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Microsomal prostaglandin E synthase-1 (mPGES-1) is the major enzyme catalyzing the isomerization of prostaglandin (PG) H(2) to PGE(2). Here we report the development of a robust and practical automated assay in a 384-well format for room temperature screening of mPGES-1 inhibitors with high precision and low reagent consumption. The assay should enable precise structure-activity relationship development. It uses acetonitrile as solvent for PGH(2), FeCl(2)/citrate as stop reagent, and a short reaction time. Combined with high-precision liquid transfer and extensive mixing after addition of reactants, these properties let the assay reach Z' &gt; 0.7 and high reproducibility of inhibitor IC(50) values. Thorough investigation of the quality of mixing in all liquid transfer steps proved crucial for reaching high-precision performance.

High-Precision, Room Temperature Screening Assay for Inhibitors of Microsomal Prostaglandin E Synthase-1