PGHS-2 inhibitors, NS-398 and DuP-697, attenuate the inhibition of PGHS-1 by aspirin and indomethacin without altering its activity Moti Rosenstock, Abraham Danon, Gilad Rimon * Department of Clinical Pharmacology, The Corob Center for Health Sciences, Ben-Gurion University and Soroka Medical Center, P.O. Box 653, Beer-Sheva 84105, Israel Received 2 March 1999; received in revised form 21 May 1999; accepted 2 June 1999 Abstract Since the discovery of the inducible form of prostaglandin (PG) H synthase (PGHS), PGHS-2, considerable effort has been made to design selective inhibitors of this isozyme. N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5- bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) have been shown to interact reversibly with PGHS-1, while irreversibly inhibiting PGHS-2 in a time-dependent manner. In the present study we have tested the effects of DuP-697 and NS-398 on the activity of PGHS-1 and further explored the interactions between these agents and the inhibition of PGHS-1 by aspirin, indomethacin and ibuprofen. Three independent experimental systems, namely bovine aortic endothelial cells (BAEC), human fibroblasts and ram seminal vesicle microsomes were used to investigate the effects of DuP-697 and NS- 398 on PGHS-1. The results show that DuP-697 and NS-398, at concentrations ranges which do not inhibit PGHS-1 activity, significantly attenuated the inhibition of PGHS-1 that was caused by aspirin and indomethacin. The same concentrations of DuP-697 and NS-398 did not affect the inhibition of PGHS-1 that was induced by the competitive reversible inhibitors ibuprofen and naproxen. Similar effects of DuP-697 and NS-393 were obtained with ram seminal vesicle microsomes. These results suggest that PGHS-2 inhibitors DuP-697 and NS-398 possibly interact with PGHS-1 at a site different from the enzyme's catalytic site, thus causing attenuation of PGHS-1 inhibition by aspirin and indomethacin without altering PGHS-1 basal activity or the ibuprofen-induced inhibition. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : DuP-697; NS-398; Aspirin; Indomethacin; Ibuprofen; Naproxen; PGHS-1; PGHS-2; Endothelial cell; Human ¢broblast; Ram seminal vesicle microsome 1. Introduction It is well established that two distinct isoforms of prostaglandin (PG) H synthase (PGHS) are involved in the catalytic conversion of arachidonic acid (AA) to PGH 2 , the committed step in PG biosynthesis [1,2]. The expression of these two isoforms is regu- lated di¡erently: PGHS-1 has been observed in a variety of prostanoid-producing cells to be constitu- tively expressed, while PGHS-2 is inducible in re- sponse to agents such as growth factors, cytokines and lipopolysaccharide (LPS) [3]. This di¡erence has suggested that PGHS-1 participates mainly in housekeeping processes, while PGHS-2 is involved mainly in di¡erentiative and in£ammatory responses. Non-steroidal antiin£ammatory drugs (NSAIDs) ex- 1388-1981 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S1388-1981(99)00105-5 * Corresponding author. Fax: +972-7-6477629; E-mail : grimon@bgumail.bgu.ac.il Biochimica et Biophysica Acta 1440 (1999) 127^137 www.elsevier.com/locate/bba