4-Amino Bis-Pyridinium Derivatives as Novel Antileishmanial Agents

The antileishmanial activity of a series of bis-pyridinium derivatives that are analogues of pentamidine have been investigated, and all compounds assayed were found to display activity against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania major, with 50% effective concentrations (EC₅₀s) lower than 1 μM in most cases. The majority of compounds showed similar behavior in both Leishmania species, being slightly more active against L. major amastigotes. However, compound VGP-106 {1,1′-(biphenyl-4,4′-diylmethylene)bis[4-(4-bromo-N-methylanilino)pyridinium] dibromide} exhibited significantly higher activity against L. donovani amastigotes (EC₅₀, 0.86 ± 0.46 μM) with a lower toxicity in THP-1 cells (EC₅₀, 206.54 ± 9.89 μM). As such, VGP-106 was chosen as a representative compound to further elucidate the mode of action of this family of inhibitors in promastigote forms of L. donovani. We have determined that uptake of VGP-106 in Leishmania is a temperature-independent process, suggesting that the compound crosses the parasite membrane by diffusion. Transmission electron microscopy analysis showed a severe mitochondrial swelling in parasites treated with compound VGP-106, which induces hyperpolarization of the mitochondrial membrane potential and a significant decrease of intracellular free ATP levels due to the inhibition of ATP synthesis. Additionally, we have confirmed that VGP-106 induces mitochondrial ROS production and an increase in intracellular Ca²⁺ levels. All these molecular events can activate the apoptotic process in Leishmania; however, propidium iodide assays gave no indication of DNA fragmentation. These results underline the potency of compound VGP-106, which may represent a new avenue for the development of novel antileishmanial compounds.     

In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences

The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC₅₀], 0.6 to 0.7 μM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC₅₀, 0.1 to 0.4 μM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC₅₀, 0.4 to 3.8 μM) and intracellular (IC₅₀, 0.9 to 4.3 μM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC₅₀, >64 μg Sb^V/ml) but showed good efficacy against intracellular amastigotes (IC₅₀, 22 to 28 μg Sb^V/ml); the crystalline substance was about two to three times more potent (IC₅₀, 9 to 11 μg Sb^V/ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC₅₀, 0.1 μg/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.Current first-line chemotherapy of leishmaniasis relies on a rather limited arsenal of drugs including sodium stibogluconate, meglumine antimoniate, amphotericin B, and miltefosine, but these entail either problems of emerging resistance, severe side effects, or high costs (5). Since vaccines are not yet on the horizon (23), maintenance and improvement of existing treatment regimens, combined with new drug discovery initiatives, appear to be the only ways to guarantee continued control of this important tropical disease (3, 11, 27).Both for drug screening and for determination of the susceptibility of field strains, different laboratory methods are being used that focus on the promastigote, the axenic amastigote, or the intracellular amastigote stage. However, it remains unclear how these models cross-validate each other. The differences in environmental conditions between promastigotes and amastigotes in vivo are reflected in their needs for in vitro cultivation. While promastigotes are easily cultured in suspension (8), amastigotes are more difficult to maintain in vitro, since they require macrophages as host cells to meet the highly acidic intracellular environment (15). Cultivation of axenic amastigotes in suspension has been successful, enabling promising new opportunities for drug screening and mode-of-action studies (31, 38). For field strains, infection of macrophages with metacyclic promastigotes is generally used but is subject to a high degree of variability in infectivity depending on the growth characteristics of the isolate and the level of in vitro metacyclogenesis (6). Despite the large body of literature on the subject, the central question remains which laboratory system is optimally suited for either drug-screening purposes or field strain susceptibility testing against the current first-line antileishmanial drugs.The aim of the present study was to compare the different in vitro test systems and the intrinsic susceptibilities of the various stages of a single reference strain of Leishmania donovani (MHOM/ET/67/L82) to pentavalent antimony (Sb^V), amphotericin B, miltefosine, and the experimental “antileishmanial lead” compound PX-6518, a saponin mixture isolated from the Vietnamese plant Maesa balansae (19) and included in the test to underline the importance of current findings for drug-screening and mode-of-action studies. To our knowledge, this is a first report on the in vitro sensitivities of all Leishmania stages (including freshly collected ex vivo amastigotes) to a broad range of standard reference drugs in a single integrated experimental setup.     

Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.     

Zinc(II)-Dipicolylamine Coordination Complexes as Targeting and Chemotherapeutic Agents for Leishmania major

Cutaneous leishmaniasis is a neglected tropical disease that causes painful lesions and severe disfigurement. Modern treatment relies on a few chemotherapeutics with serious limitations, and there is a need for more effective alternatives. This study describes the selective targeting of zinc(II)-dipicolylamine (ZnDPA) coordination complexes toward Leishmania major, one of the species responsible for cutaneous leishmaniasis. Fluorescence microscopy of L. major promastigotes treated with a fluorescently labeled ZnDPA probe indicated rapid accumulation of the probe within the axenic promastigote cytosol. The antileishmanial activities of eight ZnDPA complexes were measured using an in vitro assay. All tested complexes exhibited selective toxicity against L. major axenic promastigotes, with 50% effective concentration values in the range of 12.7 to 0.3 μM. Similar toxicity was observed against intracellular amastigotes, but there was almost no effect on the viability of mammalian cells, including mouse peritoneal macrophages. In vivo treatment efficacy studies used fluorescence imaging to noninvasively monitor changes in the red fluorescence produced by an infection of mCherry-L. major in a mouse model. A ZnDPA treatment regimen reduced the parasite burden nearly as well as the reference care agent, potassium antimony(III) tartrate, and with less necrosis in the local host tissue. The results demonstrate that ZnDPA coordination complexes are a promising new class of antileishmanial agents with potential for clinical translation.     

Green Synthesis of Silver and Titanium Dioxide Nanoparticles Using Euphorbia prostrata Extract Shows Shift from Apoptosis to G0/G1 Arrest followed by Necrotic Cell Death in Leishmania donovani

The aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO₂) nanoparticles (NPs) using green synthesis from aqueous leaf extract of Euphorbia prostrata as antileishmanial agents and to explore the underlying molecular mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamarBlue and propidium iodide uptake assays. Antileishmanial activity of synthesized NPs on intracellular amastigotes was assessed by Giemsa staining. The leishmanicidal effect of synthesized Ag NPs was further confirmed by DNA fragmentation assay and by cell cycle progression and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs showed a spherical shape with an average size of 12.82 ± 2.50 nm, and in comparison to synthesized TiO₂ NPs, synthesized Ag NPs were found to be most active against Leishmania parasites after 24 h exposure, with 50% inhibitory concentrations (IC₅₀) of 14.94 μg/ml and 3.89 μg/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G₀/G₁ phase of the cell cycle with a subsequent decrease in S (synthesis) and G₂/M phases compared to controls was observed. The growth-inhibitory effect of synthesized Ag NPs was attributed to increased length of S phase. A decreased reactive oxygen species level was also observed, which could be responsible for the caspase-independent shift from apoptosis (G₀/G₁ arrest) to massive necrosis. High-molecular-weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. We also report that the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs. The green-synthesized Ag NPs may provide promising leads for the development of cost-effective and safer alternative treatment against visceral leishmaniasis.     

Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 half-knockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.     

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

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 Ca²⁺ 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 Ca²⁺ homeostasis, followed by nicking and fragmentation of DNA in Leishmania promastigotes leading to an apoptosis-like death. Our results provide the first insight into the mechanistic lethal pathway of an 8-aminoquinoline in Leishmania. This information may be useful for the design of more specific and less toxic compounds against leishmaniasis.     

N-Cinnamoylated Aminoquinolines as Promising Antileishmanial Agents

A series of cinnamic acid conjugates of primaquine and chloroquine were evaluated for their in vitro antileishmanial activities. Although primaquine derivatives had modest activity, chloroquine conjugates exhibited potent activity against both promastigotes (50% inhibitory concentration [IC₅₀] = 2.6 to 21.8 μM) and intramacrophagic amastigotes (IC₅₀ = 1.2 to 9.3 μM) of Leishmania infantum. Both the high activity of these chloroquine analogues and their mild-to-low toxicity toward host cells make them promising leads for the discovery of new antileishmanial agents.     

Dronedarone,an Amiodarone Analog with Improved Anti-Leishmania mexicana Efficacy

Dronedarone and amiodarone are cationic lipophilic benzofurans used to treat cardiac arrhythmias. They also have activity against the parasitic protozoan Trypanosoma cruzi, the causative agent of Chagas'' disease. They function by disrupting intracellular Ca²⁺ homeostasis of the parasite and by inhibiting membrane sterol (ergosterol) biosynthesis. Amiodarone also has activity against Leishmania mexicana, suggesting that dronedarone might likewise be active against this organism. This might be of therapeutic interest, since dronedarone is thought to have fewer side effects in humans than does amiodarone. We show here that dronedarone effectively inhibits the growth of L. mexicana promastigotes in culture and, more importantly, has excellent activity against amastigotes inside infected macrophages (the clinically relevant form) without affecting the host cell, with the 50% inhibitory concentrations against amastigotes being 3 orders of magnitude lower than those obtained previously with T. cruzi amastigotes (0.65 nM versus 0.75 μM). As with amiodarone, dronedarone affects intracellular Ca²⁺ homeostasis in the parasite, inducing an elevation of intracellular Ca²⁺ levels. This is achieved by rapidly collapsing the mitochondrial membrane potential and inducing an alkalinization of acidocalcisomes at a rate that is faster than that observed with amiodarone. We also show that dronedarone inhibits parasite oxidosqualene cyclase, a key enzyme in ergosterol biosynthesis known to be vital for survival. Overall, our results suggest the possibility of repurposing dronedarone as a treatment for cutaneous, and perhaps other, leishmaniases.     

N-Butyl-[1-(4-Methoxy)Phenyl-9H-β-Carboline]-3-Carboxamide Prevents Cytokinesis in Leishmania amazonensis

Leishmaniasis, a complex of diseases caused by protozoa of the genus Leishmania, is endemic in 98 countries, affecting approximately 12 million people worldwide. Current treatments for leishmaniasis have many disadvantages, such as toxicity, high costs, and prolonged treatment, making the development of new treatment alternatives highly relevant. Several studies have verified the antileishmanial activity of β-carboline compounds. In the present study, we investigated the in vitro antileishmanial activity of N-butyl-[1-(4-methoxy)phenyl-9H-β-carboline]-3-carboxamide (β-CB) against Leishmania amazonensis. The compound was active against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis, exhibiting high selectivity for the parasite. Moreover, β-CB did not exhibit hemolytic or mutagenic potential. Promastigotes treated with the alkaloid presented rounding of the body cell, cell membrane projections, an increase in the number of promastigotes presenting two flagella, and parasites of abnormal phenotype, with three or more flagella and/or nuclei. Furthermore, we observed an increase in the subpopulation of cells in the G₂/M stage of the cell cycle. Altogether, these results suggest that β-CB likely prevents cytokinesis, although it does not interfere with the duplication of cell structures. We also verified an increase in O₂^·− production and the accumulation of lipid storage bodies. Cell membrane integrity was maintained, in addition to the absence of phosphatidylserine externalization, DNA fragmentation, and autophagosomes. Although the possibility of an apoptotic process cannot be discarded, β-CB likely exerts its antileishmanial activity through a cytostatic effect, thus preventing cellular proliferation.     

In Vitro Sensitivity Testing of Leishmania Clinical Field Isolates: Preconditioning of Promastigotes Enhances Infectivity for Macrophage Host Cells

Diagnostic material from patients with leishmaniasis is generally available as promastigotes, and proper testing for susceptibility to first-line drugs by the intracellular amastigote assay is frequently hampered by the poor infectivity of the promastigotes for the macrophage host cell. Several conditions for optimization of the in vitro metacyclogenesis and cell infectivity of Leishmania donovani, L. guyanensis, and L. braziliensis field strains obtained from patients receiving standard antimony medication were investigated. Triggering log-phase promastigotes to become amastigote-like by increasing the temperature or acidifying the culture medium was not successful. Adequate metacyclogenesis and the highest levels of macrophage infection were obtained after 5-day-old late-log-phase promastigote cultures were preconditioned at 25°C to pH 5.4 for 24 h in Schneider''s medium prior to infection. The susceptibility assay with primary peritoneal mouse macrophages included pentavalent antimony (Sb^V; sodium stibogluconate), trivalent antimony (Sb^III; potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC₅₀] < 10 μM) and PX-6518 (IC₅₀ < 2 μg/ml) but showed distinct susceptibility to Sb^V and/or Sb^III, depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmania species and strains, simultaneous Sb^V-Sb^III resistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of Sb^V-Sb^III susceptibility profiling on treatment outcome. In conclusion, the proposed conditioning protocol further contributes toward a more standardized laboratory model for evaluation of the drug sensitivities of field isolates.As the failure of first-line treatment for all clinical forms of leishmaniasis is a growing problem, it is pivotal to monitor the efficacy of standard drugs and map the prevalence of drug resistance in areas where the disease is endemic (7, 9, 24). Diagnostic field isolates are mostly provided to the laboratory as promastigotes, but it remains an experimental challenge to appropriately adapt them to the amastigote-macrophage model, still considered the “gold standard” for susceptibility evaluation (23, 31). Infection of the macrophage is generally achieved with metacyclic promastigotes, but unfortunately, infection is subject to a high degree of variability, among several other factors affecting the outcome of the sensitivity test (8, 10). These factors strongly suggest the need for the further standardization of susceptibility testing of clinical field isolates.In the normal course of events, infective metacyclic promastigotes are inoculated by the sand fly into the mammalian host, where they rapidly penetrate susceptible cells, undergo intracellular transformation to the amastigote form, and start dividing. The procyclic and metacyclic phases observed during in vitro culture appear to be similar to the stages that occur in the sand fly gut. Moreover, the infectivity for the vertebrate host steadily increases from the log to the stationary phase and is linked to the progressive increase in the numbers of metacyclic promastigotes (5, 27). Metacyclic promastigotes are identified as small, slender promastigotes (≤8 μm by 1.2 to 1.5 μm) with a flagellum measuring at least twice the length of the cell body, and they occur in the greatest numbers in vitro during late stationary phase. However, the level of metacyclogenesis may vary considerably depending on the species, strain, and culture conditions (13). Since the cells in stationary-phase cultures appear to be a heterogeneous population, another relevant limitation is the incomplete transformation into actively dividing amastigotes that occurs after internalization by the macrophage host cell. Not all metacyclic promastigotes appear to have the capability to transform into amastigotes (19), thereby influencing again the outcome of sensitivity testing. The latter is particularly relevant for drugs that specifically act on intracellular amastigotes, such as pentavalent antimony (Sb^V) compounds (4) and the experimental antileishmania compound PX-6518 (22, 31).The aim of this study was to investigate how promastigotes of different Leishmania species can be effectively triggered to differentiate into highly infective metacyclic promastigotes. Metacyclogenesis has been stimulated by culturing promastigotes at acidic pH, which leads to a more homogeneous stationary-phase population of metacyclic promastigote-like cells (33, 34). However, continuous growth under acidic conditions leads to the early appearance of metacyclic promastigotes and a lower final cell density (1, 6). Therefore, we hypothesized that after growth at neutral pH, promastigotes could be more optimally conditioned by briefly exposing them to a lower pH just prior to infection, leading to a higher final cell density and more reproducible intracellular infections. Different strains could even be synchronized to infect macrophages at the same time, which may be a practical advantage in laboratories that must evaluate the sensitivities of a large number of field isolates in the context of epidemiological studies.     

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

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 β-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 (EC₅₀) of 0.3 μM 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 β-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 β-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration.     

Alkyl Galactofuranosides Strongly Interact with Leishmania donovani Membrane and Provide Antileishmanial Activity

We investigated the in vitro effects of four alkyl-galactofuranoside derivatives, i.e., octyl-β-d-galactofuranoside (compound 1), 6-amino-β-d-galactofuranoside (compound 2), 6-N-acetamido-β-d-galactofuranoside (compound 3), and 6-azido-β-d-galactofuranoside (compound 4), on Leishmania donovani. Their mechanism of action was explored using electron paramagnetic resonance spectroscopy (EPR) and nuclear magnetic resonance (NMR), and ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Compound 1 showed the most promising effects by inhibiting promastigote growth at a 50% inhibitory concentration (IC₅₀) of 8.96 ± 2.5 μM. All compounds exhibit low toxicity toward human macrophages. Compound 1 had a higher selectivity index than the molecule used for comparison, i.e., miltefosine (159.7 versus 37.9, respectively). EPR showed that compound 1 significantly reduced membrane fluidity compared to control promastigotes and to compound 3. The furanose ring was shown to support this effect, since the isomer galactopyranose had no effect on parasite membrane fluidity or growth. NMR showed a direct interaction of all compounds (greatest with compound 1, followed by compounds 2, 3, and 4, in descending order) with the promastigote membrane and with octyl-galactopyranose and octanol, providing evidence that the n-octyl chain was primarily involved in anchoring with the parasite membrane, followed by the putative crucial role of the furanose ring in the antileishmanial activity. A morphological analysis of compound 1-treated promastigotes by TEM revealed profound alterations in the parasite membrane and organelles, but this was not the case with compound 3. Quantification of annexin V binding by flow cytometry confirmed that compound 1 induced apoptosis in >90% of promastigotes. The effect of compound 1 was also assessed on intramacrophagic amastigotes and showed a reduction in amastigote growth associated with an increase of reactive oxygen species (ROS) production, thus validating its promising effect.     

Sodium-coupled Taurocholate Transport in the Proximal Convolution of the Rat Kidney In Vivo and In Vitro

Using the standing droplet technique in the renal proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of taurocholate (ΔC_TC⁻) at 45 s was determined as a measure of active bile acid reabsorption in vivo. Starting with 0.1 mmol/liter taurocholate in both perfusates the control ΔC_TC⁻ of 0.042 mmol/liter fell to 0.006 mmol/liter (P < 0.001) when the Na⁺ concentration in the perfusates was reduced to zero. Removal of bicarbonate from the perfusates to alter pH had no influence on ΔC_TC⁻. When glycocholate was added to the perfusates ΔC_TC⁻ was decreased, while probenecid increased ΔC_TC⁻.     

Identification of Metal Dithiocarbamates as a Novel Class of Antileishmanial Agents

Dithiocarbamates have emerged as potent carbonic anhydrase (CA) inhibitors in recent years. Given that CAs are important players in cellular metabolism, the objective of this work was to exploit the CA-inhibitory property of dithiocarbamates as a chemotherapeutic weapon against the Leishmania parasite. We report here strong antileishmanial activity of three hitherto unexplored metal dithiocarbamates, maneb, zineb, and propineb. They inhibited CA activity in Leishmania major promastigotes at submicromolar concentrations and resulted in a dose-dependent inhibition of parasite growth. Treatment with maneb, zineb, and propineb caused morphological deformities of the parasite and Leishmania cell death with 50% lethal dose (LD₅₀) values of 0.56 μM, 0.61 μM, and 0.27 μM, respectively. These compounds were even more effective against parasites growing in acidic medium, in which their LD₅₀ values were severalfold lower. Intracellular acidosis leading to apoptotic and necrotic death of L. major promastigotes was found to be the basis of their leishmanicidal activity. Maneb, zineb, and propineb also efficiently reduced the intracellular parasite burden, suggesting that amastigote forms of the parasite are also susceptible to these metal dithiocarbamates. Interestingly, mammalian cells were unaffected by these compounds even at concentrations which are severalfold higher than their antileishmanial LD₅₀s). Our data thus establish maneb, zineb, and propineb as a new class of antileishmanial compounds having broad therapeutic indices.     

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone,a Triterpene Isolated from the Mushroom Astraeus hygrometricus,Involves the Induction of Protective Cell-Mediated Immunity and TLR9

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 μg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo. Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.     

Axenically Grown Amastigotes of Leishmania infantum Used as an In Vitro Model To Investigate the Pentavalent Antimony Mode of Action

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC₅₀s], about 1.8 mg/ml and 22 μg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC₅₀s, of 0.54 and 1.32 μg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.     

In Vitro Synergistic Effect of Amphotericin B and Allicin on Leishmania donovani and L. infantum

Current monotherapy against visceral leishmaniasis has serious side effects, and resistant Leishmania strains have been identified. Amphotericin B (AmB) has shown an extraordinary antileishmanial efficacy without emergence of resistance; however, toxicity has limited its general use. Results obtained showed, using a fixed-ratio analysis, that the combination of diallyl thiosulfinate (allicin) and AmB ranged from moderately synergic to synergic at low concentrations (0.07 μM AmB plus 35.45 μM allicin induced 95% growth inhibition). None of the treatments, alone or in combination, had noticeable adverse effects on macrophages (Mϕ) in the concentration range examined (allicin, 0.5, 1, 5 and 10 μM; AmB, 0.05, 0.075, and 0.1 μM). Allicin, AmB, or the combination did not affect the infection rate (percentage of infected Mϕ) of Leishmania. Allicin enhanced the activity of AmB on intracellular amastigotes of Leishmania donovani and L. infantum (ca. 45% reduction of amastigote burden with 0.05 μM AmB plus 10 μM allicin); this represented nearly a 2-fold reduction in the 50% inhibitory concentration (IC₅₀) of the antibiotic added alone. Results point toward the possible utility of testing this combination in vivo to reduce the toxicity associated with monotherapy with AmB.     

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

This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes that was previously demonstrated to be safe and orally active against visceral leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and the generation of reactive oxygen species, triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in the release of cytochrome c into the cytosol, impairing ATP production. Oxidative stress caused the depletion of reduced glutathione, while pretreatment with antioxidant N-acetyl cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore the mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment, suggesting that the generation of oxidative stress is a downstream event relative to the other events. Caspase-3/-7-like protease activity and genomic DNA fragmentation were observed. Electron microscopy studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of the nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations, and the displacement of organelles. Moreover, an increased number of lipid droplets was detected after K-09 treatment, which is suggestive of altered lipid metabolism. Our results indicate that K-09 induces mitochondrial dysfunction and oxidative stress-mediated apoptotic cell death in L. donovani promastigotes, sharing many features with metazoan apoptosis. These mechanistic insights provide a basis for further investigation toward the development of K-09 as a potential drug candidate for VL.