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 leukotr iene production. To study the mechanism of action of this drug, we expresse d human prostaglandin endoperoxide synthase-l (PGHS-1) and PGHS-2 and purif ied the recombinant enzymes using buffers that contain octylglucoside. In c yclooxygenase assays following a 15-min incubation of enzyme with inhibitor , darbufelone potently inhibits PGHS-2 (IC50 = 0.19 muM) but is much less p otent with PGHS-1 (IC50 = 20 muM). Interestingly, when the assay buffer con tains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS- 2 due to a detergent interaction that is detectable by absorption spectrosc opy. 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 w ith darbufelone is time dependent: with no preincubation, darbufelone is a weak inhibitor (IC50 = 14 muM), but after a 30-min incubation it is 20-fold more potent. Plots of PGHS-2 activity vs preincubation time at various dar bufelone concentrations reach a plateau. This finding is inconsistent with irreversible or one step slow-binding inhibition. A two-step slow-binding i nhibition model is proposed in which the E.I complex (K-i = 6.2 +/- 1.9 to 14 +/- muM) slowly transforms (k(5) = 0.015-0.030 s(-1)) to a tightly bound E*.I form with K*(i) = 0.63 +/- 0.07 muM and k(6) = 0.0034 s(-1). In stead y state kinetics inhibition experiments performed with no preincubation, we find that darbufelone isa noncompetitive inhibitor of PGHS-2 (K-i = 10 +/- 5 muM). Darbufelone quenches the fluorescence of PGHS-2 at 325 nm (lambda (ex) = 280 nm) with K-d = 0.98 +/- 0 03 muM. The PGHS substrate, arachidona te, and various cyclooxygenase inhibitors do not alter this binding affinit y of darbufelone but a structural analogue of darbufelone competes directly for binding to PGHS-2. Di-tert-butyl phenols such as darbufelone may inhib it PGHS-2 by exploiting a previously unrecognized binding site on the enzym e.