The mode of reproduction in Leishmania spp has been argued to be essentially clonal. However, recent data (genetic analysis of populations and co-infections in sand flies) have proposed the existence of a non-obligate sexual cycle in the extracellular stage of the parasite within the sand fly vector. In this article we propose the existence of intraclonal genetic exchange in the natural vector of Leishmania infantum.
The use of genetically engineered pathogens that express fluorescent or luminescent proteins represents a huge stride forward in the understanding of trypanosomatid-borne tropical diseases. Nowadays, such modified microorganisms are being used to screen thousands of compounds under a target-free (phenotypic) approach. In addition, experimental infections with transgenic parasites drastically reduce the number of animals required for preclinical studies, because no animal needs to be put down to assess its parasite load. Finally, the use of fluorescent parasites is contributing to unraveling genetic exchange events between trypanosomatid strains. This phenomenon is important for understanding the mechanism by which traits such as virulence, tissue tropism, and drug resistance are transferred, as well as the emergence of novel strains.
Sponges biosynthesize ?-methoxylated fatty acids with unusual biophysical and biological properties and in some cases they display enhanced anticancer activities. However, the antiprotozoal properties of the ?-methoxylated fatty acids have been less studied. In this work, we describe the total synthesis of (5Z,9Z)-(±)-2-methoxy-5, 9-eicosadienoic acid (1) and its acetylenic analog (±)-2-methoxy-5,9-eicosadiynoic acid (2), and report that they inhibit (EC?? values between 31 and 22 µM) the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB). The inhibition of LdTopIB (EC?? = 53 µM) by the acid (±)-2-methoxy-6-icosynoic acid (12) was studied as well. The potency of LdTopIB inhibition followed the trend 2 > 1 > 12, indicating that the effectiveness of inhibition depends on the degree of unsaturation. All of the studied ?-methoxylated fatty acids failed to inhibit the human topoisomerase IB enzyme (hTopIB) at 100 µM. However, the ?-methoxylated fatty acids were capable of inhibiting an active but truncated LdTopIB with which camptothecin (CPT) cannot interact suggesting that the methoxylated fatty acids inhibit LdTopIB with a mechanism different from that of CPT. The diunsaturated fatty acids displayed low cytotoxicity towards Leishmania infantum promastigotes (EC?? values between 260 and 240 µM), but 12 displayed a better cytotoxicity towards Leishmania donovani promastigotes (EC?? = 100 µM) and a better therapeutic index.
The aim of this work is the in vitro and ex vivo assessment of the leishmanicidal activity of camptothecin and three analogues used in cancer therapy: topotecan (Hycantim®), gimatecan (ST1481) and the pro-drug irinotecan (Camptosar®) as well as its active metabolite SN-38 against Leishmania infantum. The activity of camptothecin and its derivatives was studied on extracellular L. infantum infrared-emitting promastigotes and on an ex vivo murine model of infected splenocytes with L. infantum fluorescent amastigotes. In situ formation of SDS/KCl precipitable DNA-protein complexes in Leishmania promastigotes indicated that these drugs are DNA topoisomerase IB poisons. The inhibitory potency of camptothecin derivatives on recombinant L. infantum topoisomerase IB was assessed in vitro showing that gimatecan is the most active compound preventing the relaxation of supercoiled DNA at submicromolar concentrations. Cleavage equilibrium assays in Leishmania topoisomerase IB show that gimatecan changes the equilibrium towards cleavage at much lower concentrations than the other camptothecin derivatives and that this effect persists over time. Gimatecan and camptothecin were the most powerful compounds preventing cell growth of free-living L. infantum promastigotes within the same concentration range. All these compounds killed L. infantum splenocyte-infecting amastigotes within the nanomolar range. The amastigote form showed higher sensitivity to topoisomerase IB poisons (with high therapeutic selectivity indexes) than free-living promastigotes. All the compounds assayed poisoned L. infantum DNA topoisomerase IB leading to a strong leishmanicidal effect. Camptothecin derivatives are suitable for reducing the parasitic burden of ex vivo infected splenocytes. The selectivity index of gimatecan makes it a promising drug against this neglected disease.
Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique heterodimeric DNA-topoisomerase IB (LdTopIB). LdTopIB is a heterodimer made up of a large subunit and a small subunit that must interact with each other to build an active enzyme able to solve the topological tensions on the DNA. As LdTopIB is located within the nucleus, one or more nuclear localization signals (NLS) should exist to ensure its nuclear translocation. In this report three novel NLS have been identified through a sequential deletion study of the genes encoding of both subunits fused to that encoding the green fluorescent protein (GFP). NLS1 is a highly basic sequence of 43 amino acids in the C-terminal extension of the large protomer. We found two well-defined sequences in the small protomer: NLS2 is a 10-amino acid motif located in the N-terminal extension of the protein; NLS3 consists of a complex region of 28 amino acids placed in the vicinity of the catalytic Tyr-222 included at the conserved SKINY signature within the C-terminal. Furthermore, by means of yeast cell viability assays, conducted with several LdTopIB chimeras lacking any of the NLS motives, we have revealed that both subunits are transported independently to the nucleus. There was no evidence of LdTopIB accumulation in mitochondria or association to the kinetoplast DNA network. The results rule out the former hypothesis, which attributes nucleocytoplasmic transport of LdTopIB entirely to the large subunit. The LdTopIB is localized to the nucleus only.
Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, to the extent that its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Previous studies have shown that a MAT-overexpressing strain of Leishmania donovani controls AdoMet production, keeping the intracellular AdoMet concentration at levels that are compatible with cell survival. This unexpected result, together with the fact that MAT activity and abundance changed with time in culture, suggests that different regulatory mechanisms acting beyond the post-transcriptional level are controlling this protein. In order to gain an insight into these mechanisms, several experiments were carried out to explain the MAT abundance during promastigote cell growth. Determination of MAT turnover in cycloheximide (CHX)-treated cultures resulted in a surprising 5-fold increase in MAT turnover compared to CHX-untreated cultures. This increase agrees with a stabilization of the MAT protein, whose integrity was maintained during culture. The presence of proteasome inhibitors, namely MG-132, MG-115, epoxomycin and lactacystin in the culture medium prevented MAT degradation in both MAT-overexpressing and mock-transfected leishmanial strains. The role of the ubiquitin (Ub) pathway in MAT down-regulation was supported using immunoprecipitation experiments. Immunoprecipitated MAT cross-reacted with anti-Ub antibodies, which provides evidence of a proteasome-mediated down-regulation of the leishmanial MAT abundance.
L-Arginine is one of the precursor amino acids of polyamine biosynthesis in most living organisms including Leishmania parasites. L-Arginine is enzymatically hydrolyzed by arginase producing L-ornithine and urea. In Leishmania spp. and other trypanosomatids a single gene encoding arginase has been described. The product of this gene is compartmentalized in glycosomes and is the main source of L-ornithine for polyamine synthesis in these parasites. L-Ornithine is substrate of ornithine decarboxylase (ODC) - one of the key enzymes of polyamine biosynthesis and a validated target for therapeutic intervention - producing putrescine, which in turn is converted to spermidine by condensing with an aminopropyl group from decarboxylated S-adenosylmethionine. Unlike trypanosomatids, mammalian hosts have two arginases (arginase I and II), which have close structural and kinetic resemblances, but localize in different subcellular organelles, respond to different stimuli and have different immunological reactivity. Arginase I is a cytosolic enzyme, mostly expressed in the liver as a pivotal component of the urea cycle, providing in addition L-ornithine for polyamine synthesis. In contrast, arginase II localizes inside mitochondria and is metabolically involved in L-proline and L-glutamine biosynthesis. More striking is the role played by L-arginine as substrate for nitric oxide synthase (NOS2) in macrophages, the main route of clearance of many infectious agents including Leishmania and Trypanosoma cruzi. In infected macrophages L-arginine is catalysed by NOS2 or arginase, contributing to host defense or parasite killing, respectively. A balance between NOS2 and arginase activities is a crucial factor in the progression of the Leishmania infection inside macrophages. In response to T-helper type 2 (Th2) cytokines, resident macrophages induce arginase I inhibiting NO production from L-arginine, thereby promoting parasite proliferation. Conversely, the response to T-helper type 1 (Th1) cytokines is linked to NOS2 induction and parasite death. Moreover, induction of any of these enzymes is accompanied by suppression of the other. Specifically, arginase reduces NO synthesis by substrate depletion, and N(?)-hydroxy-L-arginine, one of the intermediates of NOS2 catalysis, competitively inhibits arginase activity. In spite of abundant data concerning arginases in mammals as well their involvement in parasite killing, there are very few papers regarding the actual role of arginase in the parasite itself. This review is an update on the recent progress in research on leishmanial arginase including the role played by this enzyme in the establishment of infection in macrophages and the immune response of the host. A comparative study of arginases from other kinetoplatids is also discussed.
The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC(50) = 165.5 ± 23.4 ?M) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC(50) = 62.3 ± 0.7 ?M.
The first total synthesis of the marine cyclopropane fatty acid (±)-17-methyltrans- 4,5-methyleneoctadecanoic acid was accomplished in 8 steps and in 9.1% overall yield starting from 1-bromo-12-methyltridecane. The cis analog (±)-17- methyl-cis-4,5-methyleneoctadecanoic acid was also synthesized but in 7 steps and in 16.4% overall yield. With the two isomeric cyclopropane fatty acids at hand it was possible to unequivocally corroborate the trans relative configuration of the naturally occurring fatty acid by gas chromatographic co-elution of the corresponding methyl esters. The cis isomer was cytotoxic to Leishmania donovani promastigotes with an IC(50) of 300.2 ± 4.2 µM.
The phylum Apicomplexa includes a large group of protozoan parasites responsible for a wide range of animal and human diseases. Destructive pathogens, such as Plasmodium falciparum and Plasmodium vivax, causative agents of human malaria, Cryptosporidium parvum, responsible of childhood diarrhoea, and Toxoplasma gondii, responsible for miscarriages and abortions in humans, are frequently associated with HIV immunosuppression in AIDS patients. The lack of effective vaccines, along with years of increasing pressure to eradicate outbreaks with the use of drugs, has favoured the formation of multi-drug resistant strains in endemic areas. Almost all apicomplexan of medical interest contain two endosymbiotic organelles that contain their own mitochondrial and apicoplast DNA. Apicoplast is an attractive target for drug testing because in addition to harbouring singular metabolic pathways absent in the host, it also has its own transcription and translation machinery of bacterial origin. Accordingly, apicomplexan protozoa contain an interesting mixture of enzymes to unwind DNA from eukaryotic and prokaryotic origins. On the one hand, the main mechanism of DNA unwinding includes the scission of one-type I-or both DNA strands-type II eukaryotic topoisomerases, establishing transient covalent bonds with the scissile end. These enzymes are targeted by camptothecin and etoposide, respectively, two natural drugs whose semisynthetic derivatives are currently used in cancer chemotherapy. On the other hand, DNA gyrase is a bacterial-borne type II DNA topoisomerase that operates within the apicoplast and is effectively targeted by bacterial antibiotics like fluoroquinolones and aminocoumarins. The present review is an update on the new findings concerning topoisomerases in apicomplexan parasites and the role of these enzymes as targets for therapeutic agents.
The first total syntheses of the naturally occurring acetylenic fatty acids-6-heptadecynoic acid (59% overall yield) and 6-icosynoic acid (34% overall yield)-was accomplished in four steps. Using the same synthetic sequence the naturally occurring fatty acids (6Z)-heptadecenoic acid (46% overall yield) and (6Z)-icosenoic acid (27% overall yield) were also synthesized. The Delta(6) acetylenic fatty acids displayed good antiprotozoal activity towards Leishmania donovani promastigotes (EC(50) = 1-6 microg/mL), but the 6-icosynoic acid was the most effective in the series. In addition, the (6Z)-icosenoic acid was a much better antiprotozoal compound (EC(50) = 5-6 microg/mL) than the (6Z)-heptadecenoic acid (EC(50) > 25 microg/mL). The saturated fatty acids n-heptadecanoic acid and n-eicosanoic acid were not effective towards L. donovani, indicating that the Delta(6) unsaturation in these fatty acids is necessary for leishmanicidal activity. In addition, both the 6-icosynoic acid and the (6Z)-icosenoic acid were inhibitors of the Leishmania DNA topoisomerase IB enzyme (EC(50s) = 36-49 microM), a possible intracellular target for these compounds. This is the first study assessing fatty acids as inhibitors of the Leishmania DNA topoisomerase IB enzyme.
Trypanosomatid (order Kinetoplastida)-borne neglected tropical diseases - African and American trypanosomiasis and leishmaniasis - are amongst the most devastating health threats of underdeveloped, developing and poor countries. Climatic changes due to global warming, tourism exchange and increasing migratory fluxes are re-distributing the endemic subtropical location of these diseases to a new scenario with a rising presence in developed countries during the last decades. In addition, the proved opportunistic transmission of these diseases through contaminated syringes shared by drug users, in combination with immunosuppression processes linked to HIV infections and the poor response to the typical treatments, point to AIDS patients as a sensitive sub-population prone to suffer from these diseases. DNA topoisomerases are the "molecular engineers" that unravel the DNA during replication and transcription. The mechanism of DNA unwinding includes the scission of a single DNA strand - type I topoisomerases - or both DNA strands - type II topoisomerases - establishing transient covalent bonds with the scissile end. Camptothecin and etoposide - two natural drugs whose semi-synthetic derivatives are currently used in cancer chemotherapy - target types I and II DNA-topoisomerases respectively, stabilizing ternary topoisomerase-DNA-drug covalent complexes, which irreversibly poison the enzymes. Several differences between parasite and host DNA topoisomerases have pointed to these enzymes as potential drug targets in Trypanosomatids. The unusual localization inside the mitochondria-like organellum - the kinetoplast - linked to mini and maxicircles, as well as the uncommon heterodimeric structure of the DNA topoisomerase IB subfamily, make these proteins unquestionable targets for drug intervention against trypanosomatids.
The first total synthesis for the (Z)-17-methyl-13-octadecenoic acid was accomplished in seven steps and in a 45% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 12-bromo-1-dodecanol followed by a second acetylide coupling to the short-chain 3-methyl-1-bromobutane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid. The title compound displayed antiprotozoal activity against Leishmania donovani (EC(50) = 19.8 microg/ml) and inhibited the leishmania DNA topoisomerase IB at concentrations of 50 microM.
Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only when CpTopIB ORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38) was assessed.
Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from L-ornithine has been well studied in protozoa, the metabolic origin(s) of L-ornithine have received less attention. Arginase (EC 184.108.40.206) catalyzes the enzymatic hydrolysis of L-arginine to L-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg(?) knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or L-ornithine for growth. A complemented line (arg(?)/+ARG) expressing arginase from a multi-copy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors ?-difluoromethylornithine (DFMO) and N(?)-hydroxy-l-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg(?) parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of l-arginine metabolism in both pro- and anti-parasitic responses involving host nitric oxide synthase, which requires L-arginine to generate NO.
Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated ?hsp70-II strain of Leishmania to assess protection against this disease.
The fatty acids (±)-2-methoxy-6Z-heptadecenoic acid (1), (±)-2-methoxy-6-heptadecynoic acid (2) and (±)-2-methoxyheptadecanoic acid (3) were synthesized and their inhibitory activity against the Leishmania DNA topoisomerase IB enzyme (LdTopIB) determined. Acids 1 and 2 were synthesized from 4-bromo-1-pentanol, the former in ten steps and in 7% overall yield, while the latter in seven steps and in 14% overall yield. Acid 3 was prepared in six steps and in 42% yield from 1-hexadecanol. Acids 1-3 inhibited the LdTopIB enzyme following the order 2 > 1 ? 3, with 2 displaying an EC(50) = 16.6 ± 1.1 ?M and 3 not inhibiting the enzyme. Acid 1 preferentially inhibited the LdTopIB enzyme over the human TopIB enzyme. Unsaturation seems to be a prerequisite for effective inhibition, rationalized in terms of weak intermolecular interactions between the active site of LdTopIB and either the double or triple bonds of the fatty acids. Toxicity towards Leishmania donovani promastigotes was also investigated resulting in the same order 2 > 1 > 3, with 2 displaying an EC(50) = 74.0 ± 17.1 ?M. Our results indicate that ?-methoxylation decreases the toxicity of C(17:1) fatty acids towards L. donovani promastigotes, but improves their selectivity index.
Leishmania donovani - the causative agent of visceral leishmaniasis - has several evolutionary characteristics that make the disease difficult to combat. Among these differences, a rare heterodimeric DNA topoisomerase IB has been reported thus opening a new promising field in the therapy of leishmaniasis. Several studies of the human enzyme have pointed to the importance of the linker domain in respect to camptothecin sensitivity. At present, it has been impossible to pinpoint the regions that make up the linker domain in Leishmania.
2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7?M. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0?M), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1?M) and Trypanosoma brucei rhodesiense (IC(50)=64.5?M). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound.
Visceral leishmaniasis is an emerging neglected tropical disease (NTD) caused by the protozoan Leishmania infantum in the countries bordering the Mediterranean Basin. Currently there is no effective vaccine against this disease, and the therapeutic approach is based on toxic derivatives of Sb(V). Therefore, the discovery of new therapeutic targets and the development of drugs designed to inhibit them comprise an extremely important approach to fighting this disease. DNA topoisomerases (Top) have been identified as promising targets for therapy against leishmaniasis. These enzymes are involved in solving topological problems generated during replication, transcription, and recombination of DNA. Being unlike that of the mammalian host, type IB DNA topoisomerase (TopIB) from Leishmania spp. is a unique bisubunit protein, which makes it very interesting as a selective drug target. In the present investigation, we studied the effect of two TopIB poisons with indenoisoquinoline structure, indotecan and AM13-55, on a murine BALB/c model of infected splenocytes with L. infantum, comparing their effectiveness with that of the clinically tested leishmanicidal drug paromomycin. Both compounds have high selectivity indexes compared with uninfected splenocytes. SDS-KCl-precipitable DNA-protein complexes in Leishmania promastigotes and in vitro cleaving assays confirmed that these drugs are Top poisons. The inhibitory potency of both indenoisoquinolines on L. infantum recombinant TopIB was assessed in vitro, with results showing that indotecan was the most active compound, preventing the relaxation of supercoiled DNA. Experimental infections in susceptible BALB/c mice treated with 2.5 mg/kg body weight/day once every other day for a total of 15 days showed that indotecan cleared more than 80% of the parasite burden of the spleen and liver, indicating promising activity against visceral leishmaniasis.
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