Chagas disease is endemic to 18 Latin American countries, with 13 million people chronically infected. Approximately 30% of chronically infected patients will suffer from irreversible damage to the heart and digestive tract leading to death. There is a need to develop new drugs against Chagas disease because only two drugs with strong toxic effects are available for treatment of the initial stages of infection and no drug is available for treating the chronic stage. The causative agent of the disease is Trypanosoma cruzi, an obligated intracellular protozoan parasite that in the chronic stage of disease is found almost exclusively in the intracellular stage. High-throughput methods that can reliably detect T. cruzi are required to screen compounds with activity against this parasite. We propose to use two high-throughput screening (HTS) assays using a recombinant T. cruzi line expressing beta-galactosidase. Since these parasites can be easily cultured in vitro, we propose to perform two assays for HTS of drugs that interfere with 1) the free extracellular parasites and 2) the intracellular infection cycle of T. cruzi. Inhibition of extracellular parasites will most likely identify drugs that are active against the early stages of disease, when parasites are found frequently outside host cells. Interfering with the cycle of infection is more likely to identify drugs that inhibit intracellular parasites, which are almost the only form observed during chronic infections. Both assays are currently performed the laboratory in 96-well plate format and have optimal sensitivity and reproducibility. We intend to perform primary screenings to select positive hits that will be further selected using secondary assays to exclude compounds toxic for host cells. We will also run secondary assays to confirm positive hits and counter screenings using alternative methods of detection to eliminate false positives. Chemical optimization will be performed if necessary and a plan for animal testing and development of putative new inhibitors into medically available drugs is described.
We intend to use two different assays for high-throughput screening: the inhibition of free Trypanosoma cruzi trypomastigotes and the inhibition of the infection of host cells by this parasite, with the aim of finding new drugs that could be used to treat Chagas disease. We will use a recombinant T. cruzi expressing 2-galactosidase, which allows for highly sensitive and reliable assays for high throughput screening. ? ? ?
| Andriani, Grasiella; Chessler, Anne-Danielle C; Courtemanche, Gilles et al. (2011) Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening. PLoS Negl Trop Dis 5:e1298 |
