title: A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.
abstract: 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.
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA thomas.e.kraft@gmail.com hruz_p@kids.wustl.edu.
authors.forename: Thomas E
authors.identifier: 0000-0002-5528-3197
authors.initials: TE
authors.lastname: Kraft
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Monique R
authors.initials: MR
authors.lastname: Heitmeier
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Marina
authors.initials: M
authors.lastname: Putanko
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Edwards
authors.affiliation: High Throughput Screening Core, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Ma Xenia G
authors.initials: MX
authors.lastname: Ilagan
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Payne
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Autry
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.|Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Audrey R
authors.initials: AR
authors.lastname: Odom
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA thomas.e.kraft@gmail.com hruz_p@kids.wustl.edu.|Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Hruz
mesh_terms: D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism / Q000469:parasitology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015870:Gene Expression; D005947:Glucose / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000235:genetics / Q000378:metabolism; D051273:Glucose Transporter Type 2 / Q000235:genetics / Q000378:metabolism; D051274:Glucose Transporter Type 3 / Q000235:genetics / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014316:Tritium
chemical_list: D000962:Antimalarials; D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D015800:Protozoan Proteins; C495677:SLC2A1 protein, human; C495680:SLC2A2 protein, human; C495687:SLC2A3 protein, human; C495692:SLC2A4 protein, human; D054852:Small Molecule Libraries; C118145:hexose transporter 1 protein, Plasmodium falciparum; D014316:Tritium; D005947:Glucose