Abstract
High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.
Highlights
The leishmaniases are a group of vector-transmitted neglected tropical diseases caused by parasites of the genus Leishmania
We have developed a standardized infection and drug screening assay for Leishmania species and aimed at further exploring the differences between species by comparing the results obtained from the screening of a diversity library against three clinically relevant Leishmania species: two cutaneous species, L. amazonensis, which causes the severe syndrome diffuse cutaneous leishmaniasis, and L. braziliensis, the species most often associated with the highly disfiguring mucocutaneous leishmaniasis, as well as the visceral species L. donovani, one of the species causing the often lethal visceral leishmaniasis
High content screening (HCS) has been largely used in the interrogation of small and large compound libraries for leishmaniasis drug discovery, as it is amenable to automation, enables compound testing against intracellular amastigotes, and results in the determination of antiparasitic activity and host cell selectivity within a single assay [29,30,31,37,38]
Summary
As different species and strains within species often present phenotypic variability and require and/or allow for different culturing conditions, both in vitro and in vivo, several screening protocols have been reported using distinct host cells (both immortalized cell lines and primary cells), parasite stages for infection (promastigotes, axenic amastigotes, and ex vivo amastigotes), periods of drug incubation, and methods of detection and analysis [36] These differences, while advantageous for tailoring experimental conditions to best address particular questions, greatly complicate the comparison of drug screening data obtained with different assays, as compound activity may be due, at least in part, to divergences in experimental conditions.
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