The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved antimalarials

Abstract

AbstractThe continued specter of resistance to existing antimalarials necessitates the pursuit of novel targets and mechanisms of action for drug development. One class of promising targets consists of the 80S ribosome and its associated components comprising the parasite translational apparatus. Development of translation-targeting therapeutics requires a greater understanding of protein synthesis and its regulation in the malaria parasite. Research in this area has been limited by the lack of appropriate experimental methods, particularly a direct measure of parasite translation. We have recently developed and optimized the PfIVT assay, an in vitro method directly measuring translation in whole-cell extracts from the malaria parasite Plasmodium falciparum.Here, we present an extensive pharmacologic assessment of the PfIVT assay using a wide range of known inhibitors, demonstrating its utility for studying activity of both ribosomal and non-ribosomal elements directly involved in translation. We further demonstrate the superiority of this assay over a historically utilized indirect measure of translation, S35-radiolabel incorporation. Additionally, we utilize the PfIVT assay to investigate a panel of clinically approved antimalarial drugs, many with unknown or unclear mechanisms of action, and show that none inhibit translation, reaffirming Plasmodium translation to be a viable alternative drug target. Within this set, we unambiguously find that mefloquine lacks translation inhibition activity, despite having been recently mischaracterized as a ribosomal inhibitor. This work exploits a direct and reproducible assay for measuring P. falciparum translation, demonstrating its value in the continued study of protein synthesis in malaria and its inhibition as a drug target.Author summaryNovel antimalarial drugs are required to combat rising resistance to current therapies. The protein synthesis machinery of the malaria parasite Plasmodium falciparum is a promising unexploited target for antimalarial development, but its study has been hindered by use of indirect experimental methods which often produce misleading and inaccurate results. We have recently developed a direct method to investigate malaria protein synthesis utilizing whole-parasite extracts. In this work, we present an extensive characterization of the assay, using a panel of pharmacologic inhibitors with known mechanisms of action. We demonstrate the specificity of the assay in various stages of protein synthesis, as well as its improved accuracy and sensitivity in comparison to an indirect measure that has been the previous standard for the field. We further demonstrate that no current clinically available antimalarial drugs inhibit protein synthesis, emphasizing its potential as a target for drugs that will overcome existing resistance. Importantly, among the antimalarials tested was mefloquine, a widely used antimalarial that has recently been mischaracterized as an inhibitor protein synthesis. Our finding that mefloquine does not inhibit protein synthesis emphasizes the importance of using direct functional measurements when determining drug targets.