Trypanocidal activity of aziridinyl nitrobenzamide prodrugs

Abstract

The trypanocidal agents nifurtimox and benznidazole both function as prodrugs and must undergo enzyme-mediated activation, a reaction catalyzed by type I nitroreductase (NTR). In the search for new parasitic therapies, we have utilized this finding to investigate whether aziridinyl nitrobenzamide derivatives have activity against bloodstream-form Trypanosoma brucei and Trypanosoma cruzi amastigotes, parasite stages that replicate in the mammalian host. For T. cruzi drug screening, we generated trypanosomes that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of compounds having a 5-(aziridin-1-yl)-2,4-dinitrobenzyl structure was shown to be metabolized by purified T. brucei NTR and when screened against both parasite life cycle stages displayed significant growth-inhibitory properties: the most potent compounds generated 50% inhibitory concentrations of <1 μM. The trypanocidal activity was shown to be NTR specific, since parasites overexpressing this enzyme were hypersensitive to the aziridinyl dinitrobenzyl agents. We conclude that members of the aziridinyl nitrobenzamide class of nitroheterocycles provide new lead structures that have the potential to treat trypanosomal infections.

FIG. 1.

Luciferase expression in insect- and mammalian-stage T. cruzi. (A) The luciferase activity, in relative light units (RLU), of four recombinant T. cruzi Cl-Brener epimastigote clones was determined and compared to that of the parental line. Twenty thousand cells were used in each analysis. (B) Correlation between amastigote load (between 10 and 10,000 cells) and luciferase activity. Two of the clones noted in part A were analyzed in parallel. (C) Vero cells infected with recombinant T. cruzi Cl-Brener amastigotes were grown in the presence of different concentrations of nifurtimox, and the luciferase activity was determined (see Materials and Methods). The concentration of nifurtimox that inhibited parasite growth by 50% (IC₅₀) was established. The data are the means from three experiments ± SDs.

FIG. 2.

Activity of trypanosomal NTRs toward different aziridinyl nitrobenzamides. (A) SDS-PAGE gel (10%) stained with Coomassie blue. Lane 1, size standards. Lane 2, recombinant protein eluted from a nickel-nitrilotriacetic acid (Ni-NTA) column using 500 mM imidazole-0.5% Triton X-100. (B) Activity of purified His-tagged TbNTR was followed by monitoring of the reduction of each ANB by HPLC. Each substrate (100 μM) was incubated with enzyme (50 μg) in the presence of NADH (200 μM), glucose (3 mM), and glucose dehydrogenase (1 U). The ANBs shown are CB1954 (diamonds), NH1 (squares), and NH2 (triangles). All other substrates showed no or little reduction by TbNTR, as typified by NH3 (circles). (C) HPLC chromatogram of CB1954 reduction products following TbNTR metabolism. The parental compound CB1954 (peak 1) and the 2- and 4-hydroxyalmine derivatives (peaks 2 and 3, respectively) were detected.

FIG. 3.

Susceptibility of bloodstream-form T. brucei overexpressing TbNTR to aziridinyl nitrobenzamides. (A) Structures of the ANBs with highest trypanocidal activity (Table 2). (B) Growth-inhibitory effect (IC₅₀s in μM) of CB1954, NH1, and NH2 on T. brucei cells overexpressing TbNTR (TbNTR^RV). Data are means from 4 experiments ± SD, and the differences in susceptibility were statistically significant (P < 0.01), as assessed by Student's t test. Melarsoprol and nifurtimox were used as drug controls.