Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Miltefosine and Nifuratel Combination: A Promising Therapy for the Treatment of Leishmania donovani Visceral Leishmaniasis.

Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel-a nitrofurantoin used against vaginal infections-as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine-the only oral drug currently used against leishmaniasis-with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.

Antileishmanial Effect of 1,5- and 1,8-Substituted Fused Naphthyridines.

In the absence of a vaccine, there is a need to find new drugs for the treatment of neglected tropical diseases, such as leishmaniasis, that can overcome the many drawbacks of those currently used. These disadvantages include cost, the need to maintain a cold chain, the route of administration, the associated adverse effects and the generation of resistance. In this work we have evaluated the antileishmanial effect of 1,5- and 1,8-substituted fused naphthyridines through in vitro and ex vivo assays, using genetically modified axenic and intramacrophagic Leishmania infantum amastigotes. The toxicity of these compounds has been tested in the mammalian host cell using murine splenic macrophages, as well as in murine intestinal organoids (miniguts) in order to assess their potential for oral administration. The 1,8- derivatives showed greater leishmanicidal activity and the presence of a nitrogen atom in the fused ring to the naphthyridine was important to increase the activity of both types of molecules. The aromatization of the pyridine ring also had marked differences in the activity of the compounds.

Drug discovery technologies: Caenorhabditis elegans as a model for anthelmintic therapeutics.

Helminthiasis is one of the gravest problems worldwide. There is a growing concern on less available anthelmintics and the emergence of resistance creating a major threat to human and livestock health resources. Novel and broad-spectrum anthelmintics are urgently needed. The free-living nematode Caenorhabditis elegans could address this issue through automated high-throughput technologies for the screening of large chemical libraries. This review discusses the strong advantages and limitations for using C elegans as a screening method for anthelmintic drug discovery. C elegans is the best model available for the validation of novel effective drugs in treating most, if not all, helminth infections, and for the elucidation the mode of action of anthelmintic candidates. This review also focuses on available technologies in the discovery of anthelmintics published over the last 15 years with particular attention to high-throughput technologies over conventional screens. On the other hand, this review highlights how combinatorial and nanomedicine strategies could prolong the use of anthelmintics and control resistance problems.

Screening Marine Natural Products for New Drug Leads against Trypanosomatids and Malaria.

Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin.

Antiparasitic effect of synthetic aromathecins on Leishmania infantum.

BACKGROUND: Canine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB - one of the enzymes controlling DNA topology - has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins. RESULTS: Aromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter. CONCLUSIONS: Aromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.

Synthesis and activity of benzopiperidine, benzopyridine and phenyl piperazine based compounds against Leishmania infantum.

In the present study, anti-leishmanial evaluation of twenty four structurally diverse compounds based on benzopiperidine, benzopyridine and phenylpiperazine nucleuses against Leishmania infantum has been reported. Cytotoxicity studies of all the compounds were performed on murine non-infected splenocytes. Tested compounds exhibited weak to potent activity against promastigote (IC50 3.21 ±â€¯1.40 to >100 µM) as well as amastigote (IC50 6.84 ±â€¯2.5 to 92.47 ±â€¯17.61 µM) forms of tested strains. Moreover, two compounds F13 and F15 exhibited potent activity (IC50 < 10 µM) against both forms of the parasite with selectivity index ranges from 11.40 to 22.10. Overall, the current study afforded few hits with novel anti-leishmanial activity in low micromolar concentration, further hit optimization studies can be performed to get more potent candidates against the selected species of parasite.

New approaches from nanomedicine for treating leishmaniasis.

Leishmaniasis, a vector-borne disease caused by obligate intramacrophage protozoa, threatens 350 million people in 98 countries around the world. There are already 12 million infected people worldwide and two million new cases occur annually. Leishmaniasis has three main clinical presentations: cutaneous (CL), mucosal (ML), and visceral (VL). It is considered an opportunistic, infectious disease and the HIV-leishmaniasis correlation is well known. Antimonial compounds are used as first-line treatment drugs, but their toxicity, which can be extremely high, leads to a number of undesirable side effects and resultant failure of the patients to adhere to treatment. There is also a reported increase in Leishmania sp. resistance to these drugs. Nanotechnology has emerged as an attractive alternative because of its improved bioavailability and lower toxicity, and other characteristics that help to relieve the burden of this disease. In this review we will present some of the recent advances in the nanotechnological research regarding the treatment of leishmaniasis. The preclinical results regarding the approaches for a biomedical treatment of the disease have been encouraging, but further efforts will still be necessary for this therapy to have greater clinical applicability in humans.

Improved Efficiency of Ibuprofen by Cationic Carbosilane Dendritic Conjugates.

In order to improve the efficiency of the anti-inflammatory drug ibuprofen, cationic carbosilane dendrimers and dendrons with ibuprofen at their periphery or at their focal point, respectively, have been synthesized, and the release of the drug was studied using HPLC. Macrophages were used to evaluate the anti-inflammatory effect of the ibuprofen-conjugated dendritic systems and compared with mixtures of non-ibuprofen dendritic systems in the presence of the drug. The cationic ibuprofen-conjugated dendron was the compound that showed higher anti-inflammatory properties. It reduces the LPS-induced COX-2 expression and decreases the release of several inflammatory cytokines such as TNFα, IL-1ß, IL-6, and CCL3. These results open new perspectives in the use of these compounds as drug carriers.

Polyamine Metabolism for Drug Intervention in Trypanosomatids.

Neglected tropical diseases transmitted by trypanosomatids include three major human scourges that globally affect the world's poorest people: African trypanosomiasis or sleeping sickness, American trypanosomiasis or Chagas disease and different types of leishmaniasis. Different metabolic pathways have been targeted to find antitrypanosomatid drugs, including polyamine metabolism. Since their discovery, the naturally occurring polyamines, putrescine, spermidine and spermine, have been considered important metabolites involved in cell growth. With a complex metabolism involving biosynthesis, catabolism and interconversion, the synthesis of putrescine and spermidine was targeted by thousands of compounds in an effort to produce cell growth blockade in tumor and infectious processes with limited success. However, the discovery of eflornithine (DFMO) as a curative drug against sleeping sickness encouraged researchers to develop new molecules against these diseases. Polyamine synthesis inhibitors have also provided insight into the peculiarities of this pathway between the host and the parasite, and also among different trypanosomatid species, thus allowing the search for new specific chemical entities aimed to treat these diseases and leading to the investigation of target-based scaffolds. The main molecular targets include the enzymes involved in polyamine biosynthesis (ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine synthase), enzymes participating in their uptake from the environment, and the enzymes involved in the redox balance of the parasite. In this review, we summarize the research behind polyamine-based treatments, the current trends, and the main challenges in this field.

In Vitro and Ex Vivo Synergistic Effect of Pyrvinium Pamoate Combined with Miltefosine and Paromomycin against Leishmania.

One of the major drawbacks of current treatments for neglected tropical diseases is the low safety of the drugs used and the emergence of resistance. Leishmaniasis is a group of neglected diseases caused by protozoa of the trypanosomatidae family that lacks preventive vaccines and whose pharmacological treatments are scarce and unsafe. Combination therapy is a strategy that could solve the above-mentioned problems, due to the participation of several mechanisms of action and the reduction in the amount of drug necessary to obtain the therapeutic effect. In addition, this approach also increases the odds of finding an effective drug following the repurposing strategy. From the previous screening of two collections of repositioning drugs, we found that pyrvinium pamoate had a potent leishmanicidal effect. For this reason, we decided to combine it separately with two clinically used leishmanicidal drugs, miltefosine and paromomycin. These combinations were tested in axenic amastigotes of Leishmania infantum obtained from bone marrow cells and in intramacrophagic amastigotes obtained from primary cultures of splenic cells, both cell types coming from experimentally infected mice. Some of the combinations showed synergistic behavior, especially in the case of the combination of pyrvinium pamoate with paromomycin, and exhibited low cytotoxicity and good tolerability on intestinal murine organoids, which reveal the potential of these combinations for the treatment of leishmaniasis.

A pentapeptide signature motif plays a pivotal role in Leishmania DNA topoisomerase IB activity and camptothecin sensitivity.

BACKGROUND: 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. METHODS: Several site-directed mutations as well as internal and linear truncations involving both subunits were assayed on both, relaxation activity and sensitivity to camptothecin. RESULTS: Truncations performed on the trypanosomatids conserved motif (RPPVVRS) of the small subunit of leishmanial DNA topoisomerase IB demonstrated that elimination of pentapeptide RPPVV produced a nonfunctional enzyme. However, the removal of the dipeptide RS led to an enzyme with reduced relaxation activity and less sensitivity to camptothecin. The basic structure, both sensitive to camptothecin and able to fully relax DNA, composed of amino acids 1-592 and 175-262 in the large and small subunits, respectively. CONCLUSION: It has been established that the region between amino acids 175 and 180 (RPPVV) of the small subunit plays a pivotal role in both interaction with the large subunit and sensitivity to camptothecin in Leishmania. GENERAL SIGNIFICANCE: The present report describes a functional analysis of the leishmanial DNA topoisomerase IB regions directly involved both in sensitivity to poisons and in the conformation of the linker domain.

The transcriptome of Leishmania major in the axenic promastigote stage: transcript annotation and relative expression levels by RNA-seq.

BACKGROUND: Although the genome sequence of the protozoan parasite Leishmania major was determined several years ago, the knowledge of its transcriptome was incomplete, both regarding the real number of genes and their primary structure. RESULTS: Here, we describe the first comprehensive transcriptome analysis of a parasite from the genus Leishmania. Using high-throughput RNA sequencing (RNA-seq), a total of 10285 transcripts were identified, of which 1884 were considered novel, as they did not match previously annotated genes. In addition, our data indicate that current annotations should be modified for many of the genes. The detailed analysis of the transcript processing sites revealed extensive heterogeneity in the spliced leader (SL) and polyadenylation addition sites. As a result, around 50% of the genes presented multiple transcripts differing in the length of the UTRs, sometimes in the order of hundreds of nucleotides. This transcript heterogeneity could provide an additional source for regulation as the different sizes of UTRs could modify RNA stability and/or influence the efficiency of RNA translation. In addition, for the first time for the Leishmania major promastigote stage, we are providing relative expression transcript levels. CONCLUSIONS: This study provides a concise view of the global transcriptome of the L. major promastigote stage, providing the basis for future comparative analysis with other development stages or other Leishmania species.

Synthesis of marine α-methoxylated fatty acid analogs that effectively inhibit the topoisomerase IB from Leishmania donovani with a mechanism different from that of camptothecin.

Sponges biosynthesize α-methoxylated fatty acids with unusual biophysical and biological properties and in some cases they display enhanced anticancer activities. However, the antiprotozoal properties of the α-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 (EC50 values between 31 and 22 µM) the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB). The inhibition of LdTopIB (EC50 = 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 α-methoxylated fatty acids failed to inhibit the human topoisomerase IB enzyme (hTopIB) at 100 µM. However, the α-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 (EC50 values between 260 and 240 µM), but 12 displayed a better cytotoxicity towards Leishmania donovani promastigotes (EC50 = 100 µM) and a better therapeutic index.

Molecular-level strategic goals and repressors in Leishmaniasis - Integrated data to accelerate target-based heterocyclic scaffolds.

Leishmaniasis is a complex of neglected tropical diseases caused by various species of leishmanial parasites that primarily affect the world's poorest people. A limited number of standard medications are available for this disease that has been used for several decades, these drugs have many drawbacks such as resistance, higher cost, and patient compliance, making it difficult to reach the poor. The search for novel chemical entities to treat leishmaniasis has led to target-based scaffold research. Among several identified potential molecular targets, enzymes involved in the purine salvage pathway include polyamine biosynthetic process, such as arginase, ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, trypanothione reductase as well as enzymes in the DNA cell cycle, such as DNA topoisomerases I and II plays vital role in the life cycle survival of leishmanial parasite. This review mainly focuses on various heterocyclic scaffolds, and their specific inhibitory targets against leishmaniasis, particularly those from the polyamine biosynthesis pathway and DNA topoisomerases with estimated activity studies of various heterocyclic analogs in terms of their IC50 or EC50 value, reported molecular docking analysis from available published literatures.

Further Investigations of Nitroheterocyclic Compounds as Potential Antikinetoplastid Drug Candidates.

Due to the lack of specific vaccines, management of the trypanosomatid-caused neglected tropical diseases (sleeping sickness, Chagas disease and leishmaniasis) relies exclusively on pharmacological treatments. Current drugs against them are scarce, old and exhibit disadvantages, such as adverse effects, parenteral administration, chemical instability and high costs which are often unaffordable for endemic low-income countries. Discoveries of new pharmacological entities for the treatment of these diseases are scarce, since most of the big pharmaceutical companies find this market unattractive. In order to fill the pipeline of compounds and replace existing ones, highly translatable drug screening platforms have been developed in the last two decades. Thousands of molecules have been tested, including nitroheterocyclic compounds, such as benznidazole and nifurtimox, which had already provided potent and effective effects against Chagas disease. More recently, fexinidazole has been added as a new drug against African trypanosomiasis. Despite the success of nitroheterocycles, they had been discarded from drug discovery campaigns due to their mutagenic potential, but now they represent a promising source of inspiration for oral drugs that can replace those currently on the market. The examples provided by the trypanocidal activity of fexinidazole and the promising efficacy of the derivative DNDi-0690 against leishmaniasis seem to open a new window of opportunity for these compounds that were discovered in the 1960s. In this review, we show the current uses of nitroheterocycles and the novel derived molecules that are being synthesized against these neglected diseases.

Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds.

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.

Indotecan (LMP400) and AM13-55: two novel indenoisoquinolines show potential for treating visceral leishmaniasis.

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.

First total synthesis of the (±)-2-methoxy-6-heptadecynoic acid and related 2-methoxylated analogs as effective inhibitors of the leishmania topoisomerase IB enzyme.

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 µM 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 µM. Our results indicate that α-methoxylation decreases the toxicity of C(17:1) fatty acids towards L. donovani promastigotes, but improves their selectivity index.

2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani.

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±0.7µM. 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)=11.0µM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1µM) and Trypanosoma brucei rhodesiense (IC(50)=64.5µM). 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.