Arachidonoylserotonin and Other Novel Inhibitors of Fatty Acid Amide Hydrolase T. Bisogno,*?l D. Melck,*" L. De Petrocellis,t M. Yu. Bobrov,$ N. M. Gretskaya,$ V. V. Bezuglov,$ N. Sitachitta,§ W. H. Gerwick,§ and V. Di Mar~o*."~ *Istituto per la Chimica di Molecole di Znteresse Biologico and tlstituto di Cibernetica, C.N.R., Via Toiano 6, 80072, Arco Felice, Napoli, Italy; $Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia; and §College of Pharmacy, Oregon State University, Coruallis, Oregon Received May 25, 1998 Fatty acid amide hydrolase (FAAH)catalyzes the hy- drolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, an- andamide (N-arachidonoyl-ethanolamine) and 2-ara- chidonoylglycerol,and the putative sleep inducing fac- tor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phos- pholipase Az (cPLA2)andfor bind to the CB1 canna- binoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamen- silipin A and grenadadiene, were screened out of a se- ries of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG)and ar- achidonoyl-serotonin (AA-5-HT)were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the ICSOs for these compounds ranged from 12.0 to 26 pM, the most active compound being AA-5- HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (ICSO=5.6 pM), and inhibited [14Clanandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a com- petitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resis- tant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HTdid not interfere with cPLA2-mediated, iono- Affiliated with the National Institute for the Chemistry of Biologi- cal Systems, C.N.R. Author to whom all correspondence should be addressed. Fax: +39-81-8041770, E-mail: vdm@trinc.icmib.na.cnr.it. Abbreviations: FAAH, fatty acid amide hydrolase; AEG, arachido- noylethylene glycol; AA-5-HT, arachidonoylserotonin; cPLA2,sPLA, , cytosolic and secretory phospholipase A2; AA, arachidonic acid; MAFP, methylarachidonoylfluorophosphonate; ATFMK, arachido- noyl-trifluoromethylketone; RBL-2H3, rat basophilic leukaemia; ADMK, arachidonoyl-diazomethylketone. mycin or antigen-inducedrelease of [3H]AA from RBL- 2H3 cells, nor with cPLAz activity in cell-free experi- ments. Finally, AA-5-HT did not activate CB1 canna- binoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mglkg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Con- versely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cP& and at CB1 receptors, whereas AEG rep- resents a new type of cannabinoid receptor agonist. c? 1998 Academic Press Key Words: cannabinoids;cannabinoid receptors; an- andamide; fatty acidamide hydrolase; 2-arachidonoyl- glycerol; serotonin; polyunsaturated fatty acids; olea- mide. Recent evidence suggests that three distinct types of bioactive long chain fatty acid derivatives, i.e., the N- acylethanolamines (I), the unsaturated monoacylglyc- erols (2-4) and the fatty acid primary amides (5,6),may be degraded through the action of the same hydrolytic enzyme. A novel 'hydrolase' was cloned from rat liver in 1996 (7) and shown to recognize as preferential sub- strates the endogenous cannabimimetic metabolite N- arachidonoylethanolamine (anandamide [81) and the sleep-inducing cis-9-octadecenoamide (oleamide 151). These observations led to the proposed name of this enzyme as 'fatty acid amide hydrolase' (FAAH). The rat tissue distribution (9, 11)' substrate specificity and catalytic properties (7, 10) of FAAH strongly suggest that this enzyme is the same protein-originally named 'anandamide amidohydro1ase'-that had been previously described to catalyze anandamide hydroly- sis in mammalian brain (12-141, and to recognize also oleamide (15). More recently, recombinant FAAH was 0006-291W98 $25.00 Copyright 0 1998 by Academic Preas All rights of reproduction in any form reserved.