High-precision, room temperature screening assay for inhibitors of microsomal prostaglandin e synthase-1

Abstract

Microsomal prostaglandin E synthase-1 (mPGES-1) is the major enzyme catalyzing the isomerization of prostaglandin (PG) H2 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 PGH2, FeCl2/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 Ź; > 0.7 and high reproducibility of inhibitor IC50 values. Thorough investigation of the quality of mixing in all liquid transfer steps proved crucial for reaching high-precision performance. © 2012 Society for Laboratory Automation and Screening.