title: Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles.
abstract: There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA, USA.
authors.forename: Heather
authors.initials: H
authors.lastname: Ewing
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Virneliz
authors.initials: V
authors.lastname: Fernández-Vega
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Spicer
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Chase
authors.affiliation: The Scripps Research Institute, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Brown
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.affiliation: The Scripps Research Institute, Dept. of Chemistry, Scripps Florida, Jupiter, FL, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Roush
authors.affiliation: The Scripps Research Institute, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Riley
authors.affiliation: The Scripps Research Institute, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Hugh
authors.initials: H
authors.lastname: Rosen
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, UMR7275, Centre National de la Recherche Scientifique et Université de Nice-Sophia-Antipolis, Valbonne, France.
authors.forename: Gerard
authors.initials: G
authors.lastname: Lambeau
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA, USA gelb@chem.washington.edu.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008407:Mast Cells / Q000737:chemistry / Q000378:metabolism; D054497:Phospholipases A2, Secretory / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D054852:Small Molecule Libraries / Q000032:analysis
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010743:Phospholipids; D054852:Small Molecule Libraries; D054497:Phospholipases A2, Secretory
keywords: automation or robotics; chemistry; high-content screening; lipids or lipid metabolism