title: Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.
abstract: BACKGROUND
  Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.
  
  
  RESULTS
  Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs.
  
  
  CONCLUSIONS
  We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.
authors.affiliation: Parasitology Division, Department of Microbiology, New York University School of Medicine, New York, New York, United States of America.
authors.forename: Julio
authors.initials: J
authors.lastname: Alonso-Padilla
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Cotillo
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Jesús L
authors.initials: JL
authors.lastname: Presa
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Cantizani
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Imanol
authors.initials: I
authors.lastname: Peña
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Ana I
authors.initials: AI
authors.lastname: Bardera
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Jose J
authors.initials: JJ
authors.lastname: Martín
authors.affiliation: Parasitology Division, Department of Microbiology, New York University School of Medicine, New York, New York, United States of America.
authors.forename: Ana
authors.initials: A
authors.lastname: Rodriguez
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D002460:Cell Line; D002522:Chlorocebus aethiops; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D032446:Myoblasts / Q000469:parasitology*; D009547:Nifurtimox / Q000494:pharmacology; D009593:Nitroimidazoles / Q000494:pharmacology; D051381:Rats; D014349:Trypanosoma cruzi / Q000187:drug effects*
chemical_list: D009593:Nitroimidazoles; D009547:Nifurtimox; C009999:benzonidazole