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...

Sitamaquine (WR6026), an 8-aminoquinoline derivative, is a new antileishmanial oral drug. As a lipophilic weak base, it rapidly accumulates in acidic compartments, represented mainly by acidocalcisomes. In this work, we show that the antileishmanial action of sitamaquine is unrelated to its level of accumulation in these acidic vesicles. We have observed significant differences in sitamaquine sensitivity and accumulation between Leishmania species and strains, and interestingly, there is no correlation between them. However, there is a relationship between the levels of accumulation of sitamaquine and acidotropic probes, acidocalcisomes size, and polyphosphate levels. The Leishmania major AP3␦-null mutant line, in which acidocalcisomes are devoid of their usual polyphosphate and proton content, is unable to accumulate sitamaquine; however, both the parental strain and the AP3␦-null mutants showed similar sensitivities to sitamaquine. Our findings provide clear evidence that the antileishmanial action of sitamaquine is unrelated to its accumulation in acidocalcisomes.

Although oral miltefosine represented an important therapeutic advance in the treatment of leishmaniasis, the appearance of resistance remains a serious threat. LMDR1/LABCB4, a P-glycoprotein-like transporter included in the Leishmania ABC (ATP-binding cassette) family, was the first molecule shown to be involved in experimental miltefosine resistance. LMDR1 pumps drugs out of the parasite, thereby decreasing their intracellular accumulation. Sitamaquine, another promising oral drug for leishmaniasis, is currently in phase 2b clinical trials. The physicochemical features of this drug suggested to us that it could be considered for use as an LMDR1 inhibitor. Indeed, we report herein that nonleishmanicidal concentrations of sitamaquine reverse miltefosine resistance in a multidrug resistance Leishmania tropica line that overexpresses LMDR1. This reversal effect is due to modulation of the LMDR1-mediated efflux of miltefosine. In addition, sitamaquine is not a substrate of LMDR1, as this transporter does not affect sitamaquine accumulation or sensitivity in the parasite. Likewise, we show that ketoconazole, another oral leishmanicidal drug known to interact with ABC transporters, is also able to reverse LMDR1-mediated miltefosine resistance, although with a lower efficiency than sitamaquine. Molecular docking on a three-dimensional homology model of LMDR1 showed different preferential binding sites for each substrate-inhibitor pair, thus explaining this different behavior. Finally, we show that sitamaquine is also able to modulate the antimony resistance mediated by MRPA/LABCC3, another ABC transporter involved in experimental and clinical antimony resistance in this parasite. Taken together, these data suggest that the combination of sitamaquine with miltefosine or antimony could avoid the appearance of resistance mediated by these membrane transporters in Leishmania.