An automated multistep high-throughput screening assay for the identification of lead inhibitors of the inducible enzyme mPGES-1 |
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Authors: | Massé Frédéric Guiral Sébastien Fortin Louis-Jacques Cauchon Elizabeth Ethier Diane Guay Jocelyne Brideau Christine |
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Institution: | Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada. frederic_masse@merck.com |
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Abstract: | Prostaglandin E2 synthase (mPGES-1), the enzyme which catalyzes the synthesis of PGE2, is induced during the inflammatory response. For this reason, mPGES-1 could be a potential therapeutic target. A high-throughput screening assay was developed to identify potential inhibitors of mPGES-1. The assay consisted of a 30-s mPGES-1 enzymatic reaction followed by the detection of PGE2 by enzyme immunoassay (EIA). The enzymatic reaction was performed in a batch mode because the instability of the substrate (10 min) limited the number of plates assayed within a working day. The detection of the product by EIA was performed on 3 instruments requiring 14 different steps for complete automation. The authors describe here the optimization and implementation of a 2-part assay on a Thermo CRS robotic system. More than 315,000 compounds were tested, and a hit rate of 0.84% was obtained for this assay. Although the entire assay required multiple steps, the assay was successfully miniaturized and automated for a high-throughput screening campaign. |
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