Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isoforms which accounts for their clinical effects. The differential inhibition of COX-1 and COX-2 is not sufficient to explain the absence of a correlation between in vitro and in vivo effects, especially for 2-aryl-propionates, thus indicating the participation of metabolites. Conjugates to glucuronic acid and to coenzyme-A are mainly produced, and have been shown to be chemically reactive. Therefore, we studied the interaction of the ketoprofen metabolites with the COX enzymes. After incubation with bovine pulmonary artery endothelial cells (BPAEC), COX-1 was inhibited stereoselectively by S-ketoprofen acylglucuronide, and more significantly by CoA-thioester. After washing-out the medium, COX-1 activity was essentially recovered, indicating a reversible inhibition. In LPS-stimulated J774.2 cells, COX activity (mainly inducible COX-2) was inhibited reversibly and stereospecifically by S-ketoprofen glucuronide, whereas it disappeared totally and was not recovered after incubation with CoA-thioester. Correspondingly, inhibition of purified COX-2 with this compound was observed to be rapid and irreversible. Using an anti-ketoprofen antibody, COX immunoprecipitated from cells exhibited adduct formation for COX-2 but not for COX-1. This was observed after incubation with CoA-thioester, and, surprisingly, also with glucuronide. Molecular docking gave support to explain this discrepancy: the glucuronide was found to establish a strong interaction with Y115 located in the membrane binding domain, whereas the thioester was preferentially bound to the active site of the enzyme. Overall, our results suggest a contribution of CoA-thioester metabolites of carboxylic NSAIDs to their pharmacological action by irreversibly and selectively inhibiting COX-2.