Miltefosine (Hexadecylphosphocholine) Inhibits Cytochrome c Oxidase in Leishmania donovani Promastigotes

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Leishmaniasis is the protozoan disease second in importance for human health, superseded only by malaria; however, the options for chemotherapeutic treatment are increasingly limited due to drug resistance and toxicity. Under this perspective, a quest for new chemical compounds is urgently needed. An N-substituted 2-aminoalkan-1-ol scaffold has been shown to be a versatile scaffold for antiparasitic activity. Knowledge about its mechanism of action is still rather limited. In this work, we endeavored to define the leishmanicidal profile of such ␤-amino alkanol derivatives using a set of 15 N-mono-and disubstituted surrogates, tested on Leishmania donovani promastigotes and intracellular amastigotes. The best compound (compound 5), 2-ethylaminododecan-1-ol, had a 50% effective concentration (EC 50 ) of 0.3 M and a selectivity index of 72 for infected THP-1 cells and was selected for further elucidation of its leishmanicidal mechanism. It induced fast depletion of intracellular ATP content in promastigotes in the absence of vital dye intracellular entry, ruling out plasma membrane permeabilization as its origin. Confocal and transmission electron microscopy analyses showed that compound 5 induced severe mitochondrial swelling and vesiculation. Polarographic analysis using an oxygen electrode demonstrated that complex II of the respiratory chain (succinate reductase) was strongly inhibited by compound 5, identifying this complex as one of the primary targets. Furthermore, for other ␤-amino alkanols whose structures differed subtly from that of compound 5, plasma membrane permeabilization or interference with membrane traffic was also observed. In all, N-substituted ␤-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration.

Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Mechanisms of Action of Substituted β-Amino Alkanols on Leishmania donovani

Abengózar

Bustos

García-Hernández

et al. 2015

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“…A similar effect has been described for the mechanism of action of other potential and established leishmanicidal drugs (19)(20)(21)(22)(23). Arguably, the two most likely hypotheses to account for this result are (i) inhibition of ATP synthesis and (ii) permeabilization of the plasma membrane with release of intracellular ATP into the external medium.…”

Section: Discussionmentioning

confidence: 82%

“…The Leishmania mitochondrion is the target for a wide variety of leishmanicidal drugs; including some in clinical use such as pentamidine (27) and miltefosine (20), and others at different stages of development, such as chalcones or histatin 5, (5,23). The inhibition of complex III by TFQ led to the production of superoxide radicals at levels similar to those produced by antimycin A (24).…”

Section: Discussionmentioning

confidence: 99%

Tafenoquine, an Antiplasmodial 8-Aminoquinoline, Targets Leishmania Respiratory Complex III and Induces Apoptosis

Carvalho

Luque‐Ortega

Manzano

et al. 2010

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Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca 2؉ levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process.Leishmaniasis includes a wide variety of clinical manifestations caused by the protozoan parasite Leishmania. Visceral leishmaniasis is the most severe form of the disease and is usually fatal if not treated (http://www.who.int/leishmaniasis /burden/en/). In the absence of a reliable vaccine, leishmaniasis treatment relies exclusively on chemotherapy. Resistance to organic pentavalent antimonials (until recently considered to be the standard treatment) in northeast India (4), together with the severe side effects associated with their use, has led to the use of alternative treatments based on the incorporation of drugs such as amphotericin B, miltefosine, and paromomycin into the arsenal of antileishmanial drugs (8). Nevertheless, the limited number of active drugs has prompted the WHO to recommend a combined therapy in order to extend the life expectancy of these compounds.Among the new drugs under development, sitamaquine (WR6026; GlaxoSmithKline), an 8-aminoquinoline, currently under phase IIb clinical trials, represents a promising drug for the oral treatment of leishmaniasis (35). In addition, another 8-aminoquinolines have been synthesized and evaluated for their leishmanicidal activity (29, 36). However, the leishmanicidal mechanism of 8-aminoquinolines is still unknown. Sitamaquine, for example, accumulates in the acidocalcisomes, but this organelle has been ruled out as its final target (17). The collapse of mitochondrial potential in digitonized Leishmania donovani promastigotes has also been reported (39). Tafenoquine (TFQ), formerly known as WR238605, is an analogue of primaquine with much lower toxicity than the parental drug. It has demonstrated significant leishmanicidal activity in the mouse experimental model (41) and may represent an alternative treatment for leishmaniasis.In the present study, we have shown that TFQ inhibits the mitochondrial cytochrome c reductase of Leishmania promastigotes. This inhibition causes a drop in the intracellular ATP levels of the parasite and the loss of mitochondrial membrane potential. TFQ induces ROS production and deregulation of Ca 2ϩ homeostasis, fol...

“…This then activates caspase-like proteases (7), which play major roles in the apoptotic cascade of these parasites (19,22). We postulate that interference in the mitochondrial respiratory chain complexes of the parasite might be responsible for the lethal effects of amlodipine and lacidipine, which cause a reduction of oxygen consumption and death, apparently by apoptosis, via activation of caspase 3-like protease, with mitochondria as the key intracellular target (10).…”

mentioning

confidence: 99%

Oral Therapy with Amlodipine and Lacidipine, 1,4-Dihydropyridine Derivatives Showing Activity against Experimental Visceral Leishmaniasis

Palit

Ali

2008

Amlodipine and lacidipine, conventional antihypertensive drugs, inhibited Leishmania donovani infection in vitro and in BALB/c mice when administered orally. These 1,4-dihydropyridine derivatives functioned through dose-dependent inhibition of oxygen consumption, triggering caspase 3-like activation-mediated programmed cell death of the parasites.The recommended drugs used for the treatment of visceral leishmaniasis (VL), i.e., pentavalent antimonials, were first introduced 60 years ago. Over the last decade, alternative new drugs and newer formulations have become available or are under clinical trial to combat this deadly disease. However, they all suffer from limitations of cost, specific toxicities, parenteral administration, emergence and spread of drug resistance, or extended treatment regimens (2, 6). The most remarkable advance has been the introduction of the first effective oral treatment of VL with miltefosine, an alkyl lysophospholipid analogue. However, teratogenicity, gastrointestinal upset, potential of resistance development, and a low therapeutic window pose limitations on its use (5,20). Hence, the ambition to develop an orally effective drug or drug formulation which requires a short course of treatment without the prevalent limitations of toxicity and drug resistance remains unfulfilled. Amlodipine and lacidipine, dihydropyridine Ca 2ϩ channel blockers, are used orally for the treatment of hypertension. Previous reports suggested that amlodipine can also inhibit the proliferation of different cancer cells (9, 21). In addition, amlodipine has been reported as a potential antimicrobial agent (8). It has also been reported that lacidipine (15) and some 3-chloro-phenyl (11) and nitro aryl 1,4-dihydropyridine (16) derivatives are cytotoxic towards Trypanosoma cruzi through respiratory chain inhibition. Moreover, nifedipine, another dihydropyridine Ca 2ϩ channel blocker, can inhibit Leishmania-macrophage attachment during initiation of the disease (13). Amlodipine and lacidipine both have a phenyl-1,4-dihydropyridine moiety (Fig. 1A) and are structurally unrelated to other Ca 2ϩ channel blockers. In view of the diverse biological activities observed for amlodipine and lacidipine, we were interested in assessing their activity against Leishmania donovani (MHOH/IN/1983/AG83) parasites in vitro and in extending our observations through oral administration in vivo.To evaluate the effects of the drugs on promastigotes, freshly transformed promastigotes of L. donovani AG83 (2 ϫ 10 6 /ml) in medium 199 containing 10% fetal bovine serum were incubated with graded concentrations of drugs at 22°C for 2 h, and their viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay (14). The 50% effective concentrations of amlodipine and lacidipine were 2 and 2.5 g/ml (calculated by sigmoidal regression analysis using Microsoft Excel, 2007), respectively (Fig. 1B). Both drugs killed (98.76% for amlodipine [P Ͻ 0.0001] and 90.5% for lacidipine [P Ͻ 0.001]) promastigotes effec...