastitis. Furthermore, the neutrophils extracellular traps (NETs) were determined by Quant-iT picogreen dsDNA assay kit®, which suggested that Cl-amidine significantly inhibited the NETs in mouse serum. This study demonstrated that Cl-amidine decreased the pathological injury in LPS-induced mouse mastitis by inhibiting NF-κB, MAPK, NLRP3 signaling pathway and NETs release, which provides a potential candidate for the treatment of mastitis.
Cl-amidine attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways
Hum Cell 2023 Jan;36(1):223-233.PMID:36352311DOI:10.1007/s13577-022-00822-1.
Cl-amidine has been reported to have anti-inflammatory properties in a variety of diseases. However, the role of Cl-amidine in periodontal disease remains unclear. Here, the purpose of this study was to investigate the effect of Cl-amidine on lipopolysaccharide (LPS)-induced inflammation in human gingival fibroblasts (HGFs). The cytotoxic effect of Cl-amidine was measured with the Cell Counting Kit-8 (CCK-8) assay and Annexin V-FITC/PI staining. The protein levels of IL-6 and IL-8 in culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines, TLR4 and MyD88 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression patterns of IL-6, TNF-ɑ, and IL-1β in HGFs were tested with western blot. The levels of NF-κB, MAPK, and Nrf2 pathway-related proteins were detected by western blot. Immunofluorescence (IF) staining was used to examine the nuclear translocation of NF-κB p65. Moreover, a rat gingivitis model was established to further clarify the role of Cl-amidine. Our results showed that Cl-amidine suppressed LPS-induced gingival inflammation both in vitro and in vivo. Mechanistically, Cl-amidine inhibited LPS-induced MyD88 expression, NF-κB activation, and JNK phosphorylation. Additionally, Cl-amidine upregulated Nrf2 and Ho-1 expression both with and without LPS stimulation but did not alter ROS levels or Keap1 expression. Overall, our data suggest that Cl-amidine acts as an inhibitor of LPS-induced gingival inflammation via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.
PAD4 Deficiency Improves Bleomycin-induced Neutrophil Extracellular Traps and Fibrosis in Mouse Lung
Am J Respir Cell Mol Biol 2020 Dec;63(6):806-818.PMID:32915635DOI:10.1165/rcmb.2019-0433OC.
Excessive release of neutrophil extracellular traps (NETs) has been implicated in several organ fibrosis, including pulmonary fibrosis. NETs constitute a phenomenon in which decorated nuclear chromatin with cytosolic proteins is released into the extracellular space. PAD4 (peptidylarginine deiminase 4) plays an important role in the formation of NETs. However, the role of NETs in the pathogenesis of pulmonary fibrosis remains undefined. Here, we identified NETs in the alveolar and interstitial lung space of mice undergoing bleomycin (BLM)-induced lung fibrosis, which was suppressed by a pan-PAD inhibitor, Cl-amidine. In vitro, BLM directly induced NETs in blood neutrophils, which was also inhibited by Cl-amidine. Furthermore, Padi4 gene knockout (PAD4-KO) in mice led to the alleviation of BLM-induced NETs and pulmonary fibrosis and to the expression of inflammatory and fibrotic genes. PAD4 deficiency prevented decreases in alveolar epithelial and pulmonary vascular endothelial cell numbers and increases in ACTA2-positive mesenchymal cells and S100A4-positive fibroblasts in the lung. Hematopoietic cell grafts from PAD4-KO mice, not wild-type mice, resolved BLM-induced lung fibrosis and fibrotic gene expression in wild-type and PAD4-KO mice, suggesting that expression of PAD4 in hematopoietic cells may be involved in the development of lung fibrosis. These data suggest that PAD4 deficiency could ameliorate BLM-induced formation of NETs and lung fibrosis, suggesting that this pathway could serve as a therapeutic target for pulmonary fibrosis treatment.