Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase

Abstract

A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli ␤-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red ␤-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 M in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with ␤-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

New method for quantification of Trypanosoma cruzi in animal’s tissue in the chronic phase of experimental Chagas' disease

“…The CL Brener B5 clone strain of T. cruzi; which contains the gene of β-galactosidase which confers high specificity and sensitivity to pharmacological tests (Buckner et al 1996), cordially provided by Dr. Alicia Gómez Barrio, the Complutense University of Madrid.…”

“…Buckner et al (1996) reported an efficient method for the quantification of T. cruzi parasites in drug-screening assays. T. cruzi strain CL parasites were genetically engineered to express the Escherichia coli b-galactosidase gene, lacZ.…”

“…The efficiency of the assay facilitates the screening of a larger number of candidate compounds against T. cruzi. (Buckner et al 1996) The purpose of this study was to determine the number of parasites present in the tissues in the chronic phase disease, by quantifying the enzyme β-galactosidase expressed by the clone CL B5 strain of Trypanosoma cruzi. This would replace the method used for histological evaluation of the chronic phase with the advantage that this new methodology provide a low cost and be done in less time.…”

In vitro and in vivo activity of lignan lactones derivatives against Trypanosoma cruzi

“…Parasites and culture procedure For in vitro assays, the clone CL-B5 of T. cruzi was used (Buckner et al 1996). The parasites, stably transfected with Escherichia coli β-galactosidase gene (lacZ), were kindly provided by Dr. F. Buckner through Instituto Comemorativo Gorgas (Panama).…”

“…The activity was evaluated by modified colorimetric method (Rolon et al 2006) using CPRG as previously described by Buckner et al (1996). NCTC-929 fibroblasts were established in 24-well tissue culture plates at a previously determined optimal concentration of 2.5×10 3 cells/well.…”