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An Automated Multistep High-Throughput Screening Assay for the Identification of Lead Inhibitors of the Inducible Enzyme mPGES-1

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

Sébastien Guiral

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

Louis-Jacques Fortin

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

Elizabeth Cauchon

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

Diane Ethier

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

Jocelyne Guay

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

Christine Brideau

Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada

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.

Key Words: high-throughput screening • prostaglandin E2 synthase • enzyme immunoassay

This version was published on September 1, 2005

Journal of Biomolecular Screening, Vol. 10, No. 6, 599-605 (2005)
DOI: 10.1177/1087057105276083


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