title: Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.
abstract: 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.
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
authors.forename: Mindy I
authors.initials: MI
authors.lastname: Davis
authors.affiliation: Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Patrick
authors.affiliation: Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA.
authors.forename: Walker M
authors.initials: WM
authors.lastname: Blanding
authors.affiliation: Departments of Microbial Infection and Immunity and Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Varun
authors.initials: V
authors.lastname: Dwivedi
authors.affiliation: Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Suryadi
authors.affiliation: School of Pharmacy, Department of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Jennifer E
authors.identifier: 0000-0002-6813-3710
authors.initials: JE
authors.lastname: Golden
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Coussens
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
authors.forename: Olivia W
authors.initials: OW
authors.lastname: Lee
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Shen
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Boxer
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Hall
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Sharlow
authors.affiliation: Departments of Microbial Infection and Immunity and Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Drew
authors.affiliation: Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA jmorri2@clemson.edu.
authors.forename: James C
authors.initials: JC
authors.lastname: Morris
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects / Q000469:parasitology; D015870:Gene Expression; D017930:Genes, Reporter; D006019:Glycolysis / Q000187:drug effects; D057809:HEK293 Cells; D006367:HeLa Cells; D006593:Hexokinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D000962:Antimalarials; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D054852:Small Molecule Libraries; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008156:Luciferases; D006593:Hexokinase