Cl-Amidine (trifluoroacetate salt)
目录号 : GC11032
Cl-Amidine (trifluoroacetate salt)是一种多靶点肽精氨酸脱胺酶（PAD）抑制剂，功能基团为RC(NR)NR2，可抑制PAD1 （IC50=0.8μM）、PAD3 （IC50=6.2μM）和PAD4 (IC50=5.9μM)，半衰期为15min。
Cas No.:1043444-18-3
Sample solution is provided at 25 µL, 10mM.
Cl-Amidine (trifluoroacetate salt) is a multi-targeting peptidylarginine deminase (PAD) inhibitor with the functional group RC(NR)NR2 that inhibits PAD1 (IC50=0.8μM), PAD3 (IC50=6.2μM), and PAD4 (IC50=5.9μM) with 15min half-time[1]. PAD inhibitors inhibit PAD enzymes from blocking arginine-citrulline conversion during a post-translational modification, leading to apoptosis induction through inhibiting neutrophil extracellular trap (NET) formation, diminishing chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL2 level, and other mechanisms[2]. A structural analogue of PAD inhibitor Cl-Amidine (trifluoroacetate salt), F-amidine, inhibits PAD4 by forming AD4–F-amidine·calcium complex to modify the Cys645 of PAD4[3].
In vitro experiments show that Cl-Amidine (trifluoroacetate salt) attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signaling pathways (HGFs from healthy donors; 60μM; 24h)[4]. Cl-Amidine (trifluoroacetate salt) also inhibits mice undergoing bleomycin-induced NETs (isolated mouse Neutrophils; 100μM; 2.5h) in blood neutrophils[5]. Moreover, Cl-Amidine (trifluoroacetate salt) attenuates cornification and interferes with the regulation of autophagy (human epidermis produced from primary normal human keratinocytes; 800μM; 48h) in the reconstructed human epidermis[6].
In vivo experiments show that Cl-Amidine (trifluoroacetate salt) treatment prior to CLP improves overall survival in sepsis, and prevents histone-3 citrullination and NET formation of C57BL/6 male mice (50mg/kg with PBS; once a day for 7 days; subcutaneous injection) [7]. Cl-Amidine (trifluoroacetate salt) also normalizes exacerbated ischemia-reperfusion (I/R) injury in Il36rn-/- mice (10 mg/kg; once; i.p)[8]. Cl-Amidine (trifluoroacetate salt) improves survival and attenuates kidney injury in LPS-induced endotoxic shock (10mg/kg diluted in 1mcL/g DMSO; once; i.p) in rabbits[9].
*1mcL/g DMSO indicates DMSO with 1g/ml
References:
[1] Knight, Jason S et al. “Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice.” *Annals of the rheumatic diseases* vol. 74,12 (2015): 2199-206. doi:10.1136/annrheumdis-2014-205365
[2] Mansouri, Pegah et al. “Peptidylarginine deiminase (PAD): A promising target for chronic diseases treatment.” *International journal of biological macromolecules* vol. 278,Pt 3 (2024): 134576. doi:10.1016/j.ijbiomac.2024.134576
[3] Luo, Yuan et al. “Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.” *Biochemistry* vol. 45,39 (2006): 11727-36. doi:10.1021/bi061180d
[4] Du, Jianxin et al. “Cl-amidine attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.” *Human cell* vol. 36,1 (2023): 223-233. doi:10.1007/s13577-022-00822-1
[5] Suzuki, Masaki et al. “PAD4 Deficiency Improves Bleomycin-induced Neutrophil Extracellular Traps and Fibrosis in Mouse Lung.” *American journal of respiratory cell and molecular biology* vol. 63,6 (2020): 806-818. doi:10.1165/rcmb.2019-0433OC
[6] Cau, Laura et al. “Peptidylarginine Deiminase Inhibitor Cl-Amidine Attenuates Cornification and Interferes with the Regulation of Autophagy in Reconstructed Human Epidermis.” The Journal of investigative dermatology vol. 139,9 (2019): 1889-1897.e4. doi:10.1016/j.jid.2019.02.026
[7] Su, Yue et al. “Activation of Cholinergic Anti-Inflammatory Pathway Ameliorates Cerebral and Cardiac Dysfunction After Intracerebral Hemorrhage Through Autophagy.” Frontiers in immunology vol. 13 870174. 23 Jun. 2022, doi:10.3389/fimmu.2022.870174
[8] Tanaka, Y et al. “Cutaneous ischemia-reperfusion injury is exacerbated by IL-36 receptor antagonist deficiency.” *Journal of the European Academy of Dermatology and Venereology : JEADV* vol. 36,2 (2022): 295-304. doi:10.1111/jdv.17767
[9]Siddiqui, Ali Z et al. “Cl-Amidine Improves Survival and Attenuates Kidney Injury in a Rabbit Model of Endotoxic Shock.” *Surgical infections* vol. 22,4 (2021): 421-426. doi:10.1089/sur.2020.189
Cl-Amidine (trifluoroacetate salt)是一种多靶点肽精氨酸脱胺酶（PAD）抑制剂，功能基团为RC(NR)NR2，可抑制PAD1 （IC50=0.8μM）、PAD3 （IC50=6.2μM）和PAD4 (IC50=5.9μM)，半衰期为15min[1]。PAD抑制剂可抑制PAD酶在翻译后修饰过程中阻断精氨酸-瓜氨酸转化来抑制中性粒细胞胞外陷阱（NET）形成、降低CXCL1和CXCL2水平等机制诱导的细胞凋亡[2]。Cl-Amidine (trifluoroacetate salt)结构类似物F-amidine是一种PAD抑制剂，其通过形成AD4–F-amidine·calcium络合物修饰PAD4的Cys645来抑制PAD4 活性[3]。
体外实验表明，Cl-Amidine (trifluoroacetate salt)通过JNK/MAPK、NF-κB和Nrf2信号通路（HGFs来自健康人体; 60μM; 处理24h）减弱脂多糖诱导的人牙龈成纤维细胞炎症[4]。Cl-Amidine (trifluoroacetate salt)还能抑制小鼠血液中性粒细胞中博莱霉素对NETs 的诱导（分离小鼠中性粒细胞; 100μM; 2.5h）[5]。此外，在重建的人表皮中，Cl-Amidine (trifluoroacetate salt)可减弱角化并干扰自噬的调节（原代正常人角质形成细胞产生的人表皮; 800μM; 48小时）[6]。
体内实验表明，CLP前的Cl-Amidine (trifluoroacetate salt)治疗可提高脓毒症的总生存率，并阻止C57BL/6雄性小鼠的组蛋白-3瓜氨酸化和NET形成（50 mg/kg加PBS; 每天1次; 连续7天; 皮下注射）[7]。Cl-Amidine (trifluoroacetate salt)还能使Il36rn-/-小鼠中加重的缺血再灌注（I/R）损伤正常化 (10 mg/kg; 单次; 腹腔注射)[8]。此外，在LPS诱导的内源性休克中，Cl-Amidine (trifluoroacetate salt)（10mg/kg，用1mcL/g DMSO稀释，单次，腹腔注射）可提高兔的存活率并减轻肾损伤[9]。
*1mcL/g DMSO 意为 1g/ml 克质比的 DMSO
| Cell experiment [1]: | |
Cell lines | reconstructed human epidermis |
Preparation Method | Primary normal human keratinocytes were isolated from abdominal skin samples, from five females (24–48 years old) without any history of skin diseases undergoing plastic surgery, obtained from Genoskin (Toulouse, France), following written informed consent of the donors and as agreed by the French Ministry of Research (#AC-2017–2897). Five keratinocyte banks were thus constituted. RHEs were produced in a humidified atmosphere with 5% CO2 at 37°C. RHEs were treated with Cl-Amidine (trifluoroacetate salt) at different concentrations (0–800μM) for 48h and harvested on day 10 after air-liquid exposure. |
Reaction Conditions | 0–800μM for 48h |
Applications | Cl-Amidine (trifluoroacetate salt) treatments inhibited deimination in a dose-dependent manner and were not cytotoxic for keratinocytes. At 800μM, Cl-Amidine (trifluoroacetate salt) was shown to reduce deimination by half, alter keratinocyte differentiation, decrease the number of corneocyte layers, significantly increase the number of transitional cells, induce clustering of mitochondria and of heterogeneous vesicles in the cytoplasm of granular keratinocytes, and upregulate the expression of autophagy proteins, including LC3-II, sestrin-2 and p62/SQSTM1. |
| Animal experiment [2]: | |
Animal models | C57BL/6 male mice, aged 8–12 weeks |
Preparation Method | Adult C57BL/6 male mice were treated with Cl-Amidine (trifluoroacetate salt), 1h prior to sepsis induced by cecal ligation and puncture (CLP). Twenty-four hours after CLP, cytokine levels, H3cit protein expression, neutrophil counts, and NET production were evaluated in the peritoneal cavity. Survival studies were also performed. |
Dosage form | 50mg/kg with PBS, once a day for 7 days, s.i |
Applications | Cl-Amidine (trifluoroacetate salt) treatment prior to CLP improves overall survival in sepsis and the abrogation of PAD4 has minimal effects on the proinflammatory immune response to sepsis, while it has no effect on overall neutrophil migration to the peritoneum. |
References: | |
| Cas No. | 1043444-18-3 | SDF | |
| 化学名 | N-[(1S)-1-(aminocarbonyl)-4-[(2-chloro-1-iminoethyl)amino]butyl]-benzamide 2,2,2-trifluoroacetate | ||
| Canonical SMILES | O=C(N[C@@H](CCCNC(CCl)=N)C(N)=O)C1=CC=CC=C1.FC(F)(C(O)=O)F | ||
| 分子式 | C14H19ClN4O2 • CF3CO2H | 分子量 | 424.8 |
| 溶解度 | DMF: 14 mg/ml, DMSO: 100 mg/ml, water: 3 mg/ml | 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 | 2.354 mL | 11.7702 mL | 23.5405 mL |
| 5 mM | 0.4708 mL | 2.354 mL | 4.7081 mL |
| 10 mM | 0.2354 mL | 1.177 mL | 2.354 mL |
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| 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。
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