Mammalian brain as well as mouse
neuroblastoma (N18TG2) and rat basophilic leukaemia (RBL) cells were previously shown to contain "
anandamide amidohydrolase', a membrane-bound
enzyme sensitive to
serine and
cysteine protease inhibitors and catalyzing the hydrolysis of the endogenous cannabimimetic metabolite,
anandamide (
arachidonoyl-ethanolamide). With the aim of developing novel inhibitors of this
enzyme, we synthesized three
arachidonic acid (AA) analogues, i.e. arachidonoyl-diazo-methyl-
ketone (ADMK), ara-chidonoyl-chloro-methyl-
ketone (ACMK) and
O-acetyl-arachidonoyl-hydroxamate (AcAHA), by adding to the
fatty acid moiety three functional groups previously used to synthesize irreversible inhibitors of
serine and
cysteine proteases. The three compounds were purified and characterized by
proton nuclear magnetic resonance and electron impact mass spectrometry. Their effect was tested on
anandamide amidohydrolase partially purified from N18TG2 and RBL-1 cells and porcine brain. Pre-treatment of the
enzyme with each compound produced a significant inhibition, with ADMK being the most potent (IC50 = 3, 2 and 6 microM) and AcAHA the weakest (IC50 = 34, 15 and 25 microM) inhibitors. The inactivated
enzyme regained its full activity when chromatographed by
anion-exchange chromatography, suggesting that none of the compounds inhibited the
amidohydrolase in a covalent manner. Accordingly, Lineweaver-Burk profiles showed competitive inhibition by each compound. Conversely, the irreversible inhibitor of cytosolic
phospholipase As, methyl-arachidonoyl-fluoro-
phosphonate (MAFP), covalently inhibited the
amidohydrolase. MAFP was active at concentrations 10(3) times lower than those reported for
phospholipase A2 inhibition, and is the most potent
anandamide amidohydrolase inhibitor so far described (IC50 = 1-3 nM). MAFP, ADMK and ACMK, probably by inhibiting
anandamide degradation, produced an apparent increase of the in vitro formation of
anandamide from its biosynthetic precursor N-arachidonoyl-phosphatidyl-
ethanolamine.