Selective inhibition of cyclooxygenase-1 and -2 using intact insect cell assays.
We have utilized the baculovirus expression system to develop an in vitro intact cell assay for screening nonsteroidal anti-inflammatory drug (NSAID) inhibition of the two isozymes of human cyclooxygenase (prostaglandin endoperoxidase synthase, EC 1.14.99.1). Infected Spodoptera frugiperda (sf9) cells expressing either human cyclooxygenase-1 (hCOX-1) or human cyclooxygenase-2 (hCOX-2) were harvested 24 hr postinfection, a time point where all cells are viable and hCOX-1 or hCOX-2 are correctly processed. Cells were distributed to a 96-well plate, preincubated with various NSAIDs, and challenged with 10 microM arachidonic acid; then cyclooxygenase activity was assessed indirectly by prostaglandin E2-specific radioimmunoassay. The rank order of potency of NSAID-mediated inhibitions of hCOX-1 and hCOX-2 paralleled those that have been observed in other cell systems. This sf9 cell-based assay can be utilized for the identification of potent and selective inhibitors of hCOX-1 and/or hCOX-2. Compounds that preferentially inhibit hCOX-2 may provide novel NSAIDs that reduce inflammation while sparing the stomach and kidneys of toxic side-effects seen with current nonselective NSAIDs.
Cromlish
W A
WA
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada Inc., Point Claire-Dorval, Québec, Canada.
Kennedy
B P
BP
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D002478:Cells, Cultured; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D011451:Prostaglandin-Endoperoxide Synthases