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Antimicrob Agents Chemother. 2016 Sep 23;60(10):6023-33. doi: 10.1128/AAC.00914-16. Print 2016 Oct.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

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National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA.
Departments of Microbial Infection and Immunity and Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio, USA.
School of Pharmacy, Department of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.
Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA


Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.

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