In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.     

Pterocarpanquinone LQB-118 Induces Apoptosis in Leishmania (Viannia) braziliensis and Controls Lesions in Infected Hamsters

Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the induction of parasite apoptosis and shows promising therapeutic option by oral or local routes in leishmaniasis.     

Activity of 1-aryl-4-(naphthalimidoalkyl) piperazine derivatives against Leishmania major and Leishmania mexicana

A series of 1-aryl-4-(phthalimidoalkyl) piperazines and 1-aryl-4-(naphthalimidoalkyl) piperazines were retrieved from a proprietary library based on their high structural similarity to haloperidol, an antipsychotic with antiparasitic activity, and assessed as potential antileishmanial scaffolds. Selected compounds were tested for antileishmanial activity against promastigotes of Leishmania major and Leishmania mexicana in dose-response assays. Two of the 1-aryl-4-(naphthalimidoalkyl) piperazines (compounds 10 and 11) were active against promastigotes of both Leishmania species without being toxic to human fibroblasts. Their activity was found to correlate with the length of their alkyl chains. Further analyses showed that compound 11 was also active against intracellular amastigotes of both Leishmania species. In promastigotes of both Leishmania species, compound 11 induced collapse of the mitochondrial electrochemical potential and increased the intracellular Ca²⁺ concentration. Therefore, it may serve as a promising lead compound for the development of novel antiparasitic drugs.     

Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice

Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨ_m) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.     

Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents

Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.     

Amphotericin B inhibits entry of Leishmania donovani into primary macrophages

Visceral leishmaniasis is a vector-borne disease caused by an obligate intra-macrophage protozoan parasite Leishmania donovani. The molecular mechanisms involved in internalization of Leishmania are still poorly understood. Amphotericin B and its formulations are considered as the best existing drugs against visceral leishmaniasis and are being increasingly used. The reason for its antileishmanial activity is believed to be its ability to bind ergosterol found in parasite membranes. In case of in vivo amphotericin B treatment, both host macrophages and parasites are exposed to amphotericin B. The effect of amphotericin B treatment could therefore be due to a combination of its interaction with both sterols i.e., ergosterol of Leishmania and cholesterol of host macrophages. We report here that cholesterol complexation by amphotericin B markedly inhibits binding of L. donovani promastigotes to macrophages. These results represent one of the first reports on the effect of amphotericin B on the binding of Leishmania parasites to host macrophages. Importantly, these results offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies that employ sterol-complexing agents such as amphotericin B to treat leishmaniasis.     

Dok proteins are recruited to the phagosome and degraded in a GP63-dependent manner during Leishmania major infection

Three adaptor molecules of the Dok family, Dok-1, Dok-2 and Dok-3 are expressed in macrophages and are involved in the negative regulation of signaling in response to lipopolysaccharide and various cytokines and growth factors. We investigated the role and the fate of these proteins following infection with Leishmania major promastigotes in macrophages. The protozoan parasite L. major causes cutaneous leishmaniasis and is known for its capacity to alter host-cell signaling and function. Dok-1/Dok-2^−/− bone marrow-derived macrophages displayed normal uptake of L. major promastigotes. Following Leishmania infection, Dok-1 was barely detectable by confocal microscopy. By contrast, phagocytosis of latex beads or zymosan led to the recruitment of Dok-1 to phagosomes. In the absence of the Leishmania pathogenesis-associated metalloprotease GP63, Dok-1 was also, partially, recruited to phagosomes containing L. major promastigotes. Further biochemical analyses revealed that similar to Dok-1, Dok-2 and Dok-3 were targets of GP63. Moreover, we showed that upon infection with wild-type or Δgp63 L. major promastigotes, production of nitric oxide and tumor necrosis factor by interferon-γ-primed Dok-1/Dok-2^−/− macrophages was reduced compared to WT macrophages. These results suggest that Dok proteins may be important regulators of macrophage responses to Leishmania infection.     

Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival

Background

Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.

Methodology/Principal Findings

An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.

Conclusions/Significance

Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.     

Leishmania amazonensis Amastigotes Highly Express a Tryparedoxin Peroxidase Isoform That Increases Parasite Resistance to Macrophage Antimicrobial Defenses and Fosters Parasite Virulence

Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO⁻), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO⁻ cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania''s ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.     

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

Background

Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis.

Methodology/Principal Findings

Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes.

Conclusion/Significance

The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.     

An Essential Type I Nitroreductase from Leishmania major Can Be Used to Activate Leishmanicidal Prodrugs

Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR^+/− heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR^+/− heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function.     

Studies on the antileishmanial properties of the antimicrobial peptides temporin A,B and 1Sa

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector‐borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Structures of Leishmania major orthologues of macrophage migration inhibitory factor

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, expresses two closely related MIF-like proteins. To ascertain the roles and potential differences of these two Leishmania proteins, recombinant L. major MIF1 and MIF2 have been produced and the structures resolved by X-ray crystallography. Each has a trimeric ring architecture similar to mammalian MIF, but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response, thereby promoting parasite survival, but suggest the LmjMIFs have potentially different biological roles. Analysis of the Leishmania braziliensis genome showed that this species lacks both MIF genes. Thus MIF is not a virulence factor in all species of Leishmania.     

Momordicatin purified from fruits of Momordica charantia is effective to act as a potent antileishmania agent

Aqueous extract of the green fruits of the Indian plant Momordica charantia and purified Momordicatin structurally established as 4-(o-carboethoxyphenyl) butanol were evaluated in vitro and in vivo against kala-azar caused by Leishmania donovani. 50% inhibitory concentration (IC₅₀) against Leishmania promastigotes in vitro for the crude extract and momordicatin were 0.6 mg/L and 0.02 mg/L, respectively. When administered in the hamster model of visceral leishmaniasis, 100% parasite clearance was achieved at a dose of 300 mg/kg body weight of crude extract and 10 mg/kg body weight of Momordicatin. Fe containing parasite superoxide dismutase (SOD) was totally inhibited when treated with 0.72 mg/L crude extract and 0.20 mg/L Momordicatin, respectively, whereas Cu–Zn containing SOD present in host remained unaffected. Results reveal that the mode of action of these newly found antileishmanial agents is mediated through inhibiting parasite SOD which is one of the key enzymes of the oxidative burst. It may be proposed from the present study that both crude extract of Momordica charantia and Momordicatin obtained from the fruits of the said plant may be considered as potential candidates towards developing new chemotherapeutics against leishmaniasis.     

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC₅₀) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC₅₀ and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural^(r) (a furvina-containing antifungal ointment) for the treatment of CL.     

Synthetic Oxoisoaporphine Alkaloids: In Vitro,In Vivo and In Silico Assessment of Antileishmanial Activities

Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L. infantum, L. braziliensis, L. amazonensis and L. guyanensis, and with intracellular amastigotes of L. infantum and L. amazonensis. Finally, the most active compounds, OXO 1 (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one) and OXO 13 (2,3,8,9,10,11-hexahydro-7H-dibenzo[de,h]quinolin-7-one), were tested in BALB/c mice infected with L. infantum. Treatment of mice at a dose of 10 mg/kg with OXO 1 yielded significant reductions (p<0.05) in parasite burden in liver and spleen (99% and 78%, respectively) whereas with OXO 13 were not significant. Although previous reports suggest that this family of molecules displays inhibitory activity against monoamine oxidase A and acetylcholinesterase, these enzymes were not confirmed as targets for antileishmanial activity on the basis of the present results. However, after development of a new bioinformatics model to analyze the Leishmania proteome, we were able to identify other putative targets for these molecules. The most promising candidates were four proteins: two putative pteridine reductase 2 (1MXF and 1MXH), one N-myristoyltransferase (2WUU) and one type I topoisomerase (2B9S).     

Stage-Specific Adhesion of Leishmania Promastigotes to Sand Fly Midguts Assessed Using an Improved Comparative Binding Assay

Background

The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood.

Methodology/Principal Findings

To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.

Conclusions/Significance

The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.