Identification of Plasmodium falciparum specific translation inhibitors from the MMV Malaria Box using a high throughput in vitro translation screen

Abstract

Background: A major goal in the search for new anti-malarial compounds is to identify new mechanisms of action or new molecular targets. While cell-based, growth inhibition-based screening have enjoyed tremendous success, an alternative approach is to specifically assay a given pathway or essential cellular process.

Methods: Here, this work describes the development of a plate-based, in vitro luciferase assay to probe for inhibitors specific to protein synthesis in Plasmodium falciparum through the use of an in vitro translation system derived from the parasite.

Results: Using the Medicines for Malaria Venture's Malaria Box as a pilot, 400 bioactive compounds with minimal human cytotoxicity profiles were screened, identifying eight compounds that displayed greater potency against the P. falciparum translation machinery relative to a mammalian translation system. Dose-response curves were determined in both translation systems to further characterize the top hit compound (MMV008270).

Conclusions: This assay will be useful not only in future anti-malarial screening efforts but also in the investigation of P. falciparum protein synthesis and essential processes in P. falciparum biology.

Keywords: Anti-malarials; MMV; Malaria Box; Plasmodium falciparum; Ribosome; Screen; Translation.

Fig. 1

Development of a luciferase based in vitro translation assay in Plasmodium falciparum. a Workflow for preparing lysate for in vitro translation assay. b Plasmid construct to generate mRNA transcripts containing a P. falciparum specific 5′ and 3′ UTR with a firefly luciferase open reading frame. Maxipreps of the plasmid were digested with PvuII and BamHI to create the templates for T7 transcription to make a final mRNA with the P. falciparum UTRs and firefly luciferase. c Lysates were incubated in the presence of a 10 × magnesium-containing translation buffer and luciferase mRNAs for a time course of 30 min to 120 min followed by the addition of luciferin reagent to assay for luciferase activity. d Lysates were incubated with DMSO control only or with 5 μM cycloheximide before or after the 120-min incubation followed by assaying for luciferase output

Fig. 2

In vitro translation assay drug screens. a Lysates were incubated in the presence of anti-malarials and cycloheximide, a general eukaryotic translation inhibitor, all at 1 μM final concentration. Error bars represent the standard deviation among three biological replicates. b MMV Malaria Box compounds were added to lysates at a 1 μM final concentration. The average of three biological replicates were used to determine the extent of translation inhibition and normalized to the average of the DMSO controls present in each plate. Each point on the graph is the averaged response of a single drug. Each point is colour-coded by type of effect: top hit compound, no P. falciparum inhibition, high standard deviation, or inhibition in rabbit reticulocyte. The histogram on the right of the graph displays the total percentage of compounds with the given luciferase ratio

Fig. 3

Flow diagram and results of the Malaria Box screen. Starting with 400 compounds, each compound was tested in three independent biological replicates in in vitro translation assays with 1 μM of the compound. Of the 46 compounds that achieved at least 20 % inhibition of translation, only eight passed both the standard deviation filter, and specificity filter removing general eukaryotic translation inhibitors (as measured by translation inhibition in rabbit reticulocyte lysate) and luciferase inhibitors. a–h Structures of the eight compounds that passed the primary and secondary screens. The letter of each structure is matched to the compounds listed in Table 1

Fig. 4

Dose–response curves of MMV008270. Dose-response curves in P. falciparum lysate or rabbit reticulocyte lysate using a 12-point 1:3 titration starting at 250 μM. Data were normalized to background and DMSO-only controls. IC₅₀ values for each system are listed below the graph