Cl-amidine TFA, an ingestible inhibitor of peptidylarginine deminase (PAD), exhibits IC50 values of 0.8 μM for PAD1, 6.2 μM for PAD3, and 5.9 μM for PAD4. It prompts apoptosis in cancerous cells and stimulates the expression of microRNA-16, leading to cell cycle arrest. Additionally, Cl-amidine TFA blocks the citrullination of histone 3 and the formation of neutrophil extracellular traps, thereby enhancing survival rates in a mouse model of sepsis[1][2][3][4][5]. Cl-amidine, at varying concentrations (0, 5, 10, 15, 20, 25, 50 μg/mL) over a 24-hour period, triggers cell death through apoptosis in TK6 lymphoblastoid cells and HT29 colon cancer cells, with the effect intensifying with higher doses. Notably, HT29 cells exhibit a comparative resistance to the apoptotic effects induced by Cl-amidine[2]. Cl-amidine permanently deactivates PAD enzymes through the covalent alteration of a crucial cysteine residue within the active site, which is essential for the enzyme's catalytic function[4].
Cl-amidine TFA 细胞实验
Cell Line
Concentration
Treated Time
Description
References
CD68+VSMCs
100 µM
0.5 hours
Inhibit PAD4 activity, reduce ETs generation
Nat Commun. 2022 Dec 6;13(1):7500.
HEK293T/PAD2 cells
25 µM
1 hour
To evaluate the target engagement of Cl-amidine TFA in HEK293T/PAD2 cells, results showed that it effectively entered the cells and covalently modified PAD2 at 25 μM concentration.
J Med Chem. 2017 Apr 13;60(7):3198-3211.
NZM mouse neutrophils
200 µM
12 hours
Cl-amidine inhibited NET release from NZM mouse neutrophils
J Clin Invest. 2013 Jul;123(7):2981-93.
BALB/c mouse neutrophils
200 µM
12 hours
Cl-amidine inhibited NZM serum-induced NET release from BALB/c mouse neutrophils
J Clin Invest. 2013 Jul;123(7):2981-93.
Keratinocytes
0–800 µM
2 days
Cl-amidine inhibited deimination in a dose-dependent manner and was not cytotoxic for keratinocytes. At 800 μM, Cl-amidine 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.
J Invest Dermatol. 2019 Sep;139(9):1889-1897.e4.
Human brain microvascular endothelial cells (HBMVECs)
6 µM
24 hours
To assess the direct effect of Cl-amidine on brain endothelial cell PADs, results showed no significant effect of Cl-amidine treatment on vWF release, indicating no citrullination in brain endothelial cells.
Nat Cardiovasc Res. 2024 Dec;3(12):1549-1567.
Human brain microvascular endothelial cells (HBMVECs)
500 ng/mL
24-72 hours
To assess the effect of NETs on EndMT in brain endothelial cells, results showed that NETs promote EndMT, as evidenced by changes in cell morphology and increased Snail expression.
Nat Cardiovasc Res. 2024 Dec;3(12):1549-1567.
MCF7/TamR cells
200 µM
4 days
To evaluate the inhibitory effect of Cl-amidine on the proliferation of MCF7/TamR cells, results showed that 200 μM Cl-amidine significantly inhibited cell proliferation
J Exp Clin Cancer Res. 2019 Oct 10;38(1):414.
MCF7/TamR cells
50 µM
4 days
To evaluate the inhibitory effect of Cl-amidine combined with tamoxifen on the proliferation of MCF7/TamR cells, results showed that 50 μM Cl-amidine combined with 5 μM tamoxifen significantly inhibited cell proliferation
J Exp Clin Cancer Res. 2019 Oct 10;38(1):414.
MCF7/TamR cells
25 µM
4 days
To evaluate the effect of Cl-amidine combined with docetaxel on the viability of MCF7/TamR cells, results showed that 0.1 μM docetaxel combined with 25 μM Cl-amidine significantly decreased cell viability
J Exp Clin Cancer Res. 2019 Oct 10;38(1):414.
U2OS cells
20 µM
72 hours
To evaluate the antiproliferative activities of Cl-amidine TFA analogs on U2OS cells. Results showed that biphenyl tetrazole tert-butyl Cl-amidine (13) completely abolished the growth of U2OS cells at 20 μM.
J Med Chem. 2015 Feb 12;58(3):1337-44.
Cl-amidine TFA 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
C57BL/6 mice
Traumatic brain injury model
Intraperitoneal injection
10 mg/kg
Once daily for three consecutive days
Inhibition of PAD4 significantly ameliorated neurological deficits in mice and reduced neuronal pyroptosis by inhibiting the STING/IRE1α-NLRP1 pathway
Front Immunol. 2023 Apr 14;14:1125759
NZM mice
NZM mouse lupus model
Subcutaneous injection
10 mg/kg/d
Daily for 14 weeks
Cl-amidine significantly improved vasculogenesis, endothelial function, and prothrombotic phenotype in NZM mice
J Clin Invest. 2013 Jul;123(7):2981-93.
Mice
Ccm3-iECKO mice
Intraperitoneal injection
10 mg/kg/day
Once daily for 7 days
To assess the effect of Cl-amidine on NET formation and CCM lesions, results showed that Cl-amidine significantly reduced NET formation and lesion burden, improving endothelial activation, EndMT, coagulation, and neuroinflammation.
Nat Cardiovasc Res. 2024 Dec;3(12):1549-1567.
Pad4−/− mice
Citrobacter rodentium infection model
Intraperitoneal injection
20 mg/kg
Administered 3 hours prior to infection, followed by once daily for the next 3 consecutive days and then once every alternate day until day 11
Cl-amidine treatment modestly enhanced the susceptibility of WT mice to C. rodentium infection, as evidenced by increased CR burden in feces and worsened colonic histopathology, though to a lesser extent compared to Pad4?/? mice and DNase I-treated mice.
Mucosal Immunol. 2019 May;12(3):761-771
BALB/c Nude mice
Breast cancer xenograft model
Intraperitoneal injection
20 mg/kg/day
Every 3 days for 3 weeks
To evaluate the effect of Cl-amidine combined with docetaxel on tumor growth in a breast cancer xenograft model, results showed that the combination treatment significantly reduced tumor weight
J Exp Clin Cancer Res. 2019 Oct 10;38(1):414.
C57BL/6J mice
DSS-induced colitis model
Gavage
25mg/kg
Once daily for 7 days
To evaluate the effect of Cl-amidine on DSS-induced colitis, the results showed that Cl-amidine alleviated the severity of colitis, reduced weight loss, decreased the disease activity index (DAI) score, and increased colon length.
Front Immunol. 2023 Apr 18;14:1144976
C57BL/6 mice
Traumatic brain injury (TBI) model
Intraperitoneal injection
50 mg/kg
Once daily for three consecutive days
Cl-amidine treatment improved short-term neurological functions, reduced cerebral lesion volume, reduced brain edema, and restored cerebral blood flow (CBF) after TBI. In addition, Cl-amidine exerted neuroprotective effects by attenuating BBB disruption, inhibiting immune cell infiltration, and alleviating neuronal death after TBI.
J Neuroinflammation. 2023 Sep 30;20(1):222
Cl-amidine TFA 动物研究
Animal study
Cl-amidine, administered intraperitoneally at a dose of 75 mg/kg daily, alleviates and manages colitis in mice induced by DSS. When given orally at doses of 5, 25, and 75 mg/kg once a day, Cl-amidine consistently and dose-dependently decreases histological scores. Specifically, a dosage of 75 mg/kg given intraperitoneally each day results in the suppression of PAD activity, protein citrullination, and PAD levels within the colon in vivo[2].
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