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https://www.arca.fiocruz.br/handle/icict/59445
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ArtigoDireito Autoral
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2099-12-31
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AN ALTERNATIVE IN VITRO DRUG SCREENING TEST USING LEISHMANIA AMAZONENSIS TRANSFECTED WITH RED FLUORESCENT PROTEIN
Green fluorescent protein
Red fluorescent protein
Chemotherapy
Drug screening
Autor(es)
Afiliação
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil.
Federal de Ouro Preto. Escola de Farmácia. Laboratório de Química Farmacêutica. Ouro Preto, MG, Brazil.
Universidade Federal de Minas Gerais. Departamento de Parasitologia. Belo Horizonte, MG, Brazil.
Department of Molecular Microbiology. Washington University School of Medicine. St. Louis, MO, USA.
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil.
Universidade Federal de São João Del Rey. Departamento de Engenharia de Biossistemas. São João Del Rey, MG, Brazil.
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil.
Federal de Ouro Preto. Escola de Farmácia. Laboratório de Química Farmacêutica. Ouro Preto, MG, Brazil.
Universidade Federal de Minas Gerais. Departamento de Parasitologia. Belo Horizonte, MG, Brazil.
Department of Molecular Microbiology. Washington University School of Medicine. St. Louis, MO, USA.
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil.
Universidade Federal de São João Del Rey. Departamento de Engenharia de Biossistemas. São João Del Rey, MG, Brazil.
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil.
Resumo em Inglês
Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 213, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.
Palavras-chave em inglês
Leishmania amazonensisGreen fluorescent protein
Red fluorescent protein
Chemotherapy
Drug screening
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