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P14004
Antagonizing mPGES-1  Developing a High-Throughput Screening Assay
Presenter Charles Lunn, Schering-Plough Research Institute, USA
Additional Authors: ArtJohn Villafania, Steven Cifelli, Carol Terminelli, Mary Petro, Frederick Monsma, Chuan-Chu Chou

We have optimized a high-throughput screening protocol to identify inhibitors of mPGES-1. We developed an automation and scheduling strategy to circumvent the instability of mPGES-1 substrate PGH2, known to spontaneously hydrolyze to the desired product PGE2 in aqueous solution. Using a Velocity11 BioCel System, incubations were initiated by adding 5 ìl PGH2 substrate (freshly diluted from frozen stocks) with a Thermo Multidrop Combi. After 60 seconds at room temperature, 5ìL of the SnCl2 Stop Reagent is added using a second Multidrop Combi. Diluted reaction mixtures were analyzed for PGE2 by competitive immunoassay using a homogeneous time resolved fluorescence assay kit (CisBio International). This process allowed screening in 10-12 plate batches (before fresh ligand dilution). We successfully used this process to screen a validation library of 48 plates with a median z’-factor of 0.65.