Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Abstract

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

Fig. 1. : chemical structures of nifurtimox (1) and benznidazole (2), the drugs currently used to treat Chagas disease, and pentamidine (3), suramin (4), melarsoprol (5) and eflornithine (6), the agents currently used to treat African sleeping sickness.

Fig. 2. : chemical structures of megazol (7) and its triazole analog [5-(1-methyl-5-nitro-1H-imidazol-2-yl)-4H-1,2,4-triazol-3-amine (8)].

Fig. 3. : DNA damage induction in human whole blood as analysed using the Comet assay. A: megazol (7) (n = 3); B: triazole analog [5-(1-methyl-5-nitro-1H-imidazol-2-yl)-4H-1,2,4-triazol-3-amine (8)] (n = 2). The white bars correspond to the untreated control culture and the solvent-control [5% (v/v) dimethyl sulfoxide (DMSO)], the grey bars correspond to the tested compounds and the black bars correspond to the positive control (160 µM methyl methane-sulfonate). The bars represent the standard error of the means of the total arbitrary units (AU). For Student’s one tailed t test, the asterisks indicate significance at 1% (**) and 0.1% (***) levels. compound 7: megazol; compound 8: 5-(1-methyl-5-nitro-1H-imidazol-2-yl)-4H-1,2,4-triazol-3-amine.

Fig. 4. : intercellular distribution of DNA damage in human whole blood, as analysed using the Comet assay. A: megazol (7) at concentrations of 380, 611, 976, 1,562, 2,500 and 4,000 µM (arrows) (n = 3); B: the triazole analog [5-(1-methyl-5-nitro-1H-imidazol-2-yl)-4H-1,2,4-triazol-3-amine (8)] at concentrations of 149, 238, 382, 610, 977, 1,562, 2,500, 4,000 and 6,400 µM (arrows) (n = 2). The white bars correspond to the untreated control culture and the solvent-control [5% (v/v) dimethyl sulfoxide], the grey bars correspond to the tested compounds and the black bars correspond to the positive control (160 µM methyl methane-sulfonate). The bars represent the standard error of the mean frequency (%) of the cells in the different classes of DNA damage. For the Dunnett’s test, the asterisks indicate significance at 5% (*), 1% (**) and 0.1% (***) levels.

Fig. 5. : electronic distributions for megazol (7) and compound 8 [5-(1-methyl-5-nitro-1H-imidazol-2-yl)-4H-1,2,4-triazol-3-amine]. Red indicates an electron-rich highest occupied molecular orbital (HOMO) [or lowest unoccupied molecular orbital (LUMO)] region, while blue indicates an electron-poor region. Semi-empirical calculations were performed using the MOPAC2009 software package (OpenMOPAC.net) with the semiempirical PM3 method and full geometric optimisation. The ejection fraction routine was used for the minimum search.

Fig. 6. : putative binding modes for compound 8 [5-(1-methyl-5-nitro-1H-imidazol-2-yl)-4H-1,2,4-triazol-3-amine] (A) and megazol (B) within the modelled Trypanosoma brucei type I nitroreductase binding site. The flavin mononucleoside cofactor is also depicted in cyan. Residues Thr227 (chain A - purple) and Phe114 (chain B - yellow) are shown as lines. The H-bond distances between the ligands and proteins are shown in yellow and the units are given in angstroms (Å).