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In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D, Boehlke CL, Tarleton RL - PLoS Negl Trop Dis (2010)

Bottom Line: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use.In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato).This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

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Related in: MedlinePlus

In vitro amastigote growth assays using tdTomato parasites.(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of T. cruzi expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).
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pntd-0000740-g004: In vitro amastigote growth assays using tdTomato parasites.(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of T. cruzi expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).

Mentions: The ability of compounds to inhibit the intracellular growth of T. cruzi amastigotes is a more rigorous and relevant test of anti-T. cruzi activity, as it is applied to a stage which is the predominant form in mammals and because the killing assay requires that drug also cross the host cell membrane. Amastigote growth assays in Vero cells worked similarly to epimastigotes assays, with parasite fluorescence increasing over the 4 day culture period. BZ exhibited a dose-dependent inhibitory effect on parasite growth (Figure 4A). Furthermore, this assay produced comparable results in 96 or in 384 well plates (Figure 4B–C), demonstrating the potential utility for the testing of large compound libraries. Lastly, the expression of tdTomato in different parasite strains which may differ in susceptibility to various drugs provides an easy method to confirm the susceptibility of multiple parasite strains to potential anti-T.cruzi compounds (Figure 4C).


In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D, Boehlke CL, Tarleton RL - PLoS Negl Trop Dis (2010)

In vitro amastigote growth assays using tdTomato parasites.(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of T. cruzi expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903469&req=5

pntd-0000740-g004: In vitro amastigote growth assays using tdTomato parasites.(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of T. cruzi expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).
Mentions: The ability of compounds to inhibit the intracellular growth of T. cruzi amastigotes is a more rigorous and relevant test of anti-T. cruzi activity, as it is applied to a stage which is the predominant form in mammals and because the killing assay requires that drug also cross the host cell membrane. Amastigote growth assays in Vero cells worked similarly to epimastigotes assays, with parasite fluorescence increasing over the 4 day culture period. BZ exhibited a dose-dependent inhibitory effect on parasite growth (Figure 4A). Furthermore, this assay produced comparable results in 96 or in 384 well plates (Figure 4B–C), demonstrating the potential utility for the testing of large compound libraries. Lastly, the expression of tdTomato in different parasite strains which may differ in susceptibility to various drugs provides an easy method to confirm the susceptibility of multiple parasite strains to potential anti-T.cruzi compounds (Figure 4C).

Bottom Line: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use.In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato).This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

Show MeSH
Related in: MedlinePlus