A common feature shared by type I DNA topoisomerases is the presence of a “serine, lysine, X,X, tyrosine” motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.

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. Visceral leishmaniasis (VL), caused by Leishmania infantum or L. donovani, is still one of the most threatening diseases affecting poor people in developing countries, with a fatality rate as high as 100% in two years in infected and untreated people. With no vaccine available and ineffective and toxic chemotherapy, the search for new potential drugs that accelerate the urgent drug discovery process are highly needed. A novel technology that addresses this important issue has been developed, by performing High Throughput Screening (HTS) assays in 384-well plates format in combination with an infrared L. infantum-expressing strain. The system was further validated to identify active compounds against VL in an ex vivo splenic culture. In addition, in vivo non-invasive imaging of the visceral infection in BALB/c mice was achieved for the first time by using transgenic fluorescent parasites. These findings open up the possibility of testing vast amounts of potential compounds and allow in vivo screening of drug candidates against this severe parasitic disease in an attempt to speed up the vital drug discovery program.

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 Δhsp70-II strain of Leishmania to assess protection against this disease. We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice. In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC₅₀ values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo. A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy. Leishmaniasis is a parasitic disease that is far from eradication. The lack of an efficacious vaccine and treatment failures are major factors in its intractable worldwide prevalence. A non-invasive imaging technique using genetically engineered parasites that expressed fluorescent proteins could give to researchers a quantitative and visual tool to characterize the parasite burden in experimental infections. In addition, it can be useful for determining the efficacy of candidate vaccines or drugs using High Throughput Screening methods that allow the testing of libraries of compounds in an automated 96-well plate format. Herein, we demonstrate that there is a good correlation between fluorescence emission and the parasite load, thus permitting the use of this output to monitor the progression of the disease. In order to validate this tool we have immunized mice prior the parasite challenge with a red-emitting parasite strain, confirming the scientific suitability of this approach as a valuable alternative model.

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.

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.