Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase.
The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.
Gierse
J K
JK
Searle Inflammatory Disease Research, Monsanto Company, St. Louis, MO 63167.
Hauser
S D
SD
Creely
D P
DP
Koboldt
C
C
Rangwala
S H
SH
Isakson
P C
PC
Seibert
K
K
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D009578:Nitrobenzenes / Q000494:pharmacology; D010101:Oxygen Consumption; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013449:Sulfonamides / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D013997:Time Factors
D016861:Cyclooxygenase Inhibitors; D018076:DNA, Complementary; D009578:Nitrobenzenes; D011994:Recombinant Proteins; D013449:Sulfonamides; D013876:Thiophenes; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D016718:Arachidonic Acid; C064874:DuP 697; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin