Format

Send to

Choose Destination
See comment in PubMed Commons below
Antimicrob Agents Chemother. 2016 Nov 21;60(12):7407-7414. Print 2016 Dec.

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.

Author information

  • 1Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA thomas.e.kraft@gmail.com hruz_p@kids.wustl.edu.
  • 2Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
  • 3High Throughput Screening Core, Washington University School of Medicine, St. Louis, Missouri, USA.
  • 4Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
  • 5Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • 6Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.

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.

PMID:
27736766
PMCID:
PMC5119023
[Available on 2017-05-21]
DOI:
10.1128/AAC.00218-16
[PubMed - in process]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Support Center