High-precision, room temperature screening assay for inhibitors of microsomal prostaglandin E synthase-1.
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' > 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.


BACKGROUND
mPGES-1 (microsomal prostaglandin E synthase-1); FRET (fluorescence resonance energy transfer); HTRF (homogeneous time-resolved fluorescence); PGH2 (prostaglandin H2); PGE2 (prostaglandin E2); SAR (structure-activity relationship); COX-2 (cyclooxygenase-2); GSH (glutathione); ALP (automated labware positioner).
AstraZeneca, CNS/Pain iMED, Department of Neuroscience, Södertälje, Sweden.
Susanne
S
Andersson
Mattias
M
Norman
Rolf
R
Olsson
Robin
R
Smith
Gang
G
Liu
Johan
J
Nord
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D000071876:Prostaglandin-E Synthases; D015203:Reproducibility of Results; D012371:Robotics; D013696:Temperature
D004791:Enzyme Inhibitors; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases