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.