Ethionamide biomimetic activation and an unprecedented mechanism for its conversion into active and non-active metabolites.

Ethionamide (ETH), a second-line anti-tubercular drug that is regaining a lot of interest due to the increasing cases of drug-resistant tuberculosis, is a pro-drug that requires an enzymatic activation step to become active and to exert its therapeutic effect. The enzyme responsible for ETH bioactivation in Mycobacterium tuberculosis is a monooxygenase (EthA) that uses flavin adenine dinucleotide (FAD) as a cofactor and is NADPH- and O2-dependant to exert its catalytic activity. In this work, we investigated the activation of ETH by various oxygen-donor oxidants and the first biomimetic ETH activation methods were developed (KHSO5, H2O2, and m-CPBA). These simple oxidative systems, in the presence of ETH and NAD+, allowed the production of short-lived radical species and the first non-enzymatic formation of active and non-active ETH metabolites. The intermediates and the final compounds of the activation pathway were well characterized. Based on these results, we postulated a consistent mechanism for ETH activation, not involving sulfinic acid as a precursor of the iminoyl radical, as proposed so far, but putting forward a novel reactivity for the S-oxide ethionamide intermediate. We proposed that ETH is first oxidized into S-oxide ethionamide, which then behaves as a "ketene-like" compound via a formal [2 + 2] cycloaddition reaction with peroxide to give a dioxetane intermediate. This unstable 4-membered intermediate in equilibrium with its open tautomeric form decomposes through different pathways, which would explain the formation of the iminoyl radical and also that of different metabolites observed for ETH oxidation, including the ETH-NAD active adduct. The elucidation of this unprecedented ETH activation mechanism was supported by the application of isotopic labelling experiments.

Synthesis and mechanistic investigation of iron(II) complexes of isoniazid and derivatives as a redox-mediated activation strategy for anti-tuberculosis therapy.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by H2O2 and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using H2O2 with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.

Update of Antitubercular Prodrugs from a Molecular Perspective: Mechanisms of Action, Bioactivation Pathways, and Associated Resistance.

The place of prodrugs in the current antitubercular therapeutic arsenal is preponderant, since two of the four first-line antitubercular agents, isoniazid (INH) and pyrazinamide (PZA), need to be activated by Mycobacterium tuberculosis before exerting their activity. In addition, six other prodrugs can be found in the second- and third-line therapeutic regimens. The emergence of mycobacterial strains resistant to one or several antitubercular agents is one of the main issues of the antitubercular therapy. In the case of prodrugs, the resistance phenomenon is often related to a mutation in the gene encoding for the activation enzymes, resulting thus in a default of these enzymes that are no more able to activate prodrugs. Consequently, identification of the prodrugs targets and a better understanding of their modes of action and also of their activation mechanisms are of crucial importance. Related to their molecular mechanism of activation, these prodrugs may thus be classified in four categories: activation via oxidation (catalase-peroxidase (KatG) or flavin monooxygenase (EthA) enzymes), condensation (FolP1 and FolC), hydrolysis (by the amidase PncA) and reduction (by the nitroreductase DnD) mechanisms. For each prodrug, these mechanisms are described in details, as well as the mechanism of action of its active metabolite. Finally, the reported resistance related to these mechanisms of activation/action are also addressed in a molecular perspective.