Novel inhibitors of brain, neuronal, and basophilic anandamide amidohydrolase.
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.
De Petrocellis
L
L
Istituto di Cibernetica, C.N.R., Arco Felice (NA), Italy.
Melck
D
D
Ueda
N
N
Maurelli
S
S
Kurahashi
Y
Y
Yamamoto
S
S
Marino
G
G
Di Marzo
V
V
D000581:Amidohydrolases / Q000037:antagonists & inhibitors*; D000818:Animals; D001095:Arachidonic Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001491:Basophils / Q000201:enzymology; D001921:Brain / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D051379:Mice; D009474:Neurons / Q000201:enzymology; D063065:Organophosphonates / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D053284:Polyunsaturated Alkamides; D051381:Rats; D014105:Tosyl Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D010741:Phospholipases A; D054467:Phospholipases A2; D053284:Polyunsaturated Alkamides; D014105:Tosyl Compounds; D000099252:Fatty Acid Amide Hydrolases; C104960:O-acetyl-arachidonoyl-hydroxamate; C104958:arachidonoyl-chloromethyl ketone; C104956:arachidonyl-diazomethyl ketone; C104810:methyl arachidonylfluorophosphonate; C003614:4-toluenesulfonyl fluoride; C078814:anandamide