title: An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein.
abstract: 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 IC(50) values for 4 propranolol derivatives (1, 2b, 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.
authors: Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz/FIOCRUZ, Belo Horizonte, MG, Brazil.
authors: Marcele N
authors: MN
authors: Rocha
authors: Célia M
authors: CM
authors: Corrêa
authors: Maria N
authors: MN
authors: Melo
authors: Stephen M
authors: SM
authors: Beverley
authors: Olindo Assis
authors: OA
authors: Martins-Filho
authors: Ana Paula
authors: AP
authors: Madureira
authors: Rodrigo P
authors: RP
authors: Soares
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007891:Leishmania / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D011433:Propranolol / Q000031:analogs & derivatives / Q000494:pharmacology; D015203:Reproducibility of Results; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D000666:Amphotericin B; D000981:Antiprotozoal Agents; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011433:Propranolol; D000097573:Red Fluorescent Protein