Leishmania mexicana: chemistry and biochemistry of sodium stibogluconate (Pentostam)

The chemical properties of the primary antileishmanial agent sodium stibogluconate (Pentostam), and the interaction of Pentostam with Leishmania mexicana amastigotes, have been investigated with the aid of [125Sb]Pentostam. The molecular weight by P2 chromatography showed [125Sb]Pentostam to be of multiple species of MW = 100-4000 Da, rather than the one species of 746 Da predicted by the commonly hypothesized structural formula. Nonradioactive Pentostam had a lower osmolarity (789 mOsm for a 100 mg Sb/ml solution) than predicted (1644 mOsm), which indicates that the multiple components of Pentostam (Sb and derivatives of gluconic acid) are more closely complexed with each other than previously thought. When incubated with L. mexicana amastigotes, labeled drug was bound to at least six polypeptides of molecular weights ranging from 14,000 to 68,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Interaction with the polypeptides is presumed to contribute to the antileishmanial action of Pentostam.     

Structural characterization of the As/Sb reductase LmACR2 from Leishmania major

The arsenate/antimonate reductase LmACR2 has been recently identified in the genome of Leishmania major. Besides displaying phosphatase activity in vitro, this enzyme is able to reduce both As(V) and Sb(V) to their respective trivalent forms and is involved in the activation of Pentostan, a drug containing Sb(V) used in the treatment of leishmaniasis. LmACR2 displays sequence and functional similarity with the arsenate reductase ScACR2 from Saccharomyces cerevisiae, and both proteins are homologous to the catalytic domain of Cdc25 phosphatases, which, in turn, belong to the rhodanese/Cdc25 phosphatase superfamily. In this work, the three-dimensional structure of LmACR2 has been determined with crystallographic methods and refined at 2.15 Å resolution. The protein structure maintains the overall rhodanese fold, but substantial modifications are observed in secondary structure position and length. However, the conformation of the active-site loop and the position of the catalytic residue Cys75 are unchanged with respect to the Cdc25 phosphatases. From an evolutionary viewpoint, LmACR2 and the related arsenate reductases form, together with the known Cdc25 phosphatases, a well-defined subfamily of the rhodanese/Cdc25 phosphatase superfamily, characterized by a 7-amino-acid-long active-site loop that is able to selectively bind substrates containing phosphorous, arsenic, or antinomy. The evolutionary tree obtained for these proteins shows that, besides the active-site motif CE[F/Y]SXXR that characterizes Cdc25 phosphatase, the novel CALSQ[Q/V]R motif is also conserved in sequences from fungi and plants. Similar to Cdc25 phosphatase, these proteins are likely involved in cell cycle control. The active-site composition of LmACR2 (CAQSLVR) does not belong to either group, but gives to the enzyme a bifunctional activity of both phosphatase and As/Sb reductase. The subtle dependence of substrate specificity on the amino acid composition of the active-site loop displays the versatility of the ubiquitous rhodanese domain.     

Leishmania spp.: development of pentostam-resistant clones in vitro by discontinuous drug exposure

Antimony unresponsiveness in mucocutaneous and visceral leishmaniasis is a serious clinical problem. Information on the mechanisms and characteristics of drug resistance in parasites that suggest chemotherapeutic strategies to overcome resistance is of practical importance. We developed nine lines of Leishmania resistant to drugs, the major emphasis being on pentavalent antimony (Sb) complexed to carbohydrate in the form of sodium stibogluconate (Pentostam), one of the only two antileishmanial agents with a clearly favorable therapeutic index. Resistance to Pentostam (33- to 212-fold increase) was obtained in promastigotes of Leishmania in vitro by exposure to gradually increasing concentrations of drug over several passages. Resistance to Sb was found to be either stable or unstable. Stable resistance to Sb required (greater than 3) exposures of the initial sensitive clones to Pentostam and tended to stabilize with increased time under pressure. In general, resistance obtained in a clone after only a few (less than or equal to 3) step treatments was low and unstable. Differences in the susceptibility to Pentostam were found between strains isolated from patients with American cutaneous leishmaniasis. In addition, natural isolates of Leishmania from patients represented a heterogeneous population of parasites as demonstrated by a biphasic concentration response to Sb (typical of mixed population dynamics) and by marked differences in susceptibility to Pentostam among clones prepared from single isolates. These results suggest that the emergence of parasite resistance to antimonial treatment is a potential risk of inadequate dose therapy.     

Dual action of antimonial drugs on thiol redox metabolism in the human pathogen Leishmania donovani

Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (Sb(III)) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, Sb(III) decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, Sb(III) inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance.     

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

Reduction of Sb(V) in a Human Macrophage Cell Line Measured by HPLC-ICP-MS

Drugs based on pentavalent antimony are first-line treatment of the parasite disease leishmaniasis. It is generally believed that Sb(V) acts as a prodrug, which is activated by reduction to Sb(III); however, the site of reduction is not known. It has been hypothesised that the reduction takes place in the parasites' host cells, the macrophages. In this study, the human macrophage cell line Mono Mac 6 was exposed to Sb(V) in form of the drug sodium stibogluconate (Pentostam?). Cell extracts were analysed for Sb species by high-performance liquid chromatography with inductively coupled plasma-mass spectrometry detection. We found that Sb(V) is actually reduced to Sb(III) in the macrophages; up to 23% of the intracellular Sb was found as Sb(III). Transfer of the cells to Sb-free medium rapidly decreased their Sb(V) and Sb(III) content. Induction of the cell's production of reactive oxygen species did not have any marked effect on the intracellular amounts of Sb(III).     

Activity of antileishmanial agents against amastigotes in human monocyte-derived macrophages and in mouse peritoneal macrophages

Leishmania multiplying within either human monocyte-derived macrophages (HM) or mouse peritoneal exudate cells (PEC) have recently been shown to be susceptible to pentavalent antimony (Sb) by several investigators. The Sb susceptibilities of 5 cutaneous strains of Leishmania were compared in the 2 model systems, with infection of the macrophages initiated with either amastigotes or promastigotes. The susceptibility to Sb of amastigote-induced infections was statistically the same as the susceptibility of promastigote-induced infections for 4 strains in the PEC model, and for 3 of 4 strains in the HM model. Promastigote-induced infections with the 5th strain were non-viable in both macrophage types. The susceptibility of Leishmania to Sb within PEC was the same statistically as that of organisms within HM for amastigote-induced infections for 4 of 5 strains and for promastigote-induced infections by 3 of 4 strains. These data suggested that the susceptibilities of organisms to Sb within PEC and HM were generally comparable and that either amastigotes or viable promastigotes could be used to initiate the infection. The several technical advantages of the PEC model may make it more useful than the HM model for testing susceptibility to Sb. The modest susceptibility of some strains in both models to the peak serum amounts of antimony which may be achieved by presently recommended treatment regimes may partially explain the high current failure rate in simple cutaneous disease. The susceptibility of one strain within peritoneal cells to primaquine and WR 6026 (8-aminoquinolines), ketoconazole (an imidazole) and formycin B (an inosine analogue) was similar to that previously reported in human macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials (Sb(V)), but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory isolates, but our understanding of resistance in field isolates is lagging behind. We describe here a comparative analysis of a genetically related pair of Sb(V)-sensitive and -resistant Leishmania donovani strains isolated from kala-azar patients. The resistant isolate exhibited cross-resistance to other unrelated Leishmania drugs including miltefosine and amphotericin B. A comparative proteomics screen has highlighted a number of proteins differentially expressed suggesting that programmed cell death (PCD) is modified in the resistant parasite. Indeed drug-induced PCD progression was altered in the Sb(V)-resistant strain as determined using early and late markers of apoptosis. Two proteins, the heat shock protein HSP83 and the small kinetoplastid calpain-related protein (SKCRP14.1) were shown to be intimately implicated in the drug-induced PCD phenotype. HSP83 increased drug resistance and reduced drug-mediated PCD activation by interfering with the mitochondrial membrane potential, whereas SKCRP14.1 promoted antimonial-induced PCD but protected against miltefosine-induced PCD. This study highlights the important role of PCD in drug susceptibility/resistance in the protozoan parasite Leishmania.     

Leishmania donovani: isolation and characterization of sodium stibogluconate (Pentostam)-resistant cell lines

Leishmania donovani promastigotes were generated by virtue of their resistance to incrementing concentrations of sodium stibogluconate (Pentostam) under completely defined growth conditions. The PENT0400 and PENT03200 cell lines were isolated after prolonged exposure to 0.4 mg/ml and 3.2 mg/ml Pentostam (Sb concentration), respectively. Whereas the effective concentration of Pentostam which inhibited the growth of wild type cells by 50% (EC50 value) was 0.1-0.15 mg/ml, the EC50 values for the PENT0400 and PENT03200 cells were approximately 1 and 4 mg/ml, respectively. The decreased sensitivities of both PENT0400 and PENT03200 cells to Pentostam were maintained after 6 months of continuous culture in the absence of selective pressure, indicating that the Pentostam resistance in the mutant organisms was a stable genetic trait. Interestingly, wild type and PENT03200 cells were equally sensitive to growth inhibition and cytotoxicity caused by SbCl5 and SbCl3, as well as to a variety of other cations such as Cd, Zn, and As. Wild type and PENT03200 cells also displayed equivalent growth sensitivities to a spectrum of other antiprotozoal agents, including antimony potassium tartrate, melarsoprol, pyrimethamine, pentamidine, formycin B, and difluoromethylornithine. These results illustrate a potentially useful model system to study Pentostam resistance in Leishmania and suggest that Pentostam resistance in vitro may be independent of antimony toxicity.     

Antimonial treatment of visceral leishmaniasis: are current in vitro susceptibility assays adequate for prognosis of in vivo therapy outcome?

In most of the Indian subcontinent, the first line treatment for visceral leishmaniasis (VL) is sodium stibogluconate (SSG), an antimonial drug, but the efficacy of the drug varies according to region. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Nepalese VL patients, and to correlate this in vitro parasite phenotype to clinical therapy outcome. Thirty-three clinical isolates of L. donovani were taken from patients with known disease history. These isolates were typed and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined. We observed (i) 22 SbV-resistant isolates out of 33 tested and (ii) 3 SbIII-resistant isolates out of 12 tested. Amongst the latter, there were three combinations of in vitro phenotypes: (i) parasites sensitive (n=4) or (ii) resistant to both drugs (n=3) and (iii) resistant to SbV only (n=5). There was no geographical clustering in terms of in vitro susceptibility. The relation between the in vitro susceptibility to antimonials and the corresponding in vivo treatment outcome was ambiguous. Our results highlight the need to adjust the currently used Leishmania drug susceptibility assays if they are to be used for prognosis of in vivo SSG treatment outcome.     

Sensitivity of Leishmania braziliensis promastigotes to meglumine antimoniate (glucantime) is higher than that of other Leishmania species and correlates with response to therapy in American tegumentary leishmaniasis

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.     

Antileishmanial dinitroaniline sulfonamides with activity against parasite tubulin

Novel dinitroaniline sulfonamides based on the herbicide oryzalin 3 were synthesized and evaluated for activity against the parasitic protozoan Leishmania donovani and against leishmanial tubulin, the putative antiparasitic target of oryzalin. A subset of these compounds possess more activity against both Leishmania and the target protein in vitro. Compound 20 displays improved potency against leishmanial tubulin and is 13.4-fold more active against L. donovani axenic amastigotes than oryzalin.     

Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.     

Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

Background

In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported.

Methodology/Principal Findings

Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug was found to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF α, IFN γ and iNOS activity increased with the concomitant decrease in IL 10 and TGF β level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity.

Conclusions

This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar.     

Isolation of protective T cells from BALB/cJ mice chronically infected with Leishmania donovani

BALB/cJ mice, which are homozygous for Lshs on chromosone 1, are genetically susceptible to Leishmania donovani (S3), but spontaneously reduce their parasite burdens late in the course of infection. Spleens from chronically infected mice (5 to 8 mo) were found to have T cells that responded to leishmanial Ag by proliferating and secreting immune IFN. An IFN-secreting T cell line derived from the spleen of a chronically infected mouse, after boosting with leishmanial Ag and treatment with Pentostam to kill residual parasites, was able to activate macrophages to kill amastigotes in vitro. Furthermore, after adoptive transfer of this cell line, naive BALB/cJ mice challenged with amastigotes demonstrated a 42-fold reduction in parasite burden compared to controls. Four of five clones derived from the protective T cell line secreted IFN and proliferated between days 7 and 14 after Ag stimulation. All clones were Ly1+2-, L3T4+. Another T cell line, which had Ly1+2-, L3T4+ phenotype, was derived from the lymph nodes of s.c. immunized, uninfected mice. It multipled but never produced IFN in response to leishmanial Ag and failed to protect macrophages against L. donovani infection in vitro or in vivo. This nonprotective T cell line and the fifth clone from the protective line, which also did not secrete IFN, proliferated between days 2 and 7 after Ag stimulation. In summary, leishmania-responsive helper/inducer T cells, which produced IFN but were slow to proliferate in response to Ag in vitro and which were able to activate macrophages to reduce amastigotes in vitro and in vivo, are present in chronically infected BALB/cJ mice and may mediate the decrease in parasite burden during chronic infection.     

A novel ATP-binding cassette transporter from Leishmania is involved in transport of phosphatidylcholine analogues and resistance to alkyl-phospholipids

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present work reports the characterization of the first ABC subfamily G (ABCG)-like transporter, LiABCG4, in the protozoan parasite Leishmania. LiABCG4 localized mainly to the parasite plasma membrane. Overexpression of this half-transporter reduced the accumulation of phosphatidylcholine analogues and conferred resistance to alkyl-phospholipids. Likewise, when expressed in Saccharomyces cerevisiae, the protein localized to the yeast plasma membrane and conferred resistance to alkyl-phospholipids. Post-Golgi secretory vesicles isolated from a LiABCG4-overexpressing yeast mutant contained the leishmanial ABC transporter and exhibited ATP-dependent, vanadate-sensitive transport of phosphatidylcholine analogues from the cytosolic to the lumenal leaflet of the vesicle membrane. Cross-linking showed dimerization of LiABCG4. These results suggest that LiABCG4 is involved in the active transport of phosphatidylcholine and resistance to alkyl-phospholipids in Leishmania.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Antileishmanial activities and mechanisms of action of indole-based azoles

Two 3-(alpha-azolylbenzyl)indoles were evaluated against Leishmania amastigotes. Both compounds proved to be very active against intracellular and axenic amastigotes. The IC50 values of the imidazole derivative, PM17, and the triazole analogue, PM19, against L. mexicana axenic amastigotes, were 4.4 +/- 0.1 and 6.4 +/- 0.1 microM, respectively. Against intracellular amastigotes, PM17 produced a 66% decrease of leishmanial burden at 1 microM and PM19 had an IC50 of 1.3 microM. In a Balb/c mice model of L. major leishmaniasis, administration of PM17 led to a clear-cut parasite burden reduction: 98.9% in the spleen, 79.0% in the liver and 49.9% in the popliteal node draining the cutaneous lesion. As anticipated, it was brought to the fore that PM17 decreases ergosterol biosynthesis leading to membrane fungal cell alterations. Moreover it was proved that this imidazole antifungal agent induces a parasite burden-correlated decrease in interleukine-4 production both in the splenocyte and the popliteal node of the mouse.