Identification of new Trypanosoma cruzi-specific protein synthesis inhibitors by using a developed high-throughput in vitro translation assay

Le résumé fourni par la source

There are restricted treatment options for Chagas disease, a global health risk causing 12,000 fatalities annually. This situation is exacerbated by environmental changes and migration, which is altering patterns of disease transmission, and by expansion of the vector range. The disease is caused by the protozoan parasite Trypanosoma cruzi, which possesses a divergent translation apparatus, characterized by enlarged ribosomes and a unique mRNA cap appended to the 5' end of a 39-nucleotide spliced leader sequence at the 5' end of all its mRNAs. Given that many antimicrobial agents are protein synthesis inhibitors and that there are significant differences between the T. cruzi protein synthesis machinery and that of its mammalian host, it is likely that T. cruzi-specific protein synthesis inhibitors could be employed for the treatment of Chagas disease. Such inhibitors can be identified by screening chemical libraries in an in vitro translation assay. However, lysates of T. cruzi cannot reinitiate translation of exogenous mRNAs. Here, we demonstrate that T. cruzi extracts derived from a strain deficient for hemin accumulation efficiently translated a capped and polyadenylated reporter mRNA. We optimized assay conditions and adapted them to a 384-well format. We further miniaturized this assay to a 5 μL volume, demonstrating its suitability for ultra-high throughput screening. Further, in a proof-of-principle pilot study, we identified quinazoline compounds that inhibit translation of reporter mRNAs by T. cruzi extracts. These compounds inhibited protein synthesis and proliferation of insect-derived forms and wild-type parasites growing in mammalian cells at a low micromolar range and a selectivity index higher than 200. This first-of-its-kind T. cruzi in vitro translation system will enable high-throughput screening of very large chemical libraries, while first-in-class hit compounds identified in the pilot study can be developed into lead compounds by design and synthesis of focused libraries to develop therapeutics for Chagas disease.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.