Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol.

The antiinflammatory agent darbufelone, ((Z)-5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl] methylene]-2-imino-4-thiazolidinone, methanesulfonate salt), was discovered as a dual inhibitor of cellular prostaglandin and leukotriene production. To study the mechanism of action of this drug, we expressed human prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2 and purified the recombinant enzymes using buffers that contain octylglucoside. In cyclooxygenase assays following a 15-min incubation of enzyme with inhibitor, darbufelone potently inhibits PGHS-2 (IC50 = 0.19 μM) but is much less potent with PGHS-1 (IC50 = 20 μM). Interestingly, when the assay buffer contains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS-2 due to a detergent interaction that is detectable by absorption spectroscopy. We therefore used octylglucoside, which does not affect darbufelone in this way, in place of Tween 20 in our PGHS buffers. Inhibition of PGHS-2 with darbufelone is time dependent:  with no pr...