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