title: Evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1.
abstract: BACKGROUND
  Thioredoxin reductase (TrxR) is up-regulated in a number of human malignant cells and becomes a promising target for anticancer drug development.
  
  
  OBJECTIVE
  To evaluate N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC), a potent anticancer agent against melanoma, as an inhibitor of mammalian TrxR1.
  
  
  METHODS
  The mechanism of inhibition against TrxR1 was investigated using substrate protection, dialysis and liquid chromatography-tandem mass spectrometry.
  
  
  RESULTS
  NACC inhibits TrxR1 in a time and concentration dependent manner. The K(I) and k(inact) of NACC against TrxR1 were determined to be 80 μM and 0.178 min(-1), respectively. The inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis demonstrated that the selenocysteine rather than cysteine residue at the active site was p-chlorophenyl carbamoylated by NACC. Inhibition of intracellular TrxR by NACC in cultured melanoma cells was observed.
  
  
  CONCLUSIONS
  NACC which irreversibly inhibits TrxR1 by forming a covalent bond with selenocysteine can be an effective tool in the study of TrxR1.
authors.affiliation: a Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Zhejiang Cancer Center , Hangzhou , Zhejiang Province , China .|b Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus) , Zhejiang Cancer Hospital , Hangzhou , Zhejiang , China .
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: a Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Zhejiang Cancer Center , Hangzhou , Zhejiang Province , China .|b Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus) , Zhejiang Cancer Hospital , Hangzhou , Zhejiang , China .
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: c Institute for Individualized Medicine, Hangzhou First People's Hospital , Hangzhou , Zhejiang , China .
authors.forename: Nengming
authors.initials: N
authors.lastname: Lin
authors.affiliation: a Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Zhejiang Cancer Center , Hangzhou , Zhejiang Province , China .|b Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus) , Zhejiang Cancer Hospital , Hangzhou , Zhejiang , China .
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: a Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Zhejiang Cancer Center , Hangzhou , Zhejiang Province , China .
authors.forename: Zhongjian
authors.initials: Z
authors.lastname: Chen
authors.affiliation: d ACEA Bio Co., Ltd. , Hangzhou , Zhejiang Province , China , and.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Zhang
authors.affiliation: e Department of Pharmaceutical Sciences , South Dakota State University , Brookings , SD , USA.
authors.forename: Xiangming
authors.initials: X
authors.lastname: Guan
mesh_terms: D045744:Cell Line, Tumor; D003545:Cysteine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D003956:Dialysis; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry; D008545:Melanoma / Q000188:drug therapy / Q000473:pathology; D009249:NADP / Q000378:metabolism; D013453:Sulfonylurea Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D054481:Thioredoxin Reductase 1 / Q000037:antagonists & inhibitors; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C454682:N-acetyl-S-(4-chlorophenylcarbamoyl)cysteine; D013453:Sulfonylurea Compounds; D009249:NADP; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase; C516126:Txnrd1 protein, rat; D003545:Cysteine
keywords: Carbamoylation; NACC; thiocarbamate; thioredoxin reductase