An automated multistep high-throughput screening assay for the identification of lead inhibitors of the inducible enzyme mPGES-1.
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.
Massé
Frédéric
F
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada. frederic_masse@merck.com
Guiral
Sébastien
S
Fortin
Louis-Jacques
LJ
Cauchon
Elizabeth
E
Ethier
Diane
D
Guay
Jocelyne
J
Brideau
Christine
C
D000818:Animals; D001331:Automation; D002417:Cattle; D002626:Chemistry, Pharmaceutical / Q000379:methods; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004345:Drug Industry / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors*; D000071876:Prostaglandin-E Synthases; D012709:Serum Albumin / Q000378:metabolism; D013997:Time Factors
D012709:Serum Albumin; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases