title: HTRF-based assay for microsomal prostaglandin E2 synthase-1 activity.
abstract: Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes the formation of prostaglandin E2 (PGE2) from the endoperoxide prostaglandin H2 (PGH2). Expression of this enzyme is induced during the inflammatory response, and mouse knockout experiments suggest it may be an attractive target for antiarthritic therapies. Assaying the activity of this enzyme in vitro is challenging because of the unstable nature of the PGH2 substrate. Here, the authors present an mPGES-1 activity assay suitable for characterization of enzyme preparations and for determining the potency of inhibitor compounds. This plate-based competition assay uses homogenous time-resolved fluorescence to measure PGE2 produced by the enzyme. The assay is insensitive to DMSO concentration up to 10% and does not require extensive washes after the initial enzyme reaction is concluded, making it a simple and convenient way to assess mPGES-1 inhibition.
authors.affiliation: Abbott Bioresearch Center, Worcester, Massachusetts 01605, USA. eric.goedken@abbott.com
authors.forename: Eric R
authors.initials: ER
authors.lastname: Goedken
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Gagnon
authors.forename: Gary T
authors.initials: GT
authors.lastname: Overmeyer
authors.forename: Junjian
authors.initials: J
authors.lastname: Liu
authors.forename: Richard A
authors.initials: RA
authors.lastname: Petrillo
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Burchat
authors.forename: Medha J
authors.initials: MJ
authors.lastname: Tomlinson
mesh_terms: D000818:Animals; D001168:Arthritis / Q000188:drug therapy; D016367:Baculoviridae / Q000378:metabolism; D001667:Binding, Competitive; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007249:Inflammation; D020128:Inhibitory Concentration 50; D007313:Insecta; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D044262:Prostaglandin H2 / Q000378:metabolism; D000071876:Prostaglandin-E Synthases; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
chemical_list: D010545:Peroxides; D044262:Prostaglandin H2; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; C000605865:Ptges protein, mouse