Nguyen M, Claparols C, Bernadou J, Meunier B
Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse cedex 4, France.
Chembiochem. 2001 Dec 3;2(12):877-83. doi: 10.1002/1439-7633(20011203)2:12<877::AID-CBIC877>3.0.CO;2-V.
It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (beta-nicotinamide adenine dinucleotide), a strong inhibitor of InhA, an enzyme essential for mycolic acid biosynthesis. This work was carried out to characterise the InhA inhibitors (named INH-NAD(H) adducts) which are generated, in the presence of the nicotinamide coenzyme NAD+, by oxidation of INH with manganese(III) pyrophosphate, a nonenzymatic and efficient oxidant used to mimic INH activation by the catalase-peroxidase KatG inside M. tuberculosis. The oxidation process is almost complete in less than 15 minutes (in comparison to the slow activation obtained in the KatG-dependent process (2.5 hours) or in the nonenzymatic O2/Mn(II)-dependent activation (5 hours)). The alkylation of NAD+ by the postulated isonicotinoyl radical generates, in solution, a family of INH-NAD(H) adducts. Analyses with liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) and experiments performed with 18O- and 2H-labelled substrates allowed us to propose two open and four hemiamidal cyclised dihydropyridine structures as the main forms present in solution; these result from the combination of the isonicotinoyl radical and the nicotinamide part of NAD+. A small amount of a secondary oxidation product was also detected. Structural data on the forms present in solution should help in the design of inhibitors of enzymes involved in the biosynthesis of mycolic acids to act as potential antituberculosis drugs.
目前认为，异烟肼（INH）在结核分枝杆菌内被氧化，通过与NAD（H）（β-烟酰胺腺嘌呤二核苷酸）的烟酰胺环共价结合，生成InhA的强效抑制剂，InhA是分枝菌酸生物合成所必需的一种酶。开展这项工作的目的是对InhA抑制剂（称为INH-NAD（H）加合物）进行表征，这些加合物是在烟酰胺辅酶NAD⁺存在的情况下，通过用焦磷酸锰（III）氧化INH生成的，焦磷酸锰（III）是一种非酶促且高效的氧化剂，用于模拟结核分枝杆菌内过氧化氢酶-过氧化物酶KatG对INH的激活作用。氧化过程在不到15分钟内几乎完成（相比之下，KatG依赖过程（2.5小时）或非酶促O₂/Mn（II）依赖激活（5小时）中获得的激活速度较慢）。假定的异烟酰基自由基对NAD⁺的烷基化在溶液中生成了一系列INH-NAD（H）加合物。通过液相色谱/电喷雾电离质谱（LC/ESI-MS）分析以及使用¹⁸O和²H标记底物进行的实验，我们提出了两种开环和四种半缩醛环化二氢吡啶结构作为溶液中存在的主要形式；这些结构是异烟酰基自由基与NAD⁺的烟酰胺部分结合的结果。还检测到少量的二级氧化产物。溶液中存在形式的结构数据应有助于设计参与分枝菌酸生物合成的酶的抑制剂，以作为潜在的抗结核药物。
