Induction of Oxidative Stress in Trypanosoma brucei by the Antitrypanosomal Dihydroquinoline OSU-40

Abstract

In the present study, we sought to provide further support for the hypothesis that OSU-40 kills trypanosomes through oxidative stress. Inducible RNA interference (RNAi) was applied to downregulate key enzymes in parasite antioxidant defense, including T. brucei trypanothione synthetase (TbTryS) and superoxide dismutase B (TbSODB). Both TbTryS RNAi-induced and TbSODB RNAi-induced cells showed impaired growth and increased sensitivity toward OSU-40 by 2.4-fold and 3.4-fold, respectively. Decreased expression of key parasite antioxidant enzymes was thus associated with increased sensitivity to OSU-40, consistent with the hypothesis that OSU-40 acts through oxidative stress. Finally, the dose-dependent formation of free radicals was observed after incubation of T. brucei with OSU-40 utilizing electron spin resonance (ESR) spectroscopy. These data support the notion that the mode of antitrypanosomal action for this class of compounds is to induce oxidative stress. H uman African trypanosomiasis (HAT) is a vector-borne parasitic disease caused by the Trypanosoma brucei subspecies T. b. rhodesiense and T. b. gambiense.The trypanosome proliferates in the host's hemolymphatic system during the first stage of HAT and invades the central nervous system in the second stage. Transmitted by the tsetse fly, the disease mainly affects rural populations in sub-Saharan Africa and is fatal if untreated. Although according to the World Health Organization the number of new cases has dropped to just over 7,000 during 2010 (39), very few drugs are available to treat HAT (2, 4, 42). Pentamidine and suramin, both developed in the first half of the 20th century, are used against first-stage disease, while melarsoprol and eflornithine are used to treat second-stage disease. These drugs are far from satisfactory due to limitations such as severe toxicity, poor efficacy, acquired resistance, rising failure rates, and lack of availability (2, 4, 42). With high efficacy and a good safety profile, nifurtimox-eflornithine combination therapy (NECT) was introduced as a first-line treatment for second-stage HAT caused by T. b. gambiense in 2009 (4, 22, 42). However, administration of NECT is relatively complicated, and its efficacy toward T. b. rhodesiense is still questionable (4, 42). Thus, new drugs are needed against HAT that are safe, affordable, easy to administer, active against first-and second-stage disease, and effective against both subspecies of T. brucei (4, 42).From a previous synthetic medicinal chemistry study, we discovered several N1-substituted 1,2-dihydroquinoline-6-ols displaying nanomolar 50% inhibitory concentrations (IC 50 s) in vitro against T. b. rhodesiense and selectivity indexes (SI) up to Ͼ18,000 (11). OSU-40 (1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate) (Fig. 1) showed a potency (IC 50 , 0.014 M; SI, 1,700) close to that of melarsoprol (IC 50 , 0.008 M; SI, 1,000) against T. b. rhodesiense STIB900 in vitro. In an early treatment mouse model of acute African trypanosomiasis, OSU-40 prolonged the life s...

Metabolic reconfiguration of the central glucose metabolism: a crucial strategy ofLeishmania donovanifor its survival during oxidative stress

“…2A-C) indicating that promastigotes do not increase glucose uptake under stressful conditions. Analysis of metabolic flux revealed that H 2 O 2 , menadione, and diamide stimulated a metabolic reconfiguration from glycolysis toward PPP indicated by marked increase in 14 CO 2 evolution from [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]D-glucose whereas CO 2 evolution after utilization of [6][7][8][9][10][11][12][13][14] C]D-glucose did not significantly alter in Figure 6. Characterization of G6PDH and TAL overexpressing (OE) cell lines against wild-type (WT) cells transfected with empty vector.…”

“…Interestingly, methylene blue, known to create intracellular oxidative stress by disrupting NADPH:NADP ratio, also had an similar effect on L. mexicana promastigotes (30). Cells cotreated with NAC in case of exposure to H 2 O 2 and menadione and GSH in case of diamide showed a decrease in 14 CO 2 evolution from [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]D-glucose indicative of ROS-mediated shifting of metabolic flux toward PPP from glycolysis. The glucose consumption assay also reinforced our observation by demonstrating that the glucose uptake pattern (Fig.…”

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…Still, an increase of ROS production has been shown in sensitive L. infantum promastigotes under antimonial (SbIII), miltefosine, and amphotericin B treatments but was absent in resistant parasites, suggesting that, somehow, the resistant strains had succeeded in avoiding oxidative stress (28). ROS formation was also shown in T. brucei and in T. cruzi under dihydroquinoline derivative and nifurtimox treatment, respectively (286,287). Leishmania parasites are known to be sensitive to ROS from macrophage (288).…”

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux

“…lular stages of Plasmodium (2), Toxoplasma (3), and Trypanosoma cruzi (4) and for extracellular stages such as the Trypanosoma brucei bloodstream form (5). Consequently, selective disruption of the parasite redox balance is an effective approach to therapeutic intervention (6 -8).…”