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In vitro evaluation of antimalarial compounds using transgenic parasites

Grant number: 19/07223-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2019
Effective date (End): May 31, 2020
Field of knowledge:Biological Sciences - Parasitology - Protozoology of Parasites
Principal Investigator:Roberto Rudge de Moraes Barros
Grantee:Thafne Plastina Astro
Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
Associated research grant:18/06219-8 - Use of Plasmodium knowlesi as a model for malaria research in vitro, AP.JP

Abstract

Malaria parasites are responsible for more than 200 million cases and 400,000 deaths worldwide each year. Five species of Plasmodium cause human infections: Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum is responsible for the most lethal form of the disease and receives the major part of research investment due to the existence of long-term in vitro culture techniques. However, P. falciparum is evolutionarily and biologically distant from other species and the control measures developed does not show the same efficiency when applied against the other four species.The development of new antimalarial drugs is needed because parasites develop drug resistance mechanisms constantly. Antimalarials have stage-specific action, and only one drug (primaquine) present action against hepatic forms. Furthermore, the compounds actions may vary between species. Currently the evaluation of new compounds is performed in vitro, using P. falciparum and the SYBR Green technique. This assays consists of the use of a fluorescent molecule (SYBR), which is intercalated in the DNA double strand. The DNA multiplication/ growth of parasites in cultures can be assessed by measuring the fluorescence emitted by the SYBR Green molecules added to culture lysates. It is also possible to use bioluminescent transgenic parasites to evaluate the drug effect. The growth of parasites in culture can be assessed by the luminescence released after an enzymatic reaction. The bioluminescence reaction can be more sensitive than the SYBR reaction, detecting a smaller number of parasites, allowing smaller size assays and consequently testing a larger number of compounds simultaneously.Recently, P. knowlesi strains (evolutionarily close to P. vivax, P. malariae and P. ovale) were adapted to long term in vitro culture, offering an alternative for malaria research. The project intends to standardize the techniques of SYBR Green and bioluminescence for drug assays against P. knowlesi in vitro. We will perform the standardization of drug assays in 96 and 384-well plates using the GloMax Explorer (Promega) equipment, capable of reading fluorescence and luminescence. For this standardization 6 compounds of known effect in P. falciparum will be utilized: chloroquine, blasticidina, G418, pyrimethamine, WR99210 and methotrexate. We will test the hematocrit, culture volume, initial parasitemia, growth time, as well as parasite lysis protocols, SYBR Green and luciferase substrate concentrations. After standardization, the assays can be used for the evaluation of new antimalarial compounds and allow the comparison of results obtained in P. falciparum and P. knowlesi.