Phenotypic screening on ‘ Pathogen Box ’ yield novel antiparasitic compounds in Leishmania infantum .

Leishmanioses are zoonotic diseases caused by intracellular protozoans of the genus Leishmania. Recent research has revealed the extensive distribution and expansion of canine leishmaniosis in large areas of the world, where the high prevalence of canine infection is associated with an increased risk of human disease. There are not specific pharmacologic treatments for canine leishmaniasis. The only way to manage the situation is the euthanasia of the infected dogs. The sacrifice of the dog was used to try to control the expansion of the infection since decades without success. Also there are a lot of other Animal species that can act as host for the disease, also with human contact. Then, to achieve a solution, we must develop a vaccine or a specific drug against for canine leishmaniasis. The Pathogen Box is a project led by Medicines for Malaria Venture (MMV, Switzerland; http://www.pathogenbox.org/) that aims to identify novel drugs with activity against diseases such as tuberculosis, malaria, toxoplasmosis, and dengue, among others. The box consists of 400 mostly novel synthetic chemicals that were initially selected from a set of ~4 million compounds due to their low toxicity for mammalian cells and activity against specific microbial pathogens. In fact, the compounds display cytotoxicity at levels that are thought to be reasonable for drug discovery programs. In this study, we screened the Pathogen Box compounds for antiparasitic activity against Leishmania infantum (reference strain and clinical insolates). This screen led to the discovery of a 5 novel hits to drug development and drug design.


Introduction
Leishmaniasis is a disease caused by a protozoa parasite from over 20 different Leishmania species and is transmitted to humans and other wild or domestic animals by the bite of infected female phlebotomine sandflies.The disease is spread worldwide affecting 98 countries in five continents, is categorized as one of the "most neglected tropical diseases" and is strongly associated with poverty and affects some of the poorest people on earth.Its spread is tightly linked to environmental changes such as deforestation, building of dams and urbanization.
Visceral leishmaniasis (VL) y one of the main clinical manifestations of the disease and is caused by Leishmania infantum, whose main vector is the dipteran Lutzomya longipalpis.Infected dogs are the main urban reservoir for zoonotic visceral leishmaniasis mostly due to the high rate of canine infection in endemic areas and intense parasitism in the skin, and are the most significant risk factor predisposing humans to infection.

Canine visceral leishmaniasis (CVL)
is expanding in the American continent and a recent report of an outbreak in a northern locality of Salto in Uruguay, documents the southernmost case of the disease.
Further investment in drug development is imperative to fill the pipeline with novel compounds, as all of the current drugs have one or more drewbacks; 1) antimonials, still remain the first line of treatment in some endemic areas, are toxic drugs with frequent lifethreatening adverse side effects which are potentiated by poor health of the patient.The Pathogen Box (PHB) is a project led by Medicines for Malaria Venture (MMV, Switzerland; http://www.pathogenbox.org/)that aims to identify novel drugs with activity against diseases such as tuberculosis, malaria, toxoplasmosis, and dengue, among others.The box consists of 400 mostly novel synthetic chemicals that were initially selected from a set of ~4 million compounds due to their low toxicity for mammalian cells and activity against specific microbial pathogens.In fact, the compounds display cytotoxicity at levels that are thought to be reasonable for drug discovery programs.The PHB project seeks to exploit hits through target identification and chemical optimization to deliver series available for robust drug discovery series ready for the take up by the community.
The aim of this work is to challenge different strains of leishmania (reference strain and local isolates) against the pathogen box, in order to identify new drugs against visceral leishmaniasis and also to compare the different behavior profiles among the strains.
Behavior of the clinical insolate parasite with the reference drugs.

Results and discussion
Behavior of the clinical insolate parasite with some compounds from the pBox.Most of the active compound were reported previously with anti-leishmania activity, but we found 5 without reports in this parasite.

Not reported
Leishmania major promastigote HTS** amphotericin B has replaced treatment in areas of India where treatment failure rates for antimonials reached > 60% mostly due to resistance.Moreover, this drug si costly and requires a complicated regime of administration.3) Liposomal amphoterin B may be the best existing drug against VL and is the first line of treatment in Europe and US but has a high market price.4) Miltefosine has shown efficacy with a cure rate of 82% and low toxicity rates, but some cases of parasite resistance have been reported.