Mn(III) pyrophosphate as an efficient tool for studying the mode of action of isoniazid on the InhA protein of Mycobacterium tuberculosis
Mn(III) pyrophosphate as an efficient tool for studying the mode of action of isoniazid on the InhA protein of Mycobacterium tuberculosis
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DOI：
10.1128/aac.46.7.2137-2144.2002
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发表时间：
2002-07-01
影响因子：
4.9
通讯作者：
Meunier, B
中科院分区：
文献类型：
--
作者：
Nguyen, M;Quémard, A;Meunier, B
抗结核药物异烟肼 (INH) 会被化学计量的焦磷酸锰 (III) 快速氧化。在烟酰胺辅酶（NAD(+)、NADH、烟酰胺单核苷酸 [NMN+]）和烟酸腺嘌呤二核苷酸（DNAD(+)）存在下，除了已知的生物非活性产物（异烟酸、异烟酰胺和异烟醛）外，INH 氧化还会形成 INH-辅酶加合物。在溶液中预先形成的 INH-NAD(H) 加合物库可以快速、强烈地抑制烯酰基-酰基载体蛋白还原酶 InhA（霉菌酸生物合成途径中的 INH 靶标）的体外活性：当在 NAD(+) 或 NADH 存在下形成加合物时，抑制分别为 90% 或 60%。在相似条件下，未检测到INH-NMN(H)和INH-DNAD(H)加合物的抑制活性。当分离出的 100 nM INH-NAD(H) 加合物首次与 InhA 一起孵育时，酶活性被抑制 80%；当过量存在时，NADH 和癸烯酰辅酶 A 都能够防止这种现象。讨论了溶液中共存的几种类型的 INH-辅酶加合物对 InhA 的抑制作用，涉及辅酶的结构、加合物的立体化学及其以开放形式和环状形式存在。因此，焦磷酸锰（III）似乎是一种有效且方便的替代氧化剂，可模拟结核分枝杆菌 KatG 过氧化氢酶-过氧化物酶的活性，并将可用于 INH 活化的进一步机制研究和反应性 INH 物种的结构研究，以促进新型 InhA 抑制剂作为潜在抗结核药物的设计。
The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD(+), NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD(+)), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD(+) or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.