title: Lead discovery for microsomal prostaglandin E synthase using a combination of high-throughput fluorescent-based assays and RapidFire mass spectrometry.
abstract: Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.
authors.affiliation: Department of Screening, GlaxoSmithKline, Stevenage, UK.
authors.forename: Melanie V
authors.initials: MV
authors.lastname: Leveridge
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Bardera
authors.forename: William
authors.initials: W
authors.lastname: LaMarr
authors.forename: Andrew
authors.initials: A
authors.lastname: Billinton
authors.forename: Ben
authors.initials: B
authors.lastname: Bellenie
authors.forename: Colin
authors.initials: C
authors.lastname: Edge
authors.forename: Peter
authors.initials: P
authors.lastname: Francis
authors.forename: Erica
authors.initials: E
authors.lastname: Christodoulou
authors.forename: Anthony
authors.initials: A
authors.lastname: Shillings
authors.forename: Martin
authors.initials: M
authors.lastname: Hibbs
authors.forename: Andrew
authors.initials: A
authors.lastname: Fosberry
authors.forename: Rob
authors.initials: R
authors.lastname: Tanner
authors.forename: Philip
authors.initials: P
authors.lastname: Hardwicke
authors.forename: Peter
authors.initials: P
authors.lastname: Craggs
authors.forename: Yugesh
authors.initials: Y
authors.lastname: Sinha
authors.forename: Oluseyi
authors.initials: O
authors.lastname: Elegbe
authors.forename: Emilio
authors.initials: E
authors.lastname: Alvarez-Ruiz
authors.forename: Jose Julio
authors.initials: JJ
authors.lastname: Martin-Plaza
authors.forename: Vanessa
authors.initials: V
authors.lastname: Barroso-Poveda
authors.forename: Stuart
authors.initials: S
authors.lastname: Baddeley
authors.forename: Chun-wa
authors.initials: CW
authors.lastname: Chung
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hutchinson
mesh_terms: D051546:Cyclooxygenase 2 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D000071876:Prostaglandin-E Synthases
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D051546:Cyclooxygenase 2; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases