Lead Discovery for Microsomal Prostaglandin E Synthase Using a Combination of High-Throughput Fluorescent-Based Assays and RapidFire Mass Spectrometry

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 (PGH2) and the challenge of detection of the product (PGE2). A coupled fluorescent assay is described for mPGES-1where PGH2 is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE2 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 PGE2 and a reduced form of the unreacted substrate were detected by mass spectrometry....