Ultra-high throughput in vitro translation assay for identification of Trypanosoma cruzi-specific protein synthesis inhibitors

Abstract

Chagas disease, a global health threat exacerbated by the migration of infected individuals, raises significant concern due to its restricted treatment options, resulting in around 12,000 fatalities annually. The disease is caused by Trypanosoma cruzi, a protozoan parasite. T. cruzi 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 inserted upstream of all its mRNAs. Given that many anti-microbial agents are protein synthesis inhibitors and the divergence of T. cruzi protein synthetic machinery compared to its mammalian host, it is likely that T. cruzi-specific protein synthesis inhibitors can 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 translate a capped and polyadenylated reporter mRNA. We optimized this assays conditions and adapted it to a 384-well format. 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 in live wild-type parasites. We further miniaturized this assay to a 5 {micro}L volume, demonstrating its suitability for ultra-high throughput screening in a 1536-well format with appropriate automation. This first-of-its-kind T. cruzi ultra-high-throughput in vitro translation assay will enable screening of very large chemical libraries while hit compounds identified in the pilot study can be developed into lead compounds by synthesis and/or assembly of focused libraries to develop therapeutics for Chagas disease.