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, 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.
Recebido em 28/10/10; aceito em 11/7/11; publicado na web em 2/9/11 THE MOLECULAR BASIS OF ANTI-INFLAMMATORY ACTION OF THE OLEANOLIC AND URSOLIC ACIDS ON CYCLOOXYGENASE ISOFORMS BY DOCKING AND MOLECULAR DYNAMICS. The triterpenoids oleanolic (OA) and ursolic (UA) acids show non-selective antiinflamatory activity in vitro for cyclooxygenase (COX) isoforms. 3D conformations of OA and UA, with three possible orientations (1, 1' and 2) in the active site of isoforms COX, obtained by docking, were submitted to molecular dynamics. The results show that orientation 2 of the OA in COX-2 is more favorable because orientation 1 moved away from the active site. The carboxylate group of OA interact by hydrogen bonds with Ser353 and with Phe357 and Leu359, mediated by water, while hydroxyl in C-3 interact by hydrogen bond, mediated by water, with Tyr385.Keywords: cyclooxygenase; oleanolic/ursolic acid; docking. INTRODUÇÃOAs isoformas 1 e 2 da enzima ciclo-oxigenase (COX), uma proteína homodimérica integral de membrana, catalisam a conversão do ácido araquidônico (AA) a prostaglandina H 2 (PGH 2 ), cujos derivados prostanoides causam efeitos fisiológicos e patológicos, como a inflamação.1 COX-1 é constitutivamente expressa em vários tecidos e produz prostanoides necessários à modulação das funções gastrintestinais, renais e à homeostase vascular. COX-2 é considerada induzível porque se expressa em células inflamatórias em resposta a agentes pró-inflamatórios, citocinas, endotoxinas, fatores de crescimento e promotores de tumor, embora esteja presente constitutivamente em regiões glomerulares e em vasos sanguíneos renais, o que demonstra sua importância na manutenção das funções fisiológicas cardiovasculares e renovasculares.2 As isoformas COX têm dois sítios ativos, ciclo-oxigenase e peroxidase dependente de heme, que catalisam a conversão de AA a prostaglandina G 2 (PGG 2 ) e a conversão desta a PGH 2 , respectivamente. Diversos fármacos anti-inflamatórios não esteroidais (AINEs) inibem o sítio ativo ciclo-oxigenase das isoformas COX (a partir de agora referido simplesmente como "sítio ativo"). Alguns AINEs disponíveis no mercado, como diclofenaco e flurbiprofeno, inibem ambas as isoformas em concentrações semelhantes, enquanto outros, como o celecoxibe, inibem preferencialmente a COX-2 e são classificados como COX-2-seletivos.Através do estudo de estruturas cristalográficas de isoformas COX complexadas com o AA, com AINEs clássicos e COX-2-seletivos e com ligantes que não se tornaram fármacos como o SC-558, foi possível conhecer a arquitetura do sítio ativo, um longo canal lipofílico cuja entrada é uma constrição feita por Tyr355, Arg120 e Glu524 na superfície da enzima. A cerca de 13 Å de Arg120, no ápice do canal, encontra-se o resíduo catalítico Tyr385, responsável pela conversão de AA em prostaglandina G 2 (PGG 2 ). Em ambas as isoformas, o canal lipofílico é circundado por Gly526, Ala527, Phe381, Leu384, Trp387, Phe513 e Ser530, bem como existe uma cavidade adjacente circundada por Met113, Val116, Val349, Tyr355, Leu359...
A molecular rational basis for the COX-2/COX-1 selectivity of lumiracoxib using molecular docking approach is described. The COX-2 docking analysis for lumiracoxib and the diclofenac analogue revealed a similar binding mode, in contrast with the COX-1 docking analysis which revealed a different binding orientation for both inhibitors.
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