Synthesis and Biophysical and Biological Studies of N-Phenylbenzamide Derivatives Targeting Kinetoplastid Parasites.

The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.

Label-Free Mass Spectrometry Proteomics Reveals Different Pathways Modulated in THP-1 Cells Infected with Therapeutic Failure and Drug Resistance Leishmania infantum Clinical Isolates.

As the world is facing increasing difficulties to treat leishmaniasis with current therapies, deeper investigation into the molecular mechanisms responsible for both drug resistance and treatment failure (TF) is essential in drug discovery and development. So far, few available drugs cause severe side effects and have developed several resistance mechanisms. Drug resistance and TF parasite strains from clinical isolates may have acquired altered expression of proteins that characterize specific mechanisms leading to therapy inefficacy. This work aims to identify the biochemical pathways of THP-1 human monocytes infected by different Leishmania infantum clinical isolates from patients with either resistance or with TF outcome, using whole cell differential Mass Spectrometry proteomics. We have adopted network enrichment analysis to integrate the transcriptomics and the proteomic results of infected cells studies. Transferrin receptor C (TFRC) and nucleoside diphosphate kinase 3 (NDK3) were discovered as overexpressed proteins in THP-1 cells infected with paromomycin, antimony, and miltefosine resistant L. infantum lines. The overall achievements represent founding concepts to confirm new targets involved in the parasitic drug resistance and TF mechanisms, and to consider in perspective the importance of a dual host-guest pharmacological approach to treat the acute stage of the disease.

Transcriptome Analysis of Intracellular Amastigotes of Clinical Leishmania infantum Lines from Therapeutic Failure Patients after Infection of Human Macrophages.

Leishmaniasis is considered to be one of the most neglected tropical diseases affecting humans and animals around the world. Due to the absence of an effective vaccine, current treatment is based on chemotherapy. However, the continuous appearance of drug resistance and therapeutic failure (TF) lead to an early obsolescence of treatments. Identification of the factors that contribute to TF and drug resistance in leishmaniasis will constitute a useful tool for establishing future strategies to control this disease. In this manuscript, we evaluated the transcriptomic changes in the intracellular amastigotes of the Leishmania infantum parasites isolated from patients with leishmaniasis and TF at 96 h post-infection of THP-1 cells. The adaptation of the parasites to their new environment leads to expression alterations in the genes involved mainly in the transport through cell membranes, energy and redox metabolism, and detoxification. Specifically, the gene that codes for the prostaglandin f2α synthase seems to be relevant in the pathogenicity and TF since it appears substantially upregulated in all the L. infantum lines. Overall, our results show that at the late infection timepoint, the transcriptome of the parasites undergoes significant changes that probably improve the survival of the Leishmania lines in the host cells, contributing to the TF phenotype as well as drug therapy evasion.

Modulation of Cholesterol Pathways in Human Macrophages Infected by Clinical Isolates of Leishmania infantum.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite's ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

Transcriptomic Analysis in Human Macrophages Infected with Therapeutic Failure Clinical Isolates of Leishmania infantum.

Leishmaniasis is one of the neglected tropical diseases with a worldwide distribution, affecting humans and animals. In the absence of an effective vaccine, current treatment is through the use of chemotherapy; however, existing treatments have frequent appearance of drug resistance and therapeutic failure (TF). The identification of factors that contribute to TF in leishmaniasis will provide the basis for a future therapeutic strategy more efficient for the control of this disease. In this article, we have evaluated the transcriptomic changes in the host cells THP-1 after infection with clinical Leishmania infantum isolates from leishmaniasis patients with TF. Our results show that distinct L. infantum isolates differentially modulate host cell response, inducing phenotypic changes that probably may account for parasite survival and TF of patients. Analysis of differential expression genes (DEGs), with a statistical significance threshold of a fold change ≥ 2 and a false discovery rate value ≤ 0.05, revealed a different number of DEGs according to the Leishmanialine. Globally, there was a similar number of genes up- and downregulated in all the infected host THP-1 cells, with exception of Hi-L2221, which showed a higher number of downregulated DEGs. We observed a total of 58 DEGs commonly modulated in all infected host cells, including upregulated (log2FC ≥ 1) and downregulated (log2FC ≤ -1) genes. Based on the results obtained from the analysis of RNA-seq, volcano plot, and GO enrichment analysis, we identified the most significant transcripts of relevance for their possible contribution to the TF observed in patients with leishmaniasis.

New Insights on Drug-Resistant Clinical Isolates of Leishmania infantum-Infected Human Macrophages as Determined by Comparative Transcriptome Analyses.

Leishmaniasis is the second most important neglected tropical parasitic disease after malaria. This disease is distributed worldwide and can be present in a variety of clinical forms, depending on the parasite species and host's genetic background. As chemotherapy is the only effective weapon whose effectiveness is limited by the frequent appearance of drug resistance and therapeutic failure, new therapeutic strategies are required. To better understand the factors that contribute to therapeutic failure and drug resistance in leishmaniasis, we studied the transcriptomic changes in host THP-1 cells after infection with clinical Leishmania infantum isolates with different susceptibilities to antileishmanial drugs by RNA-seq. Analysis of the differentially expressed genes (DEGs) in infected host cells revealed variations in DEG numbers in the THP-1-infected cells depending on the Leishmania line. A key conclusion of this study is that the modulation of host cells is Leishmania line dependent. Gene ontology enrichment analyses of DEGs indicated that certain biological processes were modulated in the infected host cells, specifically related to cellular metabolism, immune response, defense response, signaling pathways, and cell proliferation and apoptosis. Furthermore, this study provides new potential therapeutic markers and insights into the THP-1 host transcriptomic changes that occur after late infection with drug-resistant L. infantum clinical isolates.

Discovery and Pharmacological Studies of 4-Hydroxyphenyl-Derived Phosphonium Salts Active in a Mouse Model of Visceral Leishmaniasis.

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.

4-Aminoquinoline-based compounds as antileishmanial agents that inhibit the energy metabolism of Leishmania.

Among neglected tropical diseases, leishmaniasis is one of the most relevant with an estimated 30,000 deaths annually. Existing therapies have serious drawbacks in safety, drug resistance, field-adapted application and cost; therefore, new safer and shorter treatments are needed for this disease. Here we report on the synthesis of novel 4-amino-7-chloroquinoline-based compounds with leishmanicidal activity, together with deeper insight into the mechanism of action of our previously published hit compound 1. New derivatives showed comparable activity to 1 against both promastigote and intracellular amastigote forms of Leishmania infantum, with IC50 < 1 µM. Furthermore, we have determined that compound 1 induced a decrease of intracellular ATP levels, as well as a mitochondrial depolarization, together with an alteration of plasma membrane permeability and a significant ROS production. The inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound. In all, these results support the consideration of compound 1 for the future development of new leishmanicidal drugs.

Leishmania LABCG2 transporter is involved in ATP-dependent transport of thiols.

The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)2] as well as their oxidized forms GSSG and TS2 (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [3H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [3H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH)2 and TS2 being the best competitors. The IC50 value for [3H]-GSH uptake in the presence of increasing concentrations of T(SH)2 was less than 100 µM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite.

ABCI3 Is a New Mitochondrial ABC Transporter from Leishmania major Involved in Susceptibility to Antimonials and Infectivity.

We have identified and characterized ABCI3 as a new mitochondrial ABC transporter from Leishmania major Localization studies using confocal microscopy, a surface biotinylation assay, and trypsin digestion after digitonin permeabilization suggested that ABCI3 presents a dual localization in both mitochondria and the plasma membrane. From studies using parasites with a single knockout of ABCI3 (ABCI3+/-), we provide evidence that ABCI3 is directly involved in susceptibility to the trivalent form of antimony (SbIII) and metal ions. Attempts to obtain parasites with a double knockout of ABCI3 were unsuccessful, suggesting that ABCI3 could be an essential gene in L. majorABCI3+/- promastigotes were 5-fold more resistant to SbIII than the wild type, while ABCI3+/- amastigotes were approximately 2-fold more resistant to pentavalent antimony (SbV). This resistance phenotype was associated with decreased SbIII accumulation due to decreased SbIII uptake. ABCI3+/- parasites presented higher ATP levels and generated less mitochondrial superoxide after SbIII incubation. Finally, we observed that ABCI3+/- parasites showed a slightly higher infection capacity than wild-type and add-back ABCI3+/-::3×FABCI3 parasites; however, after 72 h the number of ABCI3+/- intracellular parasites per macrophage increased significantly. Our results show that ABCI3 is responsible for SbIII transport inside mitochondria, where it contributes to enhancement of the general toxic effects caused by SbIII To our knowledge, ABCI3 is the first ABC transporter which is involved in susceptibility toward antimony, conferring SbIII resistance to parasites when it is partially deleted.

Leishmania LABCG1 and LABCG2 transporters are involved in virulence and oxidative stress: functional linkage with autophagy.

BACKGROUND: The G subfamily of ABC (ATP-binding cassette) transporters of Leishmania include 6 genes (ABCG1-G6), some with relevant biological functions associated with drug resistance and phospholipid transport. Several studies have shown that Leishmania LABCG2 transporter plays a role in the exposure of phosphatidylserine (PS), in virulence and in resistance to antimonials. However, the involvement of this transporter in other key biological processes has not been studied. METHODS: To better understand the biological function of LABCG2 and its nearly identical tandem-repeated transporter LABCG1, we have generated Leishmania major null mutant parasites for both genes (ΔLABCG1-2). NBD-PS uptake, infectivity, metacyclogenesis, autophagy and thiols were measured. RESULTS: Leishmania major ΔLABCG1-2 parasites present a reduction in NBD-PS uptake, infectivity and virulence. In addition, we have shown that ΔLABCG1-2 parasites in stationary phase growth underwent less metacyclogenesis and presented differences in the plasma membrane's lipophosphoglycan composition. Considering that autophagy is an important process in terms of parasite virulence and cell differentiation, we have shown an autophagy defect in ΔLABCG1-2 parasites, detected by monitoring expression of the autophagosome marker RFP-ATG8. This defect correlates with increased levels of reactive oxygen species and higher non-protein thiol content in ΔLABCG1-2 parasites. HPLC analysis revealed that trypanothione and glutathione were the main molecules accumulated in these ΔLABCG1-2 parasites. The decrease in non-protein thiol levels due to preincubation with buthionine sulphoximide (a γ-glutamylcysteine synthetase inhibitor) restored the autophagy process in ΔLABCG1-2 parasites, indicating a relationship between autophagy and thiol content. CONCLUSIONS: LABCG1-2 transporters from Leishmania could be considered as phosphatidylserine and non-protein thiol transporters. They probably accomplish transportation in conjunction with other molecules that are involved in oxidative stress, autophagy, metacyclogenesis and infectivity processes. The overall conclusion is that LABCG1-2 transporters could play a key role in Leishmania cell survival and infectivity.

Arylthiosemicarbazones as antileishmanial agents.

Based on a screening process, we targeted substituted thiosemicarbazone as potential antileishmanial agents. Our objective was to identify the key structural elements contributing to the anti-parasite activity that might be used for development of effective drugs. A series of 32 compounds was synthesized and their efficacy was evaluated against the clinically relevant intracellular amastigotes of Leishmania donovani. From these, 22 compounds showed EC50 values below 10 µM with the most active derivative (compound 14) showing an EC50 of 0.8 µM with very low toxicity on two different mammalian cell lines. The most relevant structural elements required for higher activity indicate that the presence of a fused bicyclic aromatic ring such as a naphthalene bearing an alkyl or an alkoxy group substituent are prerequisites. Owing to the easy synthesis, high activity and low toxicity, the most active compounds could be considered as a lead for further development.

The LABCG2 Transporter from the Protozoan Parasite Leishmania Is Involved in Antimony Resistance.

Treatment for leishmaniasis, which is caused by Leishmania protozoan parasites, currently relies on a reduced arsenal of drugs. However, the significant increase in the incidence of drug therapeutic failure and the growing resistance to first-line drugs like antimonials in some areas of Northern India and Nepal limit the control of this parasitic disease. Understanding the molecular mechanisms of resistance in Leishmania is now a matter of urgency to optimize drugs used and to identify novel drug targets to block or reverse resistant mechanisms. Some members of the family of ATP-binding cassette (ABC) transporters in Leishmania have been associated with drug resistance. In this study, we have focused our interest to characterize LABCG2's involvement in drug resistance in Leishmania. Leishmania major parasites overexpressing the ABC protein transporter LABCG2 were generated in order to assess how LABCG2 is involved in drug resistance. Assays of susceptibility to different leishmanicidal agents were carried out. Analysis of the drug resistance profile revealed that Leishmania parasites overexpressing LABCG2 were resistant to antimony, as they demonstrated a reduced accumulation of Sb(III) due to an increase in drug efflux. Additionally, LABCG2 was able to transport thiols in the presence of Sb(III) Biotinylation assays using parasites expressing LABCG2 fused with an N-terminal green fluorescent protein tag revealed that LABCG2 is partially localized in the plasma membrane; this supports data from previous studies which suggested that LABCG2 is localized in intracellular vesicles that fuse with the plasma membrane during exocytosis. In conclusion, Leishmania LABCG2 probably confers antimony resistance by sequestering metal-thiol conjugates within vesicles and through further exocytosis by means of the parasite's flagellar pocket.

The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca(2+), and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei.

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 Ca(2+) 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.

Identification of specific reversal agents for Leishmania ABCI4-mediated antimony resistance by flavonoid and trolox derivative screening.

OBJECTIVES: To identify reversal agents for the Leishmania ABCI4 transporter that confers resistance to antimony. METHODS: Selective ABCI4 inhibitors among a series of 15 flavonoid and trolox derivatives or analogues were investigated by evaluating their ability to reverse antimony resistance in Leishmania parasites overexpressing ABCI4. Among the compounds screened, N-ethyltrolox carboxamide (compound D2) produced the highest reversal activity. In order to optimize the activity of D2, we synthesized a series of 10 derivatives by condensation of various amines with trolox. RESULTS: Analysis of antimony resistance reversal activity showed that N-propyltrolox carboxamide (compound D4) was the most potent ABCI4 inhibitor, with reversal activity being maintained in the intracellular amastigote stage. In addition, trolox derivatives significantly reverted the resistance to zinc protoporphyrin. The mechanism of action of these active derivatives was found to be related to significant reversion of Sb(III) and zinc protoporphyrin accumulation and to a decrease in drug efflux. CONCLUSIONS: Our findings suggest that trolox derivatives D2 and D4 could be considered to be specific reversal agents targeting the Leishmania ABCI4 transporter. The structure-activity relationship obtained in the present study highlights the importance of the size and length of the alkyl substituent linked to trolox. Furthermore, the structural data obtained provide valuable information for the further development of new, even more specific and potent Leishmania ABCI4 reversal agents.

Oxazolo[3,2-a]pyridine. A new structural scaffold for the reversal of multi-drug resistance in Leishmania.

Compounds belonging to three different classes of fused heterocyclic systems, structurally related to Calcium-channel blockers of the 1,4-dihydropyridine family, were evaluated in their ability to overcome leishmanial resistance to common drugs in a MDR Leishmania tropica strain. Compounds with the skeletal basis of oxazolo[3,2-a]pyridine displayed significant reversion of resistance to daunomycin and miltefosine, with reversion indexes up to 6.7-fold and 8.7-fold, respectively. Most interestingly, the enantiopure compound 20S attained to revert the resistance to both drugs and fairly more significantly than its enantiomer 20R.

Leishmania donovani develops resistance to drug combinations.

Drug combinations for the treatment of leishmaniasis represent a promising and challenging chemotherapeutic strategy that has recently been implemented in different endemic areas. However, the vast majority of studies undertaken to date have ignored the potential risk that Leishmania parasites could develop resistance to the different drugs used in such combinations. As a result, this study was designed to elucidate the ability of Leishmania donovani to develop experimental resistance to anti-leishmanial drug combinations. The induction of resistance to amphotericin B/miltefosine, amphotericin B/paromomycin, amphotericin B/Sb(III), miltefosine/paromomycin, and Sb(III)/paromomycin was determined using a step-wise adaptation process to increasing drug concentrations. Intracellular amastigotes resistant to these drug combinations were obtained from resistant L. donovani promastigote forms, and the thiol and ATP levels and the mitochondrial membrane potential of the resistant lines were analysed. Resistance to drug combinations was obtained after 10 weeks and remained in the intracellular amastigotes. Additionally, this resistance proved to be unstable. More importantly, we observed that promastigotes/amastigotes resistant to one drug combination showed a marked cross-resistant profile to other anti-leishmanial drugs. Additionally, the thiol levels increased in resistant lines that remained protected against the drug-induced loss of ATP and mitochondrial membrane potential. We have therefore demonstrated that different resistance patterns can be obtained in L. donovani depending upon the drug combinations used. Resistance to the combinations miltefosine/paromomycin and Sb(III)/paromomycin is easily obtained experimentally. These results have been validated in intracellular amastigotes, and have important relevance for ensuring the long-term efficacy of drug combinations.

Uptake of the antileishmania drug tafenoquine follows a sterol-dependent diffusion process in Leishmania.

OBJECTIVES: The present study was designed to elucidate the mechanism of tafenoquine uptake in Leishmania and its sterol dependence. METHODS: Because tafenoquine is a fluorescent compound, spectrofluorimetric analysis allowed us to monitor its uptake by Leishmania promastigotes and intracellular amastigotes, and to evaluate the effect of temperature, energy and H+ gradient on drug entry. The influence of sterols on tafenoquine uptake in Leishmania parasites was determined in experiments using sterol-depleting agents such as methyl-ß-cyclodextrin or cholesterol oxidase. RESULTS: Tafenoquine exhibited fast entry kinetics into Leishmania in an energy-independent, but pH- and temperature-dependent, non-saturable process. Furthermore, sterol depletion decreased tafenoquine uptake. CONCLUSIONS: These findings suggest that Leishmania takes up tafenoquine by a diffusion process and that decreases in membrane sterol content may induce a decrease in drug uptake.

Increased glycolytic ATP synthesis is associated with tafenoquine resistance in Leishmania major.

Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.