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: Melanie V Leveridge, Ana Isabel Bardera, William LaMarr, Andrew Billinton, Ben Bellenie, Colin Edge, Peter Francis, Erica Christodoulou, Anthony Shillings, Martin Hibbs, Andrew Fosberry, Rob Tanner, Philip Hardwicke, Peter Craggs, Yugesh Sinha, Oluseyi Elegbe, Emilio Alvarez-Ruiz, Jose Julio Martin-Plaza, Vanessa Barroso-Poveda, Stuart Baddeley, Chun-wa Chung, Jonathan Hutchinson
• Journal: Journal of biomolecular screening (J Biomol Screen) Vol. 17 Issue 5 Pg. 641-50 (Jun 2012) ISSN: 1552-454X [Electronic] United States
• PMID: 22337655 (Publication Type: Journal Article)

Chemical References

• Enzyme Inhibitors
• Fluorescent Dyes
• Cyclooxygenase 2
• Intramolecular Oxidoreductases
• PTGES protein, human
• Prostaglandin-E Synthases

Topics

• Cyclooxygenase 2 (metabolism)
• Dose-Response Relationship, Drug
• Drug Discovery (methods)
• Enzyme Inhibitors (pharmacology)
• Fluorescent Dyes (chemistry, metabolism)
• High-Throughput Screening Assays (methods)
• Humans
• Inhibitory Concentration 50
• Intramolecular Oxidoreductases (antagonists & inhibitors, metabolism)
• Mass Spectrometry (methods)
• Prostaglandin-E Synthases