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Diseases caused by trypanosomatids are serious public health concerns in low-income endemic countries. Leishmaniasis is presented in two main clinical forms, visceral leishmaniasis—caused by L . infantum and L . donovani —and cutaneous leishmaniasis—caused by many species, including L . major , L . tropica and L . braziliensis . As for certain other trypanosomatids, sexual reproduction has been confirmed in these parasites, and formation of hybrids can contribute to virulence, drug resistance or adaptation to the host immune system. In the present work, the capability of intraclonal and interspecies genetic exchange has been investigated using three parental strains: L . donovani , L . tropica and L . major , which have been engineered to express different fluorescent proteins and antibiotic resistance markers in order to facilitate the phenotypic selection of hybrid parasites after mating events. Stationary and exponential-phase promastigotes of each species were used, in in vitro experiments , some of them containing LULO cells (an embryonic cell line derived from Lutzomyia longipalpis ). Several intraclonal hybrids were obtained with L . tropica as crossing progenitor, but not with L . donovani or L . major . In interspecies crossings, three L . donovani x L . major hybrids and two L . donovani x L . tropica hybrids were isolated, thereby demonstrating the feasibility to obtain in vitro hybrids of parental lines causing different tropism of leishmaniasis. Ploidy analysis revealed an increase in DNA content in all hybrids compared to the parental strains, and nuclear analysis showed that interspecies hybrids are complete hybrids, i.e. each of them showing at least one chromosomal set from each parental. Regarding kDNA inheritance, discrepancies were observed between maxi and minicircle heritage. Finally, phenotypic studies showed either intermediate phenotypes in terms of growth profiles, or a decreased in vitro infection capacity compared to the parental cells. To the best of our knowledge, this is the first time that in vitro interspecies outcrossing has been demonstrated between Leishmania species with different tropism, thus contributing to shed light on the mechanisms underlying sexual reproduction in these parasites.

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.

Visceral leishmaniasis caused by Leishmania donovani is one of the major neglected tropical diseases attributable to parasitic protozoa. In the absence of an effective vaccine, chemotherapy remains the only available therapeutic option. However, current treatments rely on a limited number of drugs that are largely obsolete, highly toxic or require intravenous administration, and their extensive use has led to the emergence of drug resistance. Consequently, the discovery of new antileishmanial agents is an urgent priority. In this study, a commercial library of 449 alkaloids in a high-throughput screening format was evaluated against both axenic bone marrow-derived amastigotes and intramacrophagic amastigotes from mice infected with L. donovani IRFP, a strain engineered to emit infrared fluorescence in its viable form. Six isoquinoline-type alkaloids showed the best antileishmanial efficacy against intramacrophagic amastigotes while exhibiting minimal cytotoxicity toward RAW 264.7 and HepG2 cell lines, with a promising selective index higher than four, and good mouse intestinal tolerance in mouse organoids. Among these compounds, the protoberberine scaffold emerged as the most promising candidate for further drug development.

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.

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.

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. We have developed transgenic L. infantum lines expressing drug resistance markers linked to green and red fluorescent reporters. We hypothesized whether those cells with identical genotype can recognize each other and mate. Both types of markers were successfully exchanged within the sand fly midgut of the natural vector Phlebotomus perniciosus when individuals from these species were fed with a mixture of parental clones. Using the yellow phenotype and drug resistance markers, we provide evidence for genetic exchange in L. infantum. The hybrid progeny appeared to be triploid based on DNA content analysis. The hybrid clone analyzed was stable throughout the complete parasite life cycle. The progress of infections by the hybrid clone in BALB/c mice caused a reduction in parasite loads in both spleen and liver, and provided weight values similar to those obtained with uninfected mice. Spleen arginase activity was also significantly reduced relative to parental strains. A L. infantum hybrid lineage was obtained from intraclonal genetic exchange within the midgut of the natural vector, suggesting the ability of this parasite to recognize the same genotype and mate. The yellow hybrid progeny is stable throughout the whole parasite life cycle but with a slower virulence, which correlates well with the lower arginase activity detected both in vitro and in vivo infections.

Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years. We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks. The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

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.

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.

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.