A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing ?-galactosidase

Abstract

We have developed a new pharmacological screening assay for epimastigotes of Trypanosoma cruzi (clone CL-B5) that express the Escherichia coli LacZ gene. The assay is based on determining the activity of the cytoplasmic beta-galactosidase released into the culture on membrane lysis in the presence of the substrate chlorophenol red beta-D-galactopyranoside (CPRG). The experimental conditions were adjusted to find those in which the relationship between epimastigote number and CPRG absorbance was linear over the widest possible range. Absorbance was significantly correlated with the number epimastigote from 5x10(3) to 1.2x10(6) parasites/ml (r=0.98, P<0.01). The optimal final concentration of CPRG was 200 microM and the optimal incubation period was 6 h when parasites were incubated for 3 days. Once the assay was standardized, the trypanocidal activities of nifurtimox and benznidazole were determined both by CPRG assay and microscopic counting, demonstrating the methods utility for drug-screening. The efficacy obtained was comparable to that obtained with the manual method.

Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes

“…Several methods for antitrypanosomal screening on epimastigotes have been described: counting live cells (Gomez-Barrio et al 1997), measuring enzyme activities (Vega et al 2005), and cleavage of the tetrazolium salt (MTT) (Muelas-Serrano et al 2000). However, all these methods are time-consuming because they required a subsequent manipulation step or transfected parasites.…”

“…Some colorimetric assays for evaluation of trypanocidal activity against epimastigotes using a tetrazolium compound (MTT) (Muelas-Serrano et al 2000) and chlorophenol red-β-D-galactopyranoside (CPRG) (Vega et al 2005) have been reported, and these demonstrate more objectivity than microscopic counting. Resazurin is a colored compound that changes color by cell activity, from blue to pink, but does not precipitate upon reduction, and this is a direct correlation between the reduction of resazurin in the growth media and the range of proliferation of living organisms.…”

Benznidazole Nanoformulates: A Chance to Improve Therapeutics for Chagas Disease

“…40 Formulations with a low cytotoxicity for mammalian cells (< 20% inhibition) 41 were tested for their activity against epimastigotes chosen for preliminary screening because of its simple maintenance and relatively high drug sensitivity. 42 Drug activity was evaluated using the colorimetric method 43,44 . Absorbances and cell counts were compared to determine their correlation under the conditions of our laboratory.…”

New carbohydrazide derivatives of 1H-pyrazolo[3,4-b]pyridine and trypanocidal activity

“…Afterwards, these compounds were subjected to in vitro biological evaluation against clone CL Brener strain B5 of T. cruzi parasite (trypomastigote and amastigote forms), using the Benznidazole drug as positive control (Table I) (Brener 1962, Vega et al 2005.…”