A new tool for high-throughput assays to screen potential drugs against Trypanosoma cruzi

CANAVACI AMC, PEREZ BRANDAN CM, XU D, TARLETON RL

Buzios – Rio de Janeiro, Brasil,

Congreso; XIII International Congress of Protistology, XXV Meeting of the Brazilian Society of Protozoology and XXXVI Annual on Basic Research in Chagas Disease; 2009

Currently there are the two drugs available for the treatment of T. cruzi infection, nifurtimox and benznidazole. These compounds have several toxic side effects and variable efficacy (Docampo, 2001; Filardi and Brener, 1987), contributing in part to their low rate of use in the treatment of Chagas disease. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput assays to screen potential new drugs and existing compound libraries are essential. The objective of this work was to develop and validate an improved method for growth and drug assays in T. cruzi. Toward that end, we generated parasite lines expressing the tandem tomato fluorescent protein (tdTomato) by transfection of T. cruzi epimastigotes with a pTREX-Neo-tdTomato plasmid. Tomato red parasites were easily observed by flow cytometry or fluorescence microscopy. For drug assays, parasites were plated in 96 well black plates with or without drug and the change in fluorescence intensity as a measure of growth was determined by a plate reader for 3 consecutive days. An IC50 for each compound tested was then calculated. The fluorescence assay gave similar IC50s for control compounds (benznidazole) as did other previously described methods (e.g. visual counting by hemacytometer and colorimetric assays using AlamarBlue). However the fluorescence-based assay had the added advantages of requiring significantly lower amount of parasites (1x104parasites/well vs. 106 per well for the Alamar Blue assay), and, because it is not an endpoint assay, parasite growth can be monitored daily over the course of the experiment, giving more accurate estimation of drug activity. Inter- and intra-assay variation for determination of the IC50 for Benznidazole was low. Finally, the fluorescence based assay was used to identify new candidate drugs with IC50s equal or below to that of benznidazole. In conclusion, we have developed a consistent and low cost drug testing method for T. cruzi which does not require any additional indicator reagent, enzymatic process or laborious visual counting. These and similar fluorescent parasite lines should constitute a new tool for faster and high-throughput assays to screen potential new drugs.Supported by NIH grant P01 AI44979 to RLT