The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z′ factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.
Bibliographical noteFunding Information:
We thank Kurt Peterson and Prachi Bawaskar from the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, for productive discussions and their work on the PfHT screening assay using fluorescence lifetime measurement. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. T.E.K. conceived and designed the research, with oversight by P.W.H. Creation and optimization of the cell lines and screening conditions were performed by T.E.K. together with M.P. IC50 determinations for parasite growth inhibition and parasite culture were performed by R.L.E., supervised by A.R.O. Radiolabel uptake experiments with HEK293 cells were performed by M.R.H. Radiolabel uptake experiments with parasites were performed by T.E.K. together with M.R.H. qPCR experiments were performed by M.A.P. A.R.O. provided expertise for parasite culture and drug mechanism. High-throughput screening protocols were designed and executed by M.X.G.I. and T.E.K. Data were analyzed by T.E.K. The paper was written by T.E.K. with contributions from all coauthors, and T.E.K. produced the figures. We declare no competing financial interests. This work, including the efforts of Paul W. Hruz, was funded by Washington University Institute of Clinical and Translational Sciences (UL1 TR000448). This work, including the efforts of Audrey R. Odom, was funded by HHS | National Institutes of Health (NIH) (R01 AI103280).
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