A high-throughput in vitro translation screen towards discovery of novel antimalarial protein translation inhibitors

Drugs that target protein synthesis are well-validated for use as antimicrobials, yet specific high throughput (HTP) methods to screen for those targeting malaria are lacking. Here, we have developed a cell free in vitro translation (IVT) assay for the human malaria parasite, Plasmodium falciparum, which reconstitutes the native parasite protein translation machinery. Combining clarified IVT lysate with a click beetle luciferase reporter gene fused to untranslated regions of Pf histidine-rich proteins (hrp)-2 and 3, the HTP IVT assay accurately reports protein translation in a 384-well plate format using a standard spectrofluorometer. We validate the assay as effective in detecting compounds targeting the ribosome, ribosome co-factors (elongation factor 2) and cytosolic tRNA synthetases as well as its ability to find translation inhibitors in a blind screen using a high-density assay format amenable for high throughput. This demonstrates an ability to reconstitute the breadth of the parasite eukaryotic protein translation machinery in vitro and use it as a powerful platform for antimalarial drug discovery.