RESUMO
Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is a cofactor used in different anabolic reactions, such as lipid and nucleic acid synthesis, and for oxidative stress defense. NADPH is essential for parasite growth and viability. In trypanosomatid parasites, NADPH is supplied by the oxidative branch of the pentose phosphate pathway and by enzymes associated with the citric acid cycle. The present article will review recent achievements that suggest glucose-6-phosphate dehydrogenase and the cytosolic isoform of the malic enzyme as promising drug targets for the discovery of new drugs against Trypanosoma cruzi and T. brucei. Topics involving an alternative strategy in accelerating T. cruzi drug-target validation and the concept of drug-target classification will also be revisited.
Assuntos
Doença de Chagas/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , NADP/antagonistas & inibidores , Tripanossomicidas/farmacologia , Animais , Glucosefosfato Desidrogenase/antagonistas & inibidores , Humanos , Malato Desidrogenase (NADP+)/antagonistas & inibidores , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
The location and changes in NAD(P)H have been monitored during oscillatory growth in pollen tubes of lily (Lilium formosanum) using the endogenous fluorescence of the reduced coenzyme (excitation, 360 nm; emission, >400 nm). The strongest signal resides 20 to 40 microm behind the apex where mitochondria (stained with Mitotracker Green) accumulate. Measurements at 3-s intervals reveal that NAD(P)H-dependent fluorescence oscillates during oscillatory growth. Cross-correlation analysis indicates that the peaks follow growth maxima by 7 to 11 s or 77 degrees to 116 degrees, whereas the troughs anticipate growth maxima by 5 to 10 s or 54 degrees to 107 degrees. We have focused on the troughs because they anticipate growth and are as strongly correlated with growth as the peaks. Analysis of the signal in 10-microm increments along the length of the tube indicates that the troughs are most advanced in the extreme apex. However, this signal moves basipetally as a wave, being in phase with growth rate oscillations at 50 to 60 microm from the apex. We suggest that the changes in fluorescence are due to an oscillation between the reduced (peaks) and oxidized (troughs) states of the coenzyme and that an increase in the oxidized state [NAD(P)(+)] may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium, an inhibitor of NAD(P)H dehydrogenases, causes an increase in fluorescence and a decrease in tube growth. Finally, staining with 5-(and-6)-chloromethyl-2',7'-dichlorohydrofluorescein acetate indicates that reactive oxygen species are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.
Assuntos
Lilium/crescimento & desenvolvimento , NADP/metabolismo , Tubo Polínico/metabolismo , Fluorescência , Lilium/efeitos dos fármacos , Lilium/metabolismo , Lilium/ultraestrutura , Mitocôndrias/metabolismo , NADP/análise , NADP/antagonistas & inibidores , NADP/fisiologia , Oniocompostos/farmacologia , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/ultraestrutura , Espécies Reativas de Oxigênio/metabolismoRESUMO
The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 M, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 M, indicating a good selectivity against the parasite enzyme.