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Development of a new in vitro assay for screening malaria transmission blocking compounds
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A malária é uma doença parasitária que afecta 214 milhões de pessoas no mundo inteiro. Uma das maiores dificuldades na luta contra este parasita reside no combate aos seus estadios esporogónicos. São necessários novos compostos que bloqueiem a transmissão do parasita entre seres humanos e o mosquito vector da doença. A redução natural decorrente da transição de gametócitos maduros no hospedeiro humano através de fertilização na refeição sanguínea do mosquito até ao oocisto no mosquito representa um potencial alvo de intervenção. Nesta tese, foi desenvolvido e optimizado um ensaio in vitro que permite determinar a actividade de compostos nos diversos estadios de desenvolvimento do ciclo esporogónico de P. berghei. Este processo inclui a formação de oocinetos e oocistos e maturação de oocistos. Um total de dez compostos, atovaquona, azitromicina, ciclohexamida, cloroquina, dihidroartemisinina, lumefantrina, halofantrina, pirimetamina, pironaridina e thiostrepton foram testados in vitro em termos da sua actividade sobre gametócitos, oocinetos e oocistos. A actividade dos compostos e a validade do ensaio no desenvolvimento de oocistos foi confirmada in vivo em mosquitos Anopheles stephensi. Foram identificados a atovaquona, ciclohexamida e o thiostrepton como sendo altamente potentes na redução da transformação de gametócitos para oócinetos e no desenvolvimento de oocistos. No geral, este ensaio inovador é rápido, relativamente fácil e pouco dispendioso, permitindo fazer um rastreio de uma biblioteca abrangente de moléculas para determinar o seu efeito nas fases de desenvolvimento de Plasmodium no mosquito, minimizando a necessidade de um modelo in vivo. É urgente desenvolver e validar ensaios de elevada produtividade permitindo que novas bibliotecas de compostos sejam testadas não só para P. berghei mas também para P. falciparum e P. vivax. Estes ensaios podem identificar compostos para estudos pré-clinicos permitindo encontrar drogas com actividade que bloqueia a transmissão quer em campo quer numa situação clínica. No futuro, novas combinações de fármacos devem ter atividade não só contra a fase sanguínea dos parasitas, mas também destinar-se a bloquear a transmissão.
Malaria is a parasitic disease that affects 214 million people worldwide. One of the greatest struggles of the fight against this parasite resides in its sporogonic stages. Transmission blocking compounds are necessary to prevent transmission of the Plasmodium parasite between humans and mosquitoes. The natural bottleneck created by the transitions from a mature gametocyte in the human host, through fertilization in the mosquito blood meal, to the oocyst in the mosquito, presents a potential target for intervention. In this thesis, we have developed and optimized an in vitro assay for evaluating drug activity in each of the stages of the sporogonic cycle of P. berghei. It includes ookinete and oocyst formation, and oocyst development. A total of ten compounds, atovaquone, azithromycin, chloroquine, cycloheximide, dihydroartemisinin, lumefantrine, halofantrine, pyrimethamine, pyronaridine, and thiostrepton were tested in vitro for their activity on gametocytes and ookinetes and oocysts. The assay was validated in vivo in Anopheles stephensi mosquitoes. We have identified atovaquone, cycloheximide and thiostrepton as three very potent molecules leading to a significant impairment on gametocyte to ookinete conversion and of oocyst development. Overall, this innovative assay is a fast, easy and affordable method for screening large libraries of molecules at a wide range of concentrations for their effect on the development stages of Plasmodium parasites in the mosquito, minimizing the need for an in vivo model. It is urgent to develop and validate high throughput assays allowing for new libraries of compounds to be tested against not only P. berghei, but also P. vivax and also P. falciparum. These assays could be indicative of compounds for preclinical studies to find drugs blocking transmission in the field and in a clinical situation. In the future, new drugs combinations should have not only blood stage activity, but also transmission blocking components.
Malaria is a parasitic disease that affects 214 million people worldwide. One of the greatest struggles of the fight against this parasite resides in its sporogonic stages. Transmission blocking compounds are necessary to prevent transmission of the Plasmodium parasite between humans and mosquitoes. The natural bottleneck created by the transitions from a mature gametocyte in the human host, through fertilization in the mosquito blood meal, to the oocyst in the mosquito, presents a potential target for intervention. In this thesis, we have developed and optimized an in vitro assay for evaluating drug activity in each of the stages of the sporogonic cycle of P. berghei. It includes ookinete and oocyst formation, and oocyst development. A total of ten compounds, atovaquone, azithromycin, chloroquine, cycloheximide, dihydroartemisinin, lumefantrine, halofantrine, pyrimethamine, pyronaridine, and thiostrepton were tested in vitro for their activity on gametocytes and ookinetes and oocysts. The assay was validated in vivo in Anopheles stephensi mosquitoes. We have identified atovaquone, cycloheximide and thiostrepton as three very potent molecules leading to a significant impairment on gametocyte to ookinete conversion and of oocyst development. Overall, this innovative assay is a fast, easy and affordable method for screening large libraries of molecules at a wide range of concentrations for their effect on the development stages of Plasmodium parasites in the mosquito, minimizing the need for an in vivo model. It is urgent to develop and validate high throughput assays allowing for new libraries of compounds to be tested against not only P. berghei, but also P. vivax and also P. falciparum. These assays could be indicative of compounds for preclinical studies to find drugs blocking transmission in the field and in a clinical situation. In the future, new drugs combinations should have not only blood stage activity, but also transmission blocking components.
Descrição
Palavras-chave
Malaria Plasmodium berghei Parasitologia Bloqueio de transmissão Anopheles stephensi Ciclo esporogónico Mosquito
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical