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The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.

In this study we utilized the concept of rational drug design to identify novel compounds with optimal selectivity, efficacy and safety, which would bind to the target enzyme pteridine reductase 1 (PTR1) in Leishmania parasites. Twelve compounds afforded from Baylis-Hillman chemistry were docked by using the QUANTUM program into the active site of Leishmania donovani PTR1 homology model. The biological activity for these compounds was estimated in green fluorescent protein-transfected L. donovani promastigotes, and the most potential analogue was further investigated in intracellular amastigotes. Structure-activity relationship based on homology model drawn on our recombinant enzyme was substantiated by recombinant enzyme inhibition assay and growth of the cell culture. Flow cytometry results indicated that 7-(4-chlorobenzyl)-3-methyl-4-(4-trifluoromethyl-phenyl)-3,4,6,7,8,9-hexahydro-pyrimido[1,2-a]pyrimidin-2-one (compound 7) was 10 times more active on L. donovani amastigotes (50% inhibitory concentration [IC50] = 3 μM) than on promastigotes (IC50 = 29 μM). Compound 7 exhibited a Ki value of 0.72 μM in a recombinant enzyme inhibition assay. We discovered that novel pyrimido[1,2-a]pyrimidin-2-one systems generated from the allyl amines afforded from the Baylis-Hillman acetates could have potential as a valuable pharmacological tool against the neglected disease visceral leishmaniasis.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T. brucei PTR1 (=7 nm), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km=25 nm and Ki=2.3 nm, respectively).

Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H4B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (kcat/Km) for LmPTR1 are similar with dihydrobiopterin (H2B) and quinonoid dihydrobiopterin (qH2B) as substrates and about 20-fold lower than LmQDPR with qH2B. In contrast, TbPTR1 shows a 10-fold higher kcat/Km for H2B over qH2B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H4B (430 nm, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H4B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H4B as a medium supplement. These cells grew normally with H4B, which spontaneously oxidizes to qH2B, but were unable to survive in the absence of pterin or with either biopterin or H2B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target.

The enzyme pteridine reductase 1 (PTR1) is a potential target for new compounds to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chemical series were identified containing aminobenzothiazole and aminobenzimidazole scaffolds, respectively. One of the hits (2-amino-6-chloro-benzimidazole) was subjected to crystal structure analysis and a high resolution crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogues (2-amino-benzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein−ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, derivatives were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochemical properties were obtained.

Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for ‘repurposing’ of compounds developed originally for treatment of human cancers or other infectious agents.

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase–thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.

A crystallographic and biochemical study of L. major cysteine synthase, which is a pyridoxyl phosphate-dependent enzyme, is reported. The structure was determined to 1.8 Å resolution and revealed that the cofactor has been lost and that a fragment of γ-poly-d-glutamic acid, a crystallization ingredient, was bound in the active site. The enzyme was inhibited by peptides.

Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1- null mutants were 18-fold more sensitive to H2O2 than PTR1-overproducing lines, and significant 3-5 fold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H2O2 susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1-but not dihydrofolate reductase-dependent manner, implicating H4B metabolism specifically. Neither H2O2 consumption, nor the level of intracellular oxidative stress, was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols), these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.

The detoxification enzyme glyoxalase I from L. major has been crystallized. Preliminary molecular-replacement calculations indicate the presence of three glyoxalase I dimers in the asymmetric unit.

Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (Hs1 and Hs2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. Analysis of the screening results has shown that structure-activity relationships (SAR) for Leishmania NMT are divergent from all other NMTs tested, a finding not predicted by sequence similarity calculations, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors. We found a strong overlap between the SARs for Plasmodium NMT and both human NMTs, suggesting that achieving an appropriate selectivity profile will be more challenging. However, we did discover two novel series with selectivity for Plasmodium NMT over the other NMT orthologues in this study, and an additional two structurally distinct series with selectivity over Leishmania NMT. We believe that release of results from this study into the public domain will accelerate the discovery of NMT inhibitors to treat malaria and leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as Medical Research Council and Wellcome Trust can stimulate research for neglected diseases.

Author Summary

Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (HsNMT1 and HsNMT2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. We have identified eight series of protozoan NMT inhibitors, six having good selectivity for either Plasmodium or Leishmania NMTs over the other orthologues in this study. We believe that all of these series could form the basis of medicinal chemistry programs to deliver drug candidates against either malaria or leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as the UK Medical Research Council and Wellcome Trust can stimulate research for neglected diseases.

Selection for methotrexate resistance in Leishmania spp. is often associated with amplification of the H locus short-chain dehydrogenase-reductase gene ptr1 as part of extrachromosomal elements. Extensive sequences are always coamplified and often contain inverted duplications, most likely formed by the annealing of inverted repeats present at the H locus. By gene targeting mediated by homologous recombination, several repeated sequences were introduced in the vicinity of ptr1. Selection for methotrexate resistance in these transfectants led to ptr1 amplification as part of small circles with direct or inverted duplications whether the integrated sequences consisted of direct or inverted repeats. Hence, for a region to he amplified in L. tarentolae during drug selection, a drug resistance gene is required and must be flanked by (any) homologous repeated sequences. The distance between these repeats and their orientation will determine the length of the amplicon and whether it contains direct or inverted duplications.

The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase–thymidylate synthase (DHFR–TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR–TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR–TS is essential for parasite survival and represents a promising target for drug discovery.

Background

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease characterized by the presence of one or more lesions on the skin that usually heal spontaneously after a few months. Most cases of CL worldwide occur in Southwest Asia, Africa and South America, and a number of cases have been reported among troops deployed to Afghanistan. No vaccines are available against this disease, and its treatment relies on chemotherapy. The aim of this study was to characterize parasites isolated from Canadian soldiers at the molecular level and to determine their susceptibility profile against a panel of antileishmanials to identify appropriate therapies.

Methodology/Principal Findings

Parasites were isolated from skin lesions and characterized as Leishmania tropica based on their pulsed field gel electrophoresis profiles and pteridine reductase 1 (PTR1) sequences. Unusually high allelic polymorphisms were observed at several genetic loci for the L. tropica isolates that were characterized. The drug susceptibility profile of intracellular amastigote parasites was determined using an established macrophage assay. All isolates were sensitive to miltefosine, amphotericin B, sodium stibogluconate (Pentostam) and paromomycin, but were not susceptible to fluconazole. Variable levels of susceptibility were observed for the antimalarial agent atovaquone/proguanil (Malarone). Three Canadian soldiers from this study were successfully treated with miltefosine.

Conclusions/Significance

This study shows high heterogeneity between the two L. tropica allelic versions of a gene but despite this, L. tropica isolated from Afghanistan are susceptible to several of the antileishmanial drugs available.

Author Summary

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease transmitted by the bite of sandflies, resulting in sores on the skin. No vaccines are available, and treatment relies on chemotherapy. CL has been frequently diagnosed in military personnel deployed to Afghanistan and returning from duty. The parasites isolated from Canadian soldiers were characterized by pulsed field gels and by sequencing conserved genes and were identified as Leishmania tropica. In contrast to other Leishmania species, high allelic polymorphisms were observed at several genetic loci for the L. tropica isolates that were characterized. In vitro susceptibility testing in macrophages showed that all isolates, despite their genetic heterogeneity, were sensitive to most antileishmanial drugs (antimonials, miltefosine, amphotericin B, paromomycin) but were insensitive to fluconazole. This study suggests a number of therapeutic regimens for treating cutaneous leishmaniasis caused by L. tropica among patients and soldiers returning from Afghanistan. Canadian soldiers from this study were successfully treated with miltefosine.

In the title compound, C10H10N4O4·0.5H2O, the two rings of the pteridine system are nearly coplanar [dihedral angle = 4.25 (9)°]. The atoms of the carboxyl group are also coplanar with the pteridine unit [r.m.s. deviation from the mean plane of the pteridine skeleton = 0.092 (2) Å]. In the crystal, the presence of the water molecule of crystallization (O atom site symmetry 2) leads to a hydrogen-bonding pattern different from the one shown by many carboxylic acid compounds (dimers formed through O—H⋯O hydrogen bonds between neighbouring carboxyl groups): in the present structure, the water mol­ecule, which lies on a binary axis, acts as a bridge between two mol­ecules, forming a hydrogen-bonded dimer. In addition to the hydrogen bonds, there are π–π ring stacking inter­actions involving the pyrimidine and pyrazine rings [centroid–centroid distance = 3.689 (1)Å], and two different pyrazine rings [centroid–centroid distance = 3.470 (1)Å]. Finally, there is a C—O⋯π contact involving a carboxyl­ate C—O and the pyrimidine ring with a short O⋯Cg distance of 2.738 (2) Å.

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.

Approximately 70% of the cDNA clones identified by immunoscreening Leishmania donovani expression libraries with serum from a patient with visceral leishmaniasis (kala-azar) were found to encode the highly conserved Hsp90 and Hsp70 members of the heat shock protein family. Recombinant fusion proteins containing the C-terminal portions of L. donovani Hsp90 and Hsp70 were used as target antigens in enzyme-linked immunosorbent assays of various sera. Sera from four patients with visceral leishmaniasis recognized recombinant Leishmania Hsp90 and Hsp70, while sera from seven patients with Chagas' disease did not, despite the fact that Trypanosoma cruzi Hsp90 and Hsp70 share more than 80% amino acid identity with their counterparts in Leishmania spp. Thus, Leishmania Hsp90 and Hsp70 elicit strong humoral responses and are potential candidates for specific serodiagnostic assays capable of distinguishing between L. donovani and T. cruzi infections.

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Trypanosomiasis and leishmaniasis are two debilitating disease groups caused by parasites of Trypanosoma and Leishmania spp. and affecting millions of people worldwide. A brief outline of the potential targets for rational drug design against these diseases are presented, with an emphasis placed on the enzyme trypanothione reductase. Trypanothione reductase was identified as unique to parasites and proposed to be an effective target against trypanosomiasis and leishmaniasis. The biochemical basis of selecting this enzyme as a target, with reference to the simile and contrast to human analogous enzyme glutathione reductase, and the structural aspects of its active site are presented. The process of designing selective inhibitors for the enzyme trypanothione reductase has been discussed. An overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities against the parasites and their prospects as future chemotherapeutic agents are briefly revealed.

Leishmania species of the subgenus Leishmania and especially L. donovani are responsible for a large proportion of visceral leishmaniasis cases. The debate on the mode of reproduction and population structure of Leishmania parasites remains opened. It has been suggested that Leishmania parasites could alternate different modes of reproduction, more particularly clonality and frequent recombinations either between related individuals (endogamy) or between unrelated individuals (outcrossing) within strongly isolated subpopulations. To determine whether this assumption is generalized to other species, a population genetics analysis within Leishmania donovani complex strains was conducted within a single village. The results suggest that a mixed-mating reproduction system exists, an important heterogeneity of subsamples and the coexistence of several genetic entities in Sudanese L. donovani. Indeed, results showed significant genetic differentiation between the three taxa (L. donovani, L. infantum and L. archibaldi) and between the human or canine strains of such taxa, suggesting that there may be different imbricated transmission cycles involving either dogs or humans. Results also are in agreement with an almost strict specificity of L. donovani stricto sensu to human hosts. This empirical study demonstrates the complexity of population structure in the genus Leishmania and the need to pursue such kind of analyses at the smallest possible spatio-temporal and ecological scales.

Author Summary

Leishmaniases are a serious public health problem, especially in developing countries, caused by Leishmania parasites and transmitted by sandfly bites. More information is needed on the population biology of these pathogens for diagnostic and epidemiological inquiries and for drug and vaccine elaboration. For studies dealing with the population genetics, exploring the genetic patterns of such organisms at microgeographic scales is fundamental. In this context, we made a population genetic study, based on 20 microsatellite loci, on 61 strains of Leishmania donovani complex collected in a Sudanese village, Babar El Fugara, during the epidemic of 1996–2000. Results showed that considering the whole sample as a single population was not adequate because of the coexistence of several genetic entities and a genetic differentiation between the human or canine strains. In addition, our findings suggested that clonality may have a strong impact on the L. donovani complex, unlike other Leishmania species. This study demonstrates the need to pursue population genetics studies in Leishmania species from sampling designs that control maximum possible confounding factors and to elaborate such kinds of analyses at the smallest possible spatio-temporal and ecological scales.

Given the recent rise in antimicrobial resistance, there is an urgent need to identify and characterize new antibiotic drug targets. One such target is dihydrodipicolinate reductase (DHDPR), which is an essential bacterial enzyme that catalyzes the second step in the lysine-biosynthesis pathway. In this paper, the cloning, expression, purification and crystallization of DHDPR from methicillin-resistant S. aureus are presented.

Dihydrodipicolinate reductase (DHDPR; EC 1.3.1.26) catalyzes the nucleotide (NADH/NADPH) dependent second step of the lysine-biosynthesis pathway in bacteria and plants. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPR from methicillin-resistant Staphylococcus aureus (MRSA-DHDPR) are presented. The enzyme was crystallized in a number of forms, predominantly with ammonium sulfate as a precipitant, with the best crystal form diffracting to beyond 3.65 Å resolution. Crystal structures of the apo form as well as of cofactor (NADPH) bound and inhibitor (2,6-pyridinedicarboxylate) bound forms of MRSA-DHDPR will provide insight into the structure and function of this essential enzyme and valid drug target.

Background:

Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase (DHFR). Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more.

Methods:

PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression.

Results:

Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells.

Conclusion:

Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid.

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin–trypanothione reductase–NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.