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

Title: A high-throughput in vitro translation screen towards discovery of novel antimalarial protein translation inhibitors
Authors: Baumann, H
Matthews, H
Li, M
Hu, JJ
Willison, K
Baum, J
Item Type: Working Paper
Abstract: Drugs that target protein synthesis are well-validated for use as antimicrobials, yet specific high throughput (HTP) methods to screen for those targeting malaria are lacking. Here, we have developed a cell free in vitro translation (IVT) assay for the human malaria parasite, Plasmodium falciparum, which reconstitutes the native parasite protein translation machinery. Combining clarified IVT lysate with a click beetle luciferase reporter gene fused to untranslated regions of Pf histidine-rich proteins (hrp)-2 and 3, the HTP IVT assay accurately reports protein translation in a 384-well plate format using a standard spectrofluorometer. We validate the assay as effective in detecting compounds targeting the ribosome, ribosome co-factors (elongation factor 2) and cytosolic tRNA synthetases as well as its ability to find translation inhibitors in a blind screen using a high-density assay format amenable for high throughput. This demonstrates an ability to reconstitute the breadth of the parasite eukaryotic protein translation machinery in vitro and use it as a powerful platform for antimalarial drug discovery.
Issue Date: 16-Jan-2018
URI: http://hdl.handle.net/10044/1/68424
DOI: https://dx.doi.org/10.1101/248740
Publisher: BioRxiv
Copyright Statement: The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Sponsor/Funder: Wellcome Trust
Funder's Grant Number: 105686
Open Access location: http://biorxiv.org/cgi/content/short/248740v1
Appears in Collections:Chemistry
Biological and Biophysical Chemistry



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