title: Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease.
abstract: Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Quan
authors.initials: Q
authors.lastname: Li
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Henry
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: James
authors.initials: J
authors.lastname: Hofrichter
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Smith
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Troy
authors.initials: T
authors.lastname: Cellmer
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Emily B
authors.initials: EB
authors.lastname: Dunkelberger
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Belhu B
authors.initials: BB
authors.lastname: Metaferia
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Stacy
authors.initials: S
authors.lastname: Jones-Straehle
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Sarah
authors.initials: S
authors.lastname: Boutom
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Garrott W
authors.initials: GW
authors.lastname: Christoph
authors.affiliation: Office of the Clinical Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Terri H
authors.initials: TH
authors.lastname: Wakefield
authors.affiliation: Office of the Clinical Director, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Link
authors.affiliation: Office of the Clinical Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Dwayne
authors.initials: D
authors.lastname: Staton
authors.affiliation: Office of the Clinical Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Erica R
authors.initials: ER
authors.lastname: Vass
authors.affiliation: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Miller
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Hsieh
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: John F
authors.initials: JF
authors.lastname: Tisdale
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520; eaton@helix.nih.gov.
authors.forename: William A
authors.initials: WA
authors.lastname: Eaton
mesh_terms: D000755:Anemia, Sickle Cell / Q000628:therapy; D000986:Antisickling Agents / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects*; D004912:Erythrocytes / Q000187:drug effects* / Q000502:physiology; D005662:Furaldehyde / Q000031:analogs & derivatives* / Q000494:pharmacology; D006451:Hemoglobin, Sickle / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010100:Oxygen
chemical_list: D000986:Antisickling Agents; D006451:Hemoglobin, Sickle; C008046:5-hydroxymethylfurfural; D005662:Furaldehyde; D010100:Oxygen
keywords: drugs; hemoglobin S; screening assay; sickle cell; treatment