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

Abstract

BackgroundA 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.MethodsHere, 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.ResultsUsing 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).ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1231-8) contains supplementary material, which is available to authorized users.

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

“…Leading on from this we sought to validate the previously reported activity of candidate compounds compared to a third class of translation inhibitor, DDD107498, a current antimalarial in preclinical development that targets parasite protein translation via the translation elongation factor 2 (eEF2) 8 . Contrary to published findings 12 , measurement at 120 minutes did not show any substantial translation inhibition activity from the top 8 MMV compounds previously identified ( Figure 4A, highlighted (blue labels) besides 8 further random MMV compounds (black)). Where activity was seen it was inconsistent between replicate runs (e.g.…”

“…Screening platforms to specifically identify compounds with similar activity against malaria would be a welcome addition to the antimalarial field to increase and differentiate the drug discovery pipeline. Towards this, two studies have devised formulation of a cell free in vitro translation (IVT) lysate 11 and its development towards a luciferase reporter assay 12 . This provides a foundation from which inhibitors specific to protein synthesis in the most virulent human malaria parasite, Plasmodium falciparum.…”

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“…The lysate was then passed back into the first syringe. This back and forth cycle was repeated up to 20 times per parasite pellet 1 . This process takes between 20-30 mins per parasite pellet and the resistance in the device makes it physically taxing on the wrists and thumbs.…”

“…To demonstrate the ability of the PDSP to replace manual cell lysis we compared the two methods directly. In brief, Plasmodium falciparum parasites were harvested as previously published 1 and split into two pools, one for manual lysis and one for lysis using the PDSP. We either passed the lysate back-and-forth through the Isobiotec cell homogenizer for 20 cycles by hand or the PDSP controlled by the GUI interface passed the lysate through.…”

“…One aliquot was taken for each lysis method and used in an in vitro translation reaction as previously published with a few modifications 1 . Each 10uL reaction consisted of 7uL of lysate, 10 µM amino acid mixture, 20 mM HEPES/KOH pH 8.0, 75 mM KOAc, a range of 1-5mM Mg(OAc)2, 2 mM DTT, 0.5 mM ATP, 0.1 mM GTP, 20 mM creatine phosphate, 0.2 μg/μl creatine kinase, and 0.5 pmoles of Nanoluciferase reporter RNA.…”