Evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1

Abstract

Context: 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.

Material and 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.

Discussion and conclusion: NACC which irreversibly inhibits TrxR1 by forming a covalent bond with selenocysteine can be an effective tool in the study of TrxR1.

• Carbamoylation
• NACC
• thiocarbamate
• thioredoxin reductase

Declaration of interest

The authors confirm that this article’s contents have no conflict of interest.

This work was supported by National Natural Science Foundation of China (81202553, 81302840), Medical and Health Science Foundation of Zhejiang Province (2012KYA034) and Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus).