Relevância de ensaios fenotípicos com diversas espécies de Leishmania na descoberta inicial de fármacos

Author(s)

Thalita Camêlo da Silva Ferreira

• Document type: Doctoral Thesis
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-02-27

Examining board members

Elizabeth Bilsland; André Gustavo Tempone Cardoso; Jadel Muller Kratz; Rafael Lemos Miguez Couñago

• Advisor: Lucio Holanda Gondim de Freitas Junior

Abstract

The leishmaniasis are a group of poverty-associated diseases caused by the protozoan Leishmania spp. The complexity of this kinetoplastid parasite is highlighted by biological, ecological, and epidemiological aspects. It is endemic in more than 98 countries affecting around 12 million people. Approximately 20 species can be transmitted to humans by 78 species of the phlebotomine sandfly vector. Infected people can develop one of the several pathologies from which the most common are: cutaneous lesions, the disfiguring mucocutaneous disorder, and the life-threatening visceral disease. The final outcome is multifactorial, depending on intricate host-parasite interactions and environmental factors. The few chemotherapeutic options available are mostly unsatisfactory due to low efficacy, poor safety, emergence of parasite resistance, and other factors related to treatment (such as administration route, length of treatment or high costs) that are not suitable for the socioeconomic reality of affected populations. The current scenario demands research focused on the identification of novel effective therapeutics. Modern technologies enable the screening of millions of compounds, but a more predictive in vitro assays is still lacking. It is a common practice for a research group to target a single species, usually L. dovovani, the causative agent of visceral disease. Addressing a representative sample of viscerotropic and dermotropic species/strains of Leishmania can be useful to prioritize compounds based on their spectrum of activity. This work focused on the discovery of starting points for the development of new antileishmanials using an image-based methodology. Careful standardization was carried out aiming to achieve robustness and reproducibility, based on: (i) the biological model, determining the best condition for parasite and host cell culture, infection and drug exposure, and (ii) the image analysis algorithm. The multi-species high content assay was successfully established and validated to assess compound activity against some of the most clinically relevant dermotropic (L. amazonensis and L. braziliensis) and viscerotropic agents (L. donovani and L. infantum). This semi-automated assay is based on THP-1 macrophages infected with stationary phase promastigotes and can be virtually adapted to any in vitro infective species, providing the basis for the discovery of broad-spectrum antileishmanial molecules. A species-specific profile was observed in the general activity of libraries screenings, and a reduced number of compounds were active for all species. The simultaneous screening of the Pathogen Box library against four Leishmania species along with literature data analysis resulted in the identification of 12 pan-antileishmanial compounds. Although these molecules have been reported as anti-kinetoplastid agents, their activity against different Leishmania species had not been explored yet. The results obtained from this study demonstrate the potential value of including a panel of species in early steps of the drug discovery process, which can facilitate the prospection, validation and progression of more promising compounds. The species panel, together with other reported secondary assays, is suggested as part of a cell-based assay pipeline for Leishmania spp. aiming to empower the prioritization of compounds based on crucial aspects besides the physico-chemical and toxicity filters traditionally applied (AU)

• FAPESP's process: 15/10436-6 - Discovery of new compounds for cutaneous and mucocutaneous leishmaniasis treatment from high content screening
• Grantee: Thalita Camelo da Silva Ferreira
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)