Methyl arachidonyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase

Abstract

Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonoyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF 3), have been described (Koutek et al., J Biol Chem 269: 22937–22940, 1994). In the current study, methyl arachidonyl fluorophosphonate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB 1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF 3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approximately 1000-fold more potent than Arach-CF 3. MAFP demonstrated selectivity towards anandamide amidase for which it was approximately 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, respectively. MAFP displaced [ 3H]CP-55940 binding to the CB 1 cannabinoid receptor with an IC 50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [ 3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addition, can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonoyl ethanolamide amidase.