Lead discovery for microsomal prostaglandin E synthase using a combination of high-throughput fluorescent-based assays and RapidFire mass spectrometry

Journal of Biomolecular Screening
Melanie V LeveridgeJonathan Hutchinson

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 ...Continue Reading

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Citations

Aug 15, 2014·Metabolites·Shunji TomatsuTadao Orii
Jun 22, 2013·Journal of Basic Microbiology·Alejandro Miguel Figueroa-LópezIgnacio Eduardo Maldonado-Mendoza
Jan 23, 2016·Journal of Medicinal Chemistry·Andreas KoeberleOliver Werz
Oct 3, 2015·Journal of Biomolecular Screening·Carl HaslamMelanie Leveridge
Jun 20, 2017·Expert Opinion on Therapeutic Patents·Anastasia PsarraGeorge Kokotos
Aug 17, 2012·Journal of Biomolecular Screening·Susanne AnderssonJohan Nord
Jan 2, 2014·Journal of Biomolecular Screening·Denise M LoweJonathan P Hutchinson
Aug 29, 2013·Journal of Biomolecular Screening·Rejbinder KaurSteven B Ludbrook

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Methods Mentioned

BETA
Assay

Software Mentioned

RapidFire

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