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.
Highlights
A major goal in the search for new anti-malarial compounds is to identify new mechanisms of action or new molecular targets
Ahyong et al Malar J (2016) 15:173 screening with growth media supplemented with isopentenyl pyrophosphate (IPP), narrowing hits to only those active against apicoplast targets [4]
Development of a high‐throughput, malaria‐specific, in vitro translation assay Building upon the work of Ferreras et al, an in vitro translation assay was further developed and optimized from P. falciparum cultures with the addition of exogenous firefly luciferase reporter mRNAs to allow for high-throughput plate-based luciferase assay screening (Fig. 1a) [12]
Summary
A major goal in the search for new anti-malarial compounds is to identify new mechanisms of action or new molecular targets. A common and very successful strategy relies on screening in vitro cultures of Plasmodium falciparum against large compound collections and assaying for growth inhibition in a ‘top-down’ approach to drug discovery Using this type of approach can result in the subsequent identification of drug targets by selection of resistant strains and whole genome sequencing of these resistant strains to identify mutations Ahyong et al Malar J (2016) 15:173 screening with growth media supplemented with isopentenyl pyrophosphate (IPP), narrowing hits to only those active against apicoplast targets (such as isoprenoid enzymes) [4] Facilitating these efforts, the freely available Medicines for Malaria Venture (MMV) Malaria Box has been a welcome resource, providing biologically active compounds with unknown targets and mechanisms of action [7]. The library contains 400 chemically diverse compounds that are commercially available and pre-screened for activity in the blood stages of P. falciparum with minimal human cytotoxicity
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