Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase

Abstract

Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.

Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity

“…The stability or molecular targeting efficiency of activated nitro drug radicals may be more favorable for the new compounds (22), although chemical reduction with dithionite followed by immediate antimicrobial testing led to complete activity loss, suggesting that the toxic products formed by reduction are not very stable under these conditions. Alternatively, the spectrum of nitro drug adduction targets may be different for the most potent compounds (4,23). In any case, the observation that many compounds showed improved broad-spectrum activity would suggest that the underlying mechanisms are likely to entail broadly relevant drug properties, such as improved membrane permeability, that are not limited to particular molecular targets or microbial features.…”

“…Although the precise mechanistic reasons for this counterintuitive observation remain to be determined, 5-NIs can be activated by different microbial pathways and probably target multiple microbial molecules (11,12,23) whose relative importance may depend on the compound and microbe. Because the specific target molecules of 5-NI drugs are not fully understood (23), a strategy focused on achieving effective whole-cell antimicrobial activity in the relevant systems may be the best choice for making rapid progress in developing improved antimicrobials in the clinically indispensable 5-NI class.…”

Activation of Benznidazole by Trypanosomal Type I Nitroreductases Results in Glyoxal Formation

“…These prodrugs can be activated by two mechanisms, both involving the reduction of the conserved nitro group. In one pathway, proteins such as thioredoxin reductase, cytochrome P450 reductase, and ferredoxin promote the 1-electron reduction of the nitro substituent to yield a nitro anion radical (15,18,19,49). Under normoxic conditions, the nitro anion radical can undergo futile cycling with oxygen, producing superoxide anions and regenerating the parent nitro-compound (33).…”

“…Where oxygen is limited, the nitroso form can be produced either by a further single-electron reduction step or by the interaction of two nitro anions (26). The nitroso molecule is highly reactive and can lead to cellular damage directly or indirectly through the formation a hydroxylamine derivative (16,18). This pathway is believed to underlie the selective toxicity of nitroimidazoles against infections caused by anaerobic/microaerophilic microbes and prodrug activation in hypoxic tumors.…”

Giardia , Entamoeba , and Trichomonas Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases)

“…Metronidazole resistance is a major problem in clinical isolates of Trichomonas in the United States and elsewhere, while metronidazole-resistant Entamoeba and Giardia have for the most been prepared by selection in culture (9,12,17,19,20,23,32,38,39). Recently, a ferredoxin-nitroreductase fusion protein of Giardia (called GlNR1) which resembles oxygen-sensitive nitroreductases of bacteria and which appears to have been obtained by LGT was shown to have nitroreductase ac-tivity when it was expressed as a recombinant protein in bacteria (24,27).…”