Activity of Megazol, a Trypanocidal Nitroimidazole, Is Associated with DNA Damage

Abstract

DNA damage associated with the trypanocidal activity of megazol [2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole] was shown in experiments in which DNA repair-deficient RAD51؊/؊ Trypanosoma brucei mutants were found to be hypersensitive to the drug. Parasites resistant to megazol were selected and showed modest cross-resistance to other trypanocides, although neither drug efflux nor changes to intracellular thiols correlated with resistance.New drugs are urgently required for treatment of human African trypanosomiasis (22). One compound with promise is megazol (5, 12, 13, 18), a nitroheterocyclic compound that forms a nitro radical anion upon reduction (31). Megazol appears to enter Trypanosoma brucei via passive diffusion (3), but little is known about its mode of action. We determined the effect of megazol in assays that measured oxidative and reductive stress. We also selected parasites resistant to megazol in order to assess cellular changes associated with resistance.Bloodstream forms (17) and procyclic forms (6) of T. brucei brucei (strain 427) were cultivated by using standard techniques. Assays measuring drug sensitivity were carried out to determine the drug concentration producing a 50% decrease in cell proliferation (IC 50 ) and the resistance factor (average ratio of the IC 50 for a resistant clone to the IC 50 for a wild-type clone). The Alamar Blue test (28) was used for bloodstream forms, while cell counting by using an improved Neubauer chamber was required for procyclics. For procyclic cells the IC 50 of megazol was 0.28 Ϯ 0.01 M, and for bloodstream forms the IC 50 was 0.15 Ϯ 0.02 M.To induce megazol resistance in vitro, trypanosomes were exposed to drug concentrations that doubled every 2 weeks, starting at 0.008 and 0.01 M for bloodstream forms and procyclics, respectively. After 6 months of cultivation, procyclic and bloodstream forms that tolerated 10 and 1 M megazol, respectively, were cloned by limiting dilution.The IC 50 for the cloned, resistant, procyclic line was 29.24 Ϯ 3.2 M (105-fold-reduced sensitivity), while the IC 50 for the bloodstream form line was 3.2 Ϯ 0.48 M (21-fold-reduced sensitivity).Cross-resistance to megazol and other trypanocides was also studied (Table 1). Moderate levels of cross-resistance to two nitroheterocyclic trypanocides, the nitrofuran nifurtimox and the nitroimidazole benznidazole, were detected. Significant, albeit low-level, cross-resistance to the melaminophenyl arsenical compounds cymelarsan, melarsen oxide, and melarsoprol, the diamidine compounds pentamidine and berenil, and suramin was also observed.Verapamil (20), PAK-104P (7), and two phenothiazine derivatives (prochlorperazine and trifluoperazine) (14) all inhibit cellular extrusion pumps but, when used at 5 M, failed to reverse megazol resistance in T. brucei. Efflux pumps are thus unlikely to play a significant role in the megazol resistance characterized here.It was recently shown that megazol exposure reduces trypanothione levels in Trypanosoma cruzi (23). Bloodstream form T. b...

Buthionine Sulfoximine Increases the Toxicity of Nifurtimox and Benznidazole toTrypanosoma cruzi

“…Similar approaches with an in vitro model with megazol in T. brucei (11) and with Leishmania (17) have been reported. Nevertheless, in the Leishmania model BSO was barely effective (17).…”

Trypanocidal Activity of Melamine-Based Nitroheterocycles

“…The action of nifurtimox, however, is antagonized by NAC, whereas that of megazol is not. It therefore appears that megazol acts through reductive stress, whereas nifurtimox acts through oxidative stress (16,18). Compound 3a does not have any enhanced activity against the RAD51 knockout cells, which indicates that this compound may not exert its activity through reductive stress.…”

“…Using the RAD51 knockout line and NAC assay system, we have shown that the nitroimidazole-thiadazole megazol exerts its activity through reductive stress as RAD51 knockout parasites, which are deficient in DNA repair and hypersensitive to this drug (18). These cells are not hypersensitive to the nitrofuran nifurtimox.…”

“…Megazol is positive in Ames tests (20) and mutagenic in mammalian cell tests (34). Trypanosomes deficient in their own DNA repair enzymes are also hypersensitive to megazol, indicating that the genotoxic effects of this drug are also manifest in these cells (18). A T. brucei mutant deficient in a DNA repair enzyme (RAD51) was tested for susceptibility to megazol, to nifurtimox, and to compound 3a.…”

“…Oxidative stress is believed to arise from reduced oxygen intermediates that arise once oxygen accepts electrons from the reduced nitro group (16). We have developed assays using trypanosomes that give insight as to whether reductive or oxidative stress underlies the activity of these compounds (18). Reductive stress can be assessed by measuring the susceptibility of DNA repair-deficient trypanosomes to toxic agents.…”