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锰催化氧化抗结核药物异烟肼的自由基机理研究
Alternative TitleFree Radical Mechanism for Mn Mediated Activation of the Anti tuberculosis Drug Isoniazid
秦丽
Subtype博士
Thesis Advisor朱本占
2020-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword异烟肼和 肼类化合物 Mn( 氮中心自由基 酰碳自由基, 羟基自由 基 超氧阴离子 isoniazid And Hydrazine Derivatives, Mn(Iii), n Centered Radical, c Centered Acyl Radicals, Hydroxyl Radical, Superoxide
Abstract

      结核病仍是世界上最严重的疾病之一,世界卫生组织报道称每年仍有1000万人感染结核杆菌, 200万人死于结核病。异烟肼( INH)具有特异性强、副作用小及价格低等特点,是最常用的一线抗结核药物。 然而其确切的分子作用机理目前仍然不十分清楚。 本论文主要研究了异烟肼及其他肼类和酰肼类物质被 金属离子 特别是锰 和铜 离子 催化氧化的 自由基 机理 。研究内容主要包括以下部分:
     首次捕获催化氧化 异烟肼过程中 生成的 氮中心自由基并确定其准确位点
     作为最常用的抗结核药物之一,INH可能的作用机理 被认为是通过 过氧化物酶 KatG催化氧化产生的酰碳自由基与 NAD+结合,从而抑制结核杆菌细胞壁的生成。但是 INH被氧化的具体过程仍不清楚。有趣的是,有研究表明 INH被某些 KatG酶催化氧化的过程需要 Mn的存在 。 然而,作为酰碳自由基的前体,氮中心自由 基 能否生成仍未可知;如果生成,其准确位置在肼基的前端还是末端?为了回答以上问题,本研究综合采用 ESR自旋捕获和 HPLC/MS方法,检测并确认了 INH可被醋酸锰( Mn(OAc)3)催化氧化产生氮中心自由基。采用 15N同位素标记法进一步确定了氮自由基的准确位置 位于 肼基的末端氮原子。此外还检测到一种新的反应产物, 1,2 二异烟酰基肼。 在其他的酰肼类物质反应体系中也得到类似的结果。本研究是首次捕获到 INH催化氧化过程中产生的氮自由基并确认 其 准确位置。该发现对研究 INH被催化氧化的分子机理,发现更有效的抗结核药物提供了新视角,具有广泛的生理和毒理学意义。
      综合采用ESR自旋捕获和 HPLC/MS法准确鉴定抗结核药物异烟肼及其结构类似物氧化产生的关键酰碳自由基活性中间体
      先前研究表明INH可能的作用机理是它可被过氧化物酶 KatG催化氧化产生的酰碳自由基( INH 与 NAD+结合,从而抑制结核杆菌细胞壁的生成 在这个过程中酰碳自由基发挥重要作用 。 在本研究中,选用 DMPO 5,5 二甲基 1吡咯啉 N 氧化物)为捕获剂, Cu(II)为氧化剂,用 ESR自旋捕获技术,我们可检测到 DMPO与 INH的酰碳自由基加合物 DMPO/INH CO•的 ESR信号,但该加合物却不能用 HPLC/MS方法检测和鉴定。选用 PBN N 叔丁基 α 苯基硝酮)为捕获剂,不仅可用ESR自旋捕获检测到 PBN/INH CO•的 ESR信号,而且同时也能用 HPLC/MS方法检测并鉴定该加合物。我们进而发现 INH的另外四种结构类似物(烟酸酰肼、 2 吡啶甲酰肼 、 2 Cl异烟肼 和苯酰肼)均可产生相应的酰碳自由基。烟酸酰肼和苯酰肼可被氧化形成其相应的酰碳自由基,并可与 NAD+形成加合物,但是 研究报道 NH和 BH的最低抑菌浓度 MIC 高于 INH,可降低INH的抗菌活性 ,不能用于结核病的治疗;而 2 吡啶甲酰肼不易被 Cu(II)氧化形成其相应的酰碳自由基,只有选用 MnO2作为氧化剂时才会被很好的检测到这可能是由于形成了一个氧化还原活性较低的 Cu(II)/2 NH络合物 。 而ClINH不能检测到 C中心的酰碳自由基与 NAD+生成的加合物,这可能是由于氯基吸电子的影响 。 这是首次综合采用 ESR自旋捕获和 HPLC/MS法准确鉴定抗结核药物异烟肼及其结构类似物氧化产生的关键酰碳自由基活性中间体。该发现对研究酰肼类物质被氧化的分子机理有重要意义,且对未来发现更有效性的抗结核药物提供了新视角,具有广泛的生理和毒理学意义。
      锰催化氧化异烟肼过程中发现 一类依赖于 而非 H2O2的 HO•的生成机制
      先前研究发现肼 类化合物 可 被 Mn(III)激活,生成活性最强的 HO•。然而,无论是潜在的 HO•生成的确切的分子机制,还是结构 活性关系,都没有被清楚地了解 。 在这里,我们证明了所有的肼和 酰肼类化合物( 包括 INH 与 Mn(III)确实可以生成 HO•,其中肼被发现是最 强 的 。 有趣的是,我们发现肼 /Mn(III)生成 •OH不仅依赖于超氧阴离子自由基 的生成,而且与 的生成 有很好的相关性 ,但与 H2O2无关 。 进一步的研究表明, Mn(II)和 都可在 Mn(III)/肼氧化过程中生成,但 Mn(II)与 由 X/XO系统生成 之间的相互作用 却 不能产生HO•,且不会生成 Mn(III)。 然而,我们发现只有在 肼 类化合物 共存时 Mn(II)和才能生成 •OH 同时 还 形成以 N为中心的自由基 。 利用 FAM标记的 DNA片段, 我们进而发现 肼 类化合物 /Mn(III)诱导的 DNA裂解发生在每个核苷酸的位置,没有明显的位点特异性 。 综上所述,我们提出肼 类化合物 /Mn(III)生成 HO•是由于 先生成 Mn(II)和 二者 之间的相互作用导致了瞬态中间 体 MnO2•+的形成MnO2•+可 从肼类化合物上夺取 H生成氮中心自由基和 MnOOH+,随后 MnOOH+均裂分解产生 HO•。 本研究报道了一种 依赖于 但不依赖于 H2O2 的 新型 HO•产生 机制 ,这对今后肼 类化合物 和锰的研究具有广泛而重要的化学、生物医学和毒理学意义。
      锰催化氧化乙酰肼的分子机制:氮中心自由基和乙酰碳自由基的检测和确认
      作为异烟肼(INH)的代谢产物,研究认为乙酰肼 AcHZ)是 INH治疗后引起严重肝毒性和潜在致死性肝损伤的主要原因。 AcHZ被代谢激活产生的活性自由基可以共价结合体内关键蛋白质。但是,代谢激活 AcHZ过程中产生的活性自由基种类仍不清楚。本研究综合采用 ESR自旋捕获 /HPLC/MS方法以及 TEMPO捕获法,在酶(如髓过氧物酶 /H2O2)和金属离子(如 Mn(III)和 Cu(II))催化氧化AcHZ过程中检测并确认了氮中心自由基和乙酰碳自由基的生成。采用 15N同位素标记法进一步确定了氮中心自由基的准确位置位于肼基的末端氮原子。本研究首次确认 AcHZ催化氧化过程中产生的乙酰碳自由基、氮自由基及其准确位置。该发现为探讨 AcHZ的肝毒性效应 提供了新的视角 。

Other Abstract

Tuberculosis remains to be the most serious disease around the world. According to the World Health Organization (WHO), there are about ten million people hosting the pathogen Mycobacterium tuberculosis (Mtb), and about two million deaths per year are attributed to this infection. Isoniazid (INH) has been used as a frontline anti tuberculosis drug for over 60 years due to its specificity and efficiency of action, relatively few side effects and low cost. However, it mode of action remains not fully eluciadated. In this research, we mainly focus on studies of the mechanism of catalytic oxidation by transition metal ions (especially Mn and Cu) for isoniazid and other hydrazines. The main contents of this dissertation are as follows:
     First Detection and Unequivocal Identification of the Critical N centered Isoniazidyl Radical and its Exact Location
      Isoniazid (INH), the most widely used anti tuberculosis drug, has been shown to be activated by Mn(III) to produce the reactive carbon centered isonicotinic acyl radical, which was considered to be responsible for its anti tuberculosis activity. However, it is still not clear whether the previously proposed N centered isoniazidyl radical intermediate can be initially produced or not; and if so, what is its exact location on the hydrazine group, distal or proximal N? Through complementary applications of ESR spin trapping and HPLC/MS methods, here we show that the characteristic and transient N centered isoniazidyl radical intermediate can be detected and identified from INH activation uniquely by Mn(III)Acetate not by Mn( pyrophosphate. The exact location of the radical was found to be at the distal N of the hydrazine group by 15N isotope labeling techniques via using 15N labeled INH. Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(III). Analogous results were observed with other hydrazides. This study represents the first detection and unequivocal identification of the initial N centered isoniazidyl radical and its exact location. These findings should provide a new perspective on the molecular mechanism of INH activation, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti tuberculosis drugs.
        First Unequivocal Identification of the Critical C centered Acyl Radicals from the Anti Tuberculosis Drug Isoniazid and its Close Hydrazide Analogs by Complementary Applications of ESR Spin trapping and HPLC/MS Methods
       The carbon centered isonicotinic acyl radical of isoniazid (INH), a widely used frontline anti tuberculosis drug, has been considered to play a critical role in inhibiting Mycobacterium tuberculosis, but not fully identified. Here we show that this radical intermediate can be unequivocally characterized by complementary applications of ESR spin trapping and HPLC/MS methods by employing N tert butyl α phenylnitrone (PBN) as the suitable spin trapping agent, which can form the most stable radical adduct. More importantly, for the first time, analogous carbon centered acyl radicals and their respective NAD+ adducts have also been detected and identified from its two isomers (nicotinic acid hydrazide and 2 pyridinecarbohydrazide) and benzhydrazide which are structurally related to INH, but not by 2 chloroisonicotinohydrazide. This study represents the first unequivocal identification of the carbon centered acyl radicals of INH and other hydrazide analogs by both ESR spin trapping and HPLC/MS methods, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti tuberculosis drugs.
       Molecular Mechanism of Unusual Superoxide Dependent but H2O2 Independent Production of Hydroxyl Radicals by Hydrazines and Mn(III)
       It has been shown that hydrazines can be activated by Mn(III) to generate the most reactive HO•. However, neither the exact molecular mechanism, nor the structure activity relationship underlying such HO• generation, are clearly understood. Here we show that HO• can indeed be produced by all hydrazines and hydrazides (including the anti tuberculosis drug INH) with Mn(III), among them, hydrazine was found to be the most effective. Interestingly, we found that the formation of HO• by hydrazine/Mn(III) was not only dependent on, but also well correlated to, the generation of superoxide anion radical (O2•--) , but independent on H2O2. Further studies showed that both Mn(II) and were generated during hydrazine oxidation by Mn(III), but the interaction between Mn(II) and (generated by X/XO system) cannot produce HO•, and no Mn(II) was oxidized to Mn(III) neither. However, we found that HO• can be produced by Mn(II) and only when hydrazines were simultaneously present, with the concurrent formation of N-centered radicals. We also found that DNA cleavage induced by hydrazines/Mn(III) occur at positions of every nucleotide, with no marked site specificity, using FAM labeled DNA fragments. Taken together, we proposed that HO• production from hydrazines/Mn(III) was due to the formation of Mn(II) and O2•--, and their interactions lead to the formation of a transient intermediate MnO2•+; MnO2•+ can abstract a hydrogen from the hydrazine to produce N-centered radical and the reactive MnOOH+ intermediate, which then decompose homolytically to generate HO•. This study reported a new dependent, but H2O2 independent mechanism for HO• production from hydrazines and Mn(III) via forming the reactive MnOOH+ intermediate, which should have broad and significant chemical, biomedical and toxicological implications for future research on hydrazines and manganese.
       Molecular Mechanism for the Activation of the Acethydrazide: Detection and Unequivocal Identification of the N centered Radical and CH3CO• Radical Intermediates
       Acethydrazide (AcHZ), a major human metabolite of isoniazid (INH), was considered to be responsible for the serious hepatotoxicity and potentially fatal liver injury after INH therapy. It was proposed that reactive radical species produced from metabolic activation of AcHZ can covalently bind to critical proteins. However, it is still not clear what kind of reactive radical species can be produced from AcHZ activation. Through complementary applications of ESR spin trapping and HPLC/MS methods as well as TEMPO trapping approach, here we show that both N centered radical and CH3CO• radical intermediates can be detected and identified from AcHZ activated by both enzymes (such as neutrophil myeloperoxidase (MPO)/H2O2) and metal ions (such as Mn(III) and Cu(II)). The exact location of the radical was found to be at the distal nitrogen of the hydrazine group by 15N isotope labeling techniques via using 15N labeled AcHZ. This study represents the first detection and unequivocal identification of CH3CO• radical, the initial N centered radical and its exact location. These findings should provide a new perspective on the hepatotoxic effect of AcHZ.

Pages168
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/43628
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
秦丽. 锰催化氧化抗结核药物异烟肼的自由基机理研究[D]. 北京. 中国科学院生态环境研究中心,2020.
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