High-Throughput Screening Platform To Identify Inhibitors of Protein Synthesis with Potential for the Treatment of Malaria

Abstract

Artemisinin-based combination therapies have been crucial in driving down the global burden of malaria, the world's largest parasitic killer. However, their efficacy is now threatened by the emergence of resistance in Southeast Asia and sub-Saharan Africa. Thus, there is a pressing need to develop new antimalarials with diverse mechanisms of action. One area of Plasmodium metabolism that has recently proven rich in exploitable antimalarial targets is protein synthesis, with a compound targeting elongation factor 2 now in clinical development and inhibitors of several aminoacyl-tRNA synthetases in lead optimization. Given the promise of these components of translation as viable drug targets, we rationalized that an assay containing all functional components of translation would be a valuable tool for antimalarial screening and drug discovery. Here, we report the development and validation of an assay platform that enables specific inhibitors of Plasmodium falciparum translation (PfIVT) to be identified. The primary assay in this platform monitors the translation of a luciferase reporter in a P. falciparum lysate-based expression system. Hits identified in this primary assay are assessed in a counterscreen assay that enables false positives that directly interfere with the luciferase to be triaged. The remaining hit compounds are then assessed in an equivalent human IVT assay. This platform of assays was used to screen MMV's Pandemic and Pathogen Box libraries, identifying several selective inhibitors of protein synthesis. We believe this new high-throughput screening platform has the potential to greatly expedite the discovery of antimalarials that act via this highly desirable mechanism of action.

Keywords: Plasmodium; antimalarials; drug discovery; in vitro translation; malaria; protein synthesis.

FIG 1

(A and B) Schematic representation of PfIVT (A) and luciferase counterscreen (B) assays. (C) Assay workflow and criteria for progression.

FIG 2

Established inhibitors of translation. Compounds that are known to inhibit different aspects of in vitro translation were selected to validate our IVT assay. (A) Cycloheximide is an inhibitor of the translocation step in elongation (46). (B) Borrelidin is an inhibitor of ThrRS (47). (C) DDD00197451 is an inhibitor of translation via eEF2 (44). (D) Halofuginone is an inhibitor of ProRS (11). (E) DDD001712277 is an inhibitor of LysRS. (F) Emetine is an inhibitor of the 80S ribosome (43). (G) Cladosporin is an inhibitor of LysRS (7). All curves shown are from a single technical replicate and are representative of data for at least two biological replicates. IC₅₀ values (insets) are weighted means ± SD from at least two biological replicates.

FIG 3

Assessment of the Pathogen Box and the Pandemic Box open access compound libraries against P. falciparum IVT assay. (A) Single-point (30 μM) high-throughput screen of the 400 compounds contained within the Pathogen Box against the IVT assay. Compounds demonstrating >58.02% inhibition were identified as hits (18 compounds; 4.5% hit rate) (highlighted in red). Mean Z′ = 0.855. (B) Single-point (30 μM) screen of the Pandemic Box (400 compounds) against PfIVT assay. Compounds demonstrating >47.29% inhibition were identified as hits (14 compounds; 3.5% hit rate) (highlighted in red). The mean Z′ of the assay was 0.825. (C) Assessment of selected hit compounds in 8-point potency assays (closed circles) and a luciferase counterscreen (open circles). All curves shown are from a single technical replicate but representative of data from two biological replicates. IC₅₀ values are summarized in Tables 1 and 2.