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A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.
- Source :
-
Antimicrobial agents and chemotherapy [Antimicrob Agents Chemother] 2016 Nov 21; Vol. 60 (12), pp. 7407-7414. Date of Electronic Publication: 2016 Nov 21 (Print Publication: 2016). - Publication Year :
- 2016
-
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 (IC <subscript>50</subscript> ) 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.<br /> (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Subjects :
- Antimalarials chemistry
Cells, Cultured
Erythrocytes drug effects
Erythrocytes metabolism
Erythrocytes parasitology
Gene Expression
Glucose metabolism
Glucose Transporter Type 1 genetics
Glucose Transporter Type 1 metabolism
Glucose Transporter Type 2 genetics
Glucose Transporter Type 2 metabolism
Glucose Transporter Type 3 genetics
Glucose Transporter Type 3 metabolism
Glucose Transporter Type 4 genetics
Glucose Transporter Type 4 metabolism
HEK293 Cells
Humans
Monosaccharide Transport Proteins genetics
Monosaccharide Transport Proteins metabolism
Plasmodium falciparum growth & development
Plasmodium falciparum metabolism
Protozoan Proteins genetics
Protozoan Proteins metabolism
Small Molecule Libraries chemistry
Species Specificity
Structure-Activity Relationship
Tritium
Antimalarials pharmacology
Fluorescence Resonance Energy Transfer methods
Glucose antagonists & inhibitors
High-Throughput Screening Assays
Monosaccharide Transport Proteins antagonists & inhibitors
Plasmodium falciparum drug effects
Protozoan Proteins antagonists & inhibitors
Small Molecule Libraries pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1098-6596
- Volume :
- 60
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Antimicrobial agents and chemotherapy
- Publication Type :
- Academic Journal
- Accession number :
- 27736766
- Full Text :
- https://doi.org/10.1128/AAC.00218-16