Development of the first potent and specific inhibitors of endocannabinoid biosynthesis.

Enzyrnes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alfa and beta (DAGLalfa and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalfa to screen new synthetic substances as DAGLalfa inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalfa 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 60 nM), the most potent inhibitors of DAGLalfa were 0-3640 [octadec-9-enoic acid-l-(fluoro-methyl-phospboryloxymcthyl)-propylester] (lC50=500 nM). and 0-3841 [octadec-P-enoic acid l-methoxymethyl-2-(tluoro-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-l0l [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLa and MAGL with similar potencies (JC50=O.8-0.1 and 3.7-3.2 11M, respectively). Thus, we report the first potent inhibitor of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this cannabinoid.