Evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1
Evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1
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N-乙酰基-S-(对氯苯基氨基甲酰基)半胱氨酸作为哺乳动物硫氧还蛋白还原酶不可逆抑制剂的评价1
DOI：
10.3109/14756366.2015.1016512
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发表时间：
2016-03-03
影响因子：
5.6
通讯作者：
Guan，Xiangming
中科院分区：
文献类型：
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作者：
Chen，Wei;Jiang，Zhiming;Guan，Xiangming
摘要背景：硫氧还蛋白还原酶（Thioredoxin reductase, TrxR）在许多人类恶性细胞中上调，成为抗癌药物开发的一个有希望的靶点。目的：评价n -乙酰基- s -（对氯苯氨基甲酰）半胱氨酸（NACC）作为抗黑色素瘤的有效药物对哺乳动物TrxR1的抑制作用。材料与方法：采用底物保护、透析、液相色谱-串联质谱法研究其对TrxR1的抑制作用机制。结果：NACC抑制TrxR1呈时间和浓度依赖性。测定NACC对TrxR1的Ki值为80 μM，亲和度为0.178 min−1。这种抑制仅发生在NADPH存在的情况下，并在广泛透析后持续存在。串联质谱分析表明，活性位点的硒代半胱氨酸而非半胱氨酸残基被NACC对氯苯氨基甲酰化。观察NACC对培养黑色素瘤细胞胞内TrxR的抑制作用。讨论与结论：NACC通过与硒代半胱氨酸形成共价键不可逆地抑制TrxR1，可以成为研究TrxR1的有效工具。
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 Ki and kinact 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.