The 8-Aminoquinoline Analogue Sitamaquine Causes Oxidative Stress in Leishmania donovani Promastigotes by Targeting Succinate Dehydrogenase

Abstract

The 8-aminoquinoline analogue sitamaquine (SQ) is an oral antileishmanial drug currently undergoing phase 2b clinical trials for the treatment of visceral leishmaniasis. In the present study, we investigated the mechanism of action of this drug in Leishmania donovani promastigotes. SQ causes a dose-dependent inhibition of complex II (succinate dehydrogenase) of the respiratory chain in digitonin-permeabilized promastigotes, together with a drop in intracellular ATP levels and a decrease of the mitochondrial electrochemical potential. This is associated with increases of reactive oxygen species and intracellular Ca 2؉ levels, a higher percentage of the population with sub-G 1 DNA content, and exposure of phosphatidylserine. Taken together, these results support a lethal mechanism for SQ that involves inhibition of the respiratory chain complex II, which in turn triggers oxidative stress and finally leads to an apoptosis-like death of Leishmania parasites.Leishmaniasis, a protozoal infectious disease caused by a set of 17 species of the genus Leishmania, shows a wide spectrum of clinical manifestations, including, in order of increasing severity, cutaneous (CL), mucocutaneous (MCL), and visceral (VL) leishmaniasis (3). Although chemotherapy is the only current treatment option for leishmaniasis, its efficacy is increasingly limited by growing resistance to first-line drugs, especially antimonials, by the frequent side effects associated with their use, and by the high cost of treatment (30). The paucity of new drugs in the pipeline, together with the poor definition of Leishmania targets for the drugs in current clinical use, represents an additional concern for current chemotherapy. Solutions to curb this pessimistic scenario rely on combination therapy (40) and the rescue of old drugs, such as paromomycin (17, 36) and sitamaquine (SQ) (39), that were previously discarded.8-Aminoquinolines are an important class of antiparasitic agents (37) with broad application and excellent efficacy but with limitations due to their hematological toxicity (primarily metahemoglobinemia and hemolysis). SQ, formerly known as WR6026, is an 8-aminoquinoline that was initially developed by the Walter Reed Army Institute (46). The results of phase 2b clinical trials of this drug against VL in India (16) and Kenya (45) by GlaxoSmithKline were encouraging. These results, together with its oral administration, represent a substantial advantage in terms of its future widespread implementation.The targets for SQ remain elusive. Entry of SQ into the parasite starts with an electrostatic interaction with anionic phospholipids of the plasma membrane (11). In a seminal work, Vercesi and Docampo (43) observed a loss of mitochondrial electrochemical potential in digitonin-permeabilized parasites after SQ addition, together with alkalinization of acidocalcisomes (44), which also underwent a privileged SQ accumulation, although no correlation was found with its toxicity (18).Herein we provide further insight into the leishmanicidal mechanism of S...

Induction of Mitochondrial Dysfunction and Oxidative Stress in Leishmania donovani by Orally Active Clerodane Diterpene

“…Studies have detected a significantly larger amount of active-form metacaspase in cells undergoing H 2 O 2 -induced PCD (8), as well as elevated metacaspase gene expression in miltefosine-induced PCD (42). However, studies that report caspase-independent PCD in these parasites might suggest that metacaspase involvement is not essential (13,34). In our study, we found major upregulation of caspase-3/-7-like protease levels in cells following K-09 exposure, and the pretreatment of cells with the caspase-3 inhibitor Ac-DEVD-CHO diminished caspase-3/-7-like protease activity and PS externalization.…”

Experimental Resistance to Drug Combinations in Leishmania donovani: Metabolic and Phenotypic Adaptations

“…Sytox green dye was used to assess plasma membrane integrity, as described previously, with some modifications (40). Briefly, parasites (1 ϫ 10 7 promastigotes) of the different L. donovani lines (WTM, A, M, S, AS, and SP) were left untreated or treated with increasing concentrations of their corresponding drugs (AmB, MIL, or Sb III ) for 48 h at 28°C in culture medium, washed twice with HBS buffer, and then incubated with 2 M Sytox green for 15 min at 28°C.…”

Distribution of Human Leishmaniasis (VL) and Its Associated Risk Factors, in Metemma, Ethiopia

“…A combination therapy of miltefosine with amphotericin B or paromomycin is very efficient and could be helpful to treat antimony-resistant VL infections. Sitamaquine is the second oral drug in the development of leishmaniasis treatment [8].…”