title: Development of the first potent and specific inhibitors of endocannabinoid biosynthesis.
abstract: Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.
authors.affiliation: Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, C.N.R., Via Campi Flegrei, 34-80078-Pozzuoli (NA), Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bisogno
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Cascio
authors.forename: Bijali
authors.initials: B
authors.lastname: Saha
authors.forename: Anu
authors.initials: A
authors.lastname: Mahadevan
authors.forename: Paolo
authors.initials: P
authors.lastname: Urbani
authors.forename: Alberto
authors.initials: A
authors.lastname: Minassi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Appendino
authors.forename: Carmela
authors.initials: C
authors.lastname: Saturnino
authors.forename: Billy
authors.initials: B
authors.lastname: Martin
authors.forename: Raj
authors.initials: R
authors.lastname: Razdan
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Di Marzo
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors; D000818:Animals; D019556:COS Cells; D063385:Cannabinoid Receptor Modulators / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D002522:Chlorocebus aethiops; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D063388:Endocannabinoids; D006801:Humans; D066298:In Vitro Techniques; D007783:Lactones / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors / Q000235:genetics; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D008994:Monoacylglycerol Lipases / Q000037:antagonists & inhibitors / Q000235:genetics; D009829:Oleic Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000077403:Orlistat; D010739:Phospholipase D / Q000037:antagonists & inhibitors; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D007783:Lactones; D008049:Lipase; D008071:Lipoprotein Lipase; D008994:Monoacylglycerol Lipases; D009829:Oleic Acids; D000077403:Orlistat; D010739:Phospholipase D; D011994:Recombinant Proteins; D000099252:Fatty Acid Amide Hydrolases; C480280:DAGLA protein, human