A simplified direct O2 consumption-based assay to test COX inhibition

Abstract

Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA) producing the hydroperoxide PGG2, then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions are a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors. Methods: we developed a new simplified and easy to be carried out protocol for O2 consumption measurement by using disrupted HEK293-derived adherent cells, stably transfected either with COX-1 or COX-2 genes, as a source of the COX enzymes. The Clark electrode is used to measure the O2 concentration variation during the enzyme-catalyzed reactions. Results and Discussion: the novel assay was validated by determining the IC50 values of the known inhibitors such as indomethacin, ibuprofen, SC560 and celecoxib. Indomethacin and ibuprofen are two traditional non-steroidal anti-inflammatory drugs (NSAIDs). SC560 is a commercially available reference compound used for COX-1 inhibition investigations. Celecoxib is a clinically used COXIBs. The assay was also applied to measure the kinetics and IC50 of mofezolac and P6. Mofezolac is the most potent selective COX-1 inhibitor and active principal ingredient of Disopain® used to treat the rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as reference in “in vitro” and “in vivo” COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies. The obtained results showed the suitability of the newly developed assay to measure COXs activity in the presence of inhibitors as well as the kinetics of the inhibition (i.e., Vmax and Km).