Exploiting the drug-activating properties of a novel trypanosomal nitroreductase

Abstract

Nitroheterocyclic prodrugs have been used to treat trypanosomal diseases for more than 40 years. Recently, the key step involved in the activation of these compounds has been elucidated and shown to be catalyzed by a type I nitroreductase (NTR). This class of enzyme is normally associated with bacteria and is absent from most eukaryotes, with trypanosomes being a major exception. Here we exploit this difference by evaluating the trypanocidal activity of a library of nitrobenzylphosphoramide mustards against bloodstream-form Trypanosoma brucei parasites. Biochemical screening against the purified enzyme revealed that a subset of halogenated nitroaromatic compounds were effective substrates for T. brucei NTR (TbNTR), having apparent K(cat)/K(m) values approximately 100 times greater than nifurtimox. When tested against T. brucei, cytotoxicity mirrored enzyme activity, with 50% inhibitory concentrations of the most potent substrates being less than 10 nM. T. brucei NTR plays a key role in parasite killing: heterozygous lines displayed resistance to the compounds, while parasites overexpressing the enzyme showed hypersensitivity. We also evaluated the cytotoxicities of substrates with the highest trypanocidal activities by using mammalian THP-1 cells. The relative toxicities of these newly identified compounds were much lower than that of nifurtimox. We conclude that halogenated nitrobenzylphosphoramide mustards represent a novel class of antitrypanosomal agents, and their efficacy validates the strategy of specifically targeting NTR activity to develop new therapeutics.

FIG. 1.

Proposed mechanism for the activation of nitrobenzylphosphoramide mustards. The nitroreductase-mediated reduction of the nitro group (electron withdrawing) to hydroxylamine (electron donating) causes a rearrangement of electrons within the NBPM backbone. This promotes the cleavage of a C-O bond, activating a cytotoxic phosphoramide mustard moiety (shaded). It was previously proposed that this molecule triggers DNA damage by acting as an alkylating agent (15, 16). Two types of NBPM are shown. One form contains the mustard as part of a cyclic arrangement and is analogous to cyclophosphamide. After nitroreduction, the cyclophosphamide structure is predicted to undergo ring opening, exposing the cytotoxic mustard. In the second form, the mustard is part of a linear (acyclic) structure. Here, the NBPM is postulated to fragment, releasing the cytotoxic phosphoramide mustard.

FIG. 2.

Activity of TbNTR toward different nitrobenzylphosphoramide mustards. (A) SDS-PAGE gel (10%) stained with Coomassie blue. Lane 1, size standards; lane 2, crude E. coli extract loaded onto a Ni-NTA column; lane 3, flowthrough. The column was washed extensively with 50 mM imidazole (lane 4) and 100 mM imidazole (lane 5). Recombinant protein was eluted with 500 mM imidazole containing 0.5% Triton X-100 (lanes 6 and 7). (B) The activity of purified His-tagged TbNTR was assessed by using various NBPMs as substrates at a fixed concentration of 100 μM NADH and 100 μM NBPM. The values shown are the means of data from three experiments ± standard deviations. TbNTR activity was deemed to be high if it was >500 nmol NADH oxidized min⁻¹ mg⁻¹ (dotted line). The activity obtained when using nifurtimox (NFX) as a substrate is also shown. (C) TbNTR activity was assayed by monitoring the oxidation of NADH (100 μM) in the presence of TbNTR (20 μg ml⁻¹) and nitroheterocyclic substrate (2 to 75 μM). The substrates used were nifurtimox (•), LH34 (▪), or LH37 (▴). All reactions were initiated by the addition of the recombinant enzyme. TbNTR activities are expressed as nmol NADH oxidized min⁻¹ mg⁻¹ of enzyme.

FIG. 3.

Susceptibility of bloodstream-form T. brucei with altered levels of NTR to nitrobenzylphosphoramide mustards. (A) Structures of the NBPMs with the highest trypanocidal activities (Table 3). (B) Growth-inhibitory effect of LH32, LH33, LH34, and LH37 on T. brucei NTR heterozygote (NTR^−/+) cells as judged by the IC₅₀. (B) Overexpression of TbNTR (TbNTR^RV) confers hypersensitivity to NBPMs. Data in A and B are means of data from 4 experiments ± SD, and the differences in susceptibility were statistically significant (P < 0.01), as assessed by a Student's t test. Melarsoprol and nifurtimox were used as drug controls.