Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum
Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum
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DOI：
10.1128/aac.00773-07
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发表时间：
2008-01-01
影响因子：
4.9
通讯作者：
Gromer, Stephan
中科院分区：
文献类型：
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作者：
Buchholz, Kathrin;Schirmer, R. Heiner;Gromer, Stephan
亚甲基蓝(MB)经历了复兴，主要是作为治疗恶性疟疾的药物组合的一种成分。在这里，我们报告了有关MB的生物化学相关药理数据，如在pH 7.4时MB与细胞还原剂NAD(P)H(k=4M-1 S(-1))、硫氧还蛋白(k=8.5M-1至26M-1 S(-1))、二氢硫胺(k=53M-1 S(-1))以及缓慢反应谷胱甘肽的非催化反应的速率常数。由于二硫化物还原酶是MB的主要靶标，因此在好氧条件下以NAD(P)H为代价还原MB的酶的光学测试被开发出来。产品亮甲基蓝(LeucoMB)在pH 7时自动氧化回MB，但在pH 5.0时可被酶稳定，这使这种无色化合物成为有趣的候选药物。MB是哺乳动物和恶性疟原虫二硫化物还原酶的抑制剂和/或氧化还原循环底物，在25℃时k(CAT)值在0.03 S(-1)到10 S(-1)之间。突变的谷胱甘肽还原酶的动力学光谱表明，MB的还原是通过酶结合的还原菲亚文而不是活性中心二硫醇半胱氨酸(58)/半胱氨酸(63)进行的。MB的酶催化还原和随后产物LeucoMB的自动氧化意味着MB是一个氧化还原循环剂，在每个循环中以O-2和NAD(P)H为代价产生H_2O_2，将抗氧化剂二硫键还原酶转变为促氧化酶。这解释了术语颠覆性衬底或翻转涂层阻聚剂的甲基溴。这些结果在细胞病理学和临床背景下进行了讨论。
Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M-1 s(-1)), thioredoxins (k = 8.5 to 26 M-1 s(-1)), dihydrolipoamide (k = 53 M-1 s(-1)), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the k(cat) values ranging from 0.03 s(-1) to 10 s(-1) at 25 degrees C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced fiavin rather than by the active-site dithiol Cys(58)/Cys(63). The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O-2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts.