[Growth and DNA synthesis inhibition and superoxide anion formation by iminoquinones in Trypanosoma cruzi]

Abstract

Despite its epidemiological importance, chemotherapy of Chagas' disease is still an unsolved problem. Many drugs have been assayed for their trypanocidal action on T. cruzi, but, so far, naftoquinone-imines have not been included in drug screenings. Fernández A.E. et al., prepared a series of 4-(aminomethylisoxazolyl)-1,2-naftoquinones. Some of these, namely, IIIA (2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4- imine), IIIC hydroxy-N-3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i min e, IIIE (2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-i min e), and IVD (5-methyl-3-isoxazolyl)-1,2-naftoquinone-4-imine) were assayed on T. cruzi epimastigotes. Parasite growth and DNA synthesis were inhibited 12-36% and 14-49%, respectively, by 18-19 microM quinones or 44-83% and 37-73%, respectively, by 60-78 microM quinones (Table 1). The assayed compounds (37-100 microM concentration) stimulated oxygen uptake (Figs. 2 and 3) and superoxide anion generation by epimastigotes to 0.20-0.70 nmol/min/mg of cell protein (O2- measured by the adrenochrome method, Table 2). In the presence of quinones, T. cruzi mitochondrial fraction supplemented with 0.42 mM NADH produced O2- at a rate of 1.10-1.45 nmol/min/mg of protein (31 and 75 microM quinone, respectively; Table 3) whereas, under the same experimental conditions, the microsomal fraction supplemented with NADPH produced O2- at rate of 0.85 and 1.03, respectively (same units). The kinetics of O2- generation by the mitochondrial fraction supplemented with quinone IIIC and NADH revealed two interacting sites (Fig. 5). The corresponding Km(s) were 5.2 and 17 microM and the Vm, 13 and 17 nmoles O2-/min/mg of protein, respectively. The quinone interaction with the mitochondrial membranes involved a "quinone reductase" located on the substrate site of the antimycin site, as shown by the 3.6-fold increase of the oxygen uptake rate induced by quinone IIIE in Fig. 2. Apparently, the quinone excess inhibited the reductase since by varying the quinone concentration in the 0-500 microM range, the oxygen uptake versus quinone concentration plots showed a maximum at 100-150 microM quinone (Fig. 3). O2- generation by quinones depended on the latter redox-cycling, as shown by a) the decrease of quinone absorption at 480-500 nm after incubation with T. cruzi epimastigotes for the time necessary to exhaust the reaction mixture oxygen, and b) the reappearance of the quinone absorption band after oxygenation of the anaerobic reaction mixture (Fig. 6).