Isoform-selective Inhibition of Facilitative Glucose Transporters

Hresko, Richard C.; Kraft, Thomas E.; Tzekov, Anatoly; Wildman, Scott A.; Hruz, Paul W.

doi:10.1074/jbc.m113.528430

Cited by 15 publications

(18 citation statements)

References 41 publications

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“…1a). We previously determined the inhibitory effects of several HIV protease inhibitors on human glucose transport (11,13,26). Since HIV PIs inhibit human glucose transport, we evaluated whether lopinavir might inhibit glucose uptake in isolated P. falciparum parasites.…”

Section: Resultsmentioning

confidence: 99%

“…Lopinavir and compound 3361 were added 5 min prior to the addition of [ 14 C]2DOG (final concentration, 0.2 Ci/ml). Uptake of [ 3 H]2DOG into HEK293 cells was measured in phosphate buffer at 37°C for 5 min as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

“…We compared the PfHT-lopinavir putative binding site with the binding site of the structurally related protease inhibitor indinavir in the PfHT homologue GLUT4, published by Hresko et al (Fig. 6b) (26). The docking experiments suggest that both indinavir and lopinavir bind to a structurally similar binding pocket in their respective glucose transporters.…”

Section: Fig 4 Specific Activity Of Glucose Uptake In Hek293 Cells Stmentioning

confidence: 99%

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The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir

Kraft

Armstrong

Heitmeier

et al. 2015

Antimicrob Agents Chemother

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Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of Plasmodium species to existing therapies has increased the need for new therapeutic approaches. The Plasmodium glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Fig 4 Specific Activity Of Glucose Uptake In Hek293 Cells Stmentioning

confidence: 99%

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The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir

Kraft

Armstrong

Heitmeier

et al. 2015

Antimicrob Agents Chemother

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“…For the mammalian glucose transporters, recent progress has been made in solving the crystal structures of several isoforms in both inward and outward conformations (28)(29)(30). Furthermore, our prior work has provided evidence for the binding pocket that mediates transporter inhibition by HIV protease inhibitors (12,20,31). This has been used to model lopinavir binding to a model of PfHT (12).…”

Section: Discussionmentioning

confidence: 99%

“…Data were fit by nonlinear regression analysis using GraphPad Prism software. Uptake of [ 3 H]2-DOG into HEK293-FLIP cells was measured in HEPESbuffered saline at room temperature for 4 min as described previously (20). Data were fit by nonlinear regression analysis using GraphPad Prism software.…”

Section: Materials [mentioning

confidence: 99%

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

Kraft

Heitmeier

Putanko

et al. 2016

Antimicrob Agents Chemother

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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 50 ) 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. Malaria is a major threat to the human population in large areas of the world, affecting over 200 million people per year (1, 2). Beyond the effects of this disease on human life, malaria also cripples economic development and burdens the health care systems of countries where malaria is endemic (3). The emergence of parasites with resistance to even the most potent existing antimalarial drugs, such as the artemisinins (4), has made paramount the development of novel drugs that target essential pathways for parasite survival. Blood stage parasites utilize glucose for both biomass production and ATP synthesis (5). The malarial hexose transporter, Plasmodium falciparum hexose transporter (PfHT), first cloned by Woodrow et al. in 1999 (6), mediates parasite transport of glucose, fructose, mannose, and galactose (7). Since PfHT is essential for parasite survival (8), this protein is a highly promising molecular target for antimalarial drug development. This is supported by the ability of compound 3361, a glucose analogue that inhibits PfHT with high selectivity over the human orthologue GLUT1, to inhibit asexual intraerythrocytic growth in culture (9). Compound 3361 is also active against Plasmodium berghei liver and transmission stage parasites in infected mice (10), suggesting that PfHT is a promising target for full life cycle activity. However, while compound 3361 validates efforts to target PfHT, this compound is not itself considered drug-like and is therefore not a valid candidate for lead development (11). We have recently extended these earlier find...

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Expression, purification, and functional characterization of the insulin‐responsive facilitative glucose transporter GLUT4

2015

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The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses.

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Isoform-selective Inhibition of Facilitative Glucose Transporters

Cited by 15 publications

References 41 publications

The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir

The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir

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

Expression, purification, and functional characterization of the insulin‐responsive facilitative glucose transporter GLUT4

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