Novel Inhibitors of Brain, Neuronal, and Basophilic Anandamide Amidohydrolase

• Published in: Biochemical and biophysical research communications Vol. 231; no. 1; pp. 82 - 88
• Main Authors: De Petrocellis, L., Melck, D., Ueda, N., Maurelli, S., Kurahashi, Y., Yamamoto, S., Marino, G., Di Marzo, V.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03-02-1997
• Subjects: Amidohydrolases - antagonists & inhibitors; Animals; Arachidonic Acids - chemical synthesis; Arachidonic Acids - chemistry; Arachidonic Acids - metabolism; Arachidonic Acids - pharmacology; Basophils - enzymology; Brain - enzymology; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - pharmacology; Mice; Neurons - enzymology; Organophosphonates - pharmacology; Phospholipases A - antagonists & inhibitors; Phospholipases A2; Polyunsaturated Alkamides; Rats; Tosyl Compounds - pharmacology; Tumor Cells, Cultured
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1997.6000

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), arachidonoyl-chloro-methyl-ketone (ACMK) andO-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 N18TG2and 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 μM) and AcAHA the weakest (IC50= 34, 15 and 25 μM) 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 A2, methyl-arachidonoyl-fluoro-phosphonate (MAFP), covalently inhibited the amidohydrolase. MAFP was active at concentrations 103times lower than those reported for phospholipase A2inhibition, 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 thein vitroformation of anandamide from its biosynthetic precursorN-arachidonoyl-phosphatidyl-ethanolamine.