Detergents profoundly affect inhibitor potencies against both cyclo-oxygenase isoforms

Abstract

The sensitivity of Coxs (cyclo-oxygenases) to inhibition is known to be highly dependent on assay conditions. In the present study, the inhibitor sensitivities of purified Cox-1 and -2 were determined in a colorimetric assay using the reducing agent N, N, N ', N '-tetramethyl- p -phenylenediamine. With the detergent genapol X-100 (2 mM) present, the potencies of nimesulide, ibuprofen, flufenamic acid, niflumic acid and naproxen were increased over 100-fold against Cox-2 and titration curve shapes changed, so that maximal inhibition now approached 100%. Indomethacin, diclofenac and flosulide were not changed in potency. Similar effects of genapol were observed with inhibitors of Cox-1. DuP-697 and two analogues became more than 10-fold less potent against Cox-2 with genapol present. Tween-20, Triton X-100 and phosphatidylcholine, but not octylglucoside, gave qualitatively similar effects as genapol. Similar detergent-dependent changes in inhibitor potency were also observed using a [(14)C]arachidonic acid HPLC assay. The increases in potency of ibuprofen, flufenamic acid, isoxicam and niflumic acid towards Cox-2 and ibuprofen towards Cox-1 were accompanied by a change from time-independent to time-dependent inhibition. The interactions of Cox inhibitors has been described in terms of multiple binding step mechanisms. The genapol-dependent increase in inhibitor potency for ketoprofen was associated with an increase in the rate constant for the conversion of the initial enzyme-inhibitor complex to a second, more tightly bound form. The loss of potency for some inhibitors is probably due to inhibitor partitioning into detergent micelles. The present study identifies detergents as another factor that must be considered when determining inhibitor potencies against both Cox isoforms.

Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

“…The effects of the detergents on the inhibitors' potency were complex and were not interpreted in terms of a unified mechanism. The PC was reported to increase the inhibitory potency of ibuprofen and flufenamic acid, fitting the pattern observed with several nonionic detergents (7). The present results with structurally defined DOPC liposomes and a simple, reversible competitive inhibitor (ibuprofen) showed roughly 5-fold increases in the inhibitor's potency against both COX-1 and -2 (Fig.…”

“…This interpretation envisages that, unlike the case of ibuprofen, some inhibitors may actually partition into phospholipid bilayers more effectively than arachidonate and thus have their potency decreased. This provides a potential explanation for the otherwise puzzling observation that detergent micelles decreased the potency of some COX inhibitors (7). There are also kinetic considerations.…”

“…PGG 2 has been found to channel between the cyclooxygenase and peroxidase active sites in microsomal PGHS-1 but not in the detergent-solubilized protein (5). Further, detergent micelles, which mimic some aspects of phospholipid membranes, are known to markedly alter the effectiveness of several COX inhibitors (6,7), although the large variation between detergents made mechanistic interpretation difficult. PC was reported to partially inactivate COX-2 and to increase the potency of two COX inhibitors (7), but the structure of the PC was not described and the details of the mechanism were not investigated.…”

“…Further, detergent micelles, which mimic some aspects of phospholipid membranes, are known to markedly alter the effectiveness of several COX inhibitors (6,7), although the large variation between detergents made mechanistic interpretation difficult. PC was reported to partially inactivate COX-2 and to increase the potency of two COX inhibitors (7), but the structure of the PC was not described and the details of the mechanism were not investigated. In the present study, we used purified enzymes to study the effects of PC molecules of varying chain length and saturation on the catalytic properties of both COX isoforms.…”

New Evidence of H₁-Receptor Independent COX-2 Inhibition by Fexofenadine HCl in vitro

“…In contrast, LOR inhibited ovine COX-1 activity at 10 -8 mol/l but had little inhibitory effect on COX-1 at high concentrations. Although the effects of the reference compound DuP-679 were not investigated on ovine COX-1 in this study, it has previously been shown to inhibit COX-1 at high doses [19] . Following the studies on ovine COX-1 and COX-2 enzymes with FEX, isolated human COX-2 became available for testing.…”