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

Abstract

Benznidazole, a 2-nitroimidazole, is the front-line treatment used against American trypanosomiasis, a parasitic infection caused by Trypanosoma cruzi. Despite nearly 40 years of use, the trypanocidal activity of this prodrug is not fully understood. It has been proposed that benznidazole activation leads to the formation of reductive metabolites that can cause a series of deleterious effects, including DNA damage and thiol depletion. Here, we show that the key step in benznidazole activation involves an NADH-dependent trypanosomal type I nitroreductase. This catalyzes an oxygen-insensitive reaction with the interaction of enzyme, reductant, and prodrug occurring through a ping-pong mechanism. Liquid chromatography/mass spectrometry (LC/MS) analysis of the resultant metabolites identified 4,5-dihydro-4,5-dihydroxyimidazole as the major product of a reductive pathway proceeding through hydroxylamine and hydroxy intermediates. The breakdown of this product released the reactive dialdehyde glyoxal, which, in the presence of guanosine, generated guanosine-glyoxal adducts. These experiments indicate that the reduction of benznidazole by type I nitroreductase activity leads to the formation of highly reactive metabolites and that the expression of this enzyme is key to the trypanocidal properties displayed by the prodrug.

An Essential Type I Nitroreductase from Leishmania major Can Be Used to Activate Leishmanicidal Prodrugs

“…We have now shown that L. major expresses a type I NTR that in the related trypanosomal parasites activates the clinically used prodrugs nifurtimox and benznidazole (26,49,50).…”

“…6B). For both compounds, cells with reduced levels of the nitroreductase were up to 3-fold more resistant to the agent than wild type controls; L. major wild type cells exhibited IC 50 Based on our findings, NBPMs that contain the phosphoramide mustard as part of an acyclic structure and have halogen substituents on the nitrobenzyl ring are the most readily metabolized by the LmNTR enzyme and represent the most potent agents against both L. major promastigotes and amastigotes. Some (LH32, -33, and -34) have IC 50 values of 1 M or less against the intracellular stage without inducing mammalian cell toxicity.…”

“…For the remaining leishmanicidal compounds, growth inhibition assays were performed to determine their IC 50 values (Table 4). Many of these displayed appreciable leishmanicidal properties, with five NBPMs (LH31-34 and -37) having significant activity (IC 50 values of Ͻ10 M) against both parasite forms. These five com-FIGURE 5.…”

Targeting the Substrate Preference of a Type I Nitroreductase To Develop Antitrypanosomal Quinone-Based Prodrugs

“…3B). This mechanism of kinetics, typical for oxidoreductase cascades, has previously been observed when using benznidazole as an electron acceptor under normoxic conditions but not when employing nifurtimox as the substrate (26,27). When reduced by the trypanosomal NTRs, this 5-nitrofuran forms a cytotoxic open chain nitrile while undergoing limited futile cycling.…”

“…It is now clear that trypanosomes activate such nitrobased drugs using a mitochondrially targeted, oxygen-insensitive type I NTR, leading to the formation of cytotoxic reduction products (26,27,65). This class of oxidoreductase is largely restricted to bacteria and absent from most eukaryotes, with a subset of protozoan parasites being major exceptions.…”