Bisogno T, Melck D, De Petrocellis L, Gretskaya N M, Bezuglov V V, Sitachitta N, Gerwick W H, Di Marzo V
Istituto per la Chimica di Molecole di Interesse Biologico, C.N.R., Napoli, Italy.
Biochem Biophys Res Commun. 1998 Jul 30;248(3):515-22. doi: 10.1006/bbrc.1998.8874.
Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and 2-arachidonoylglycerol, and the putative sleep inducing factor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A2 (cPLA2) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 12.0 to 26 microM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (IC50 = 5.6 microM), and inhibited [14C]anandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA2-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-2H3 cells, nor with cPLA2 activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cPLA2 and at CB1 receptors, whereas AEG represents a new type of cannabinoid receptor agonist.
脂肪酸酰胺水解酶（FAAH）催化生物活性脂肪酸酰胺和酯的水解，如内源性大麻素受体配体花生四烯酸乙醇胺（N-花生四烯酰基乙醇胺）和2-花生四烯酸甘油酯，以及假定的睡眠诱导因子顺-9-十八碳烯酰胺（油酰胺）。迄今为止开发的大多数FAAH阻滞剂也抑制胞质磷脂酶A2（cPLA2）和/或与CB1大麻素受体亚型结合。在此我们报告发现了四种新型FAAH抑制剂，其中两种，malhamensilipin A和格氏双烯，是从32种不同的藻类天然产物中筛选出来的，另外两种，花生四烯酸乙二醇（AEG）和花生四烯酰基-5-羟色胺（AA-5-HT）是从五种人工功能化的多不饱和脂肪酸中挑选出来的。当使用来自小鼠神经母细胞瘤N18TG2细胞的FAAH制剂和[14C]花生四烯酸乙醇胺作为底物时，这些化合物的IC50值在12.0至26μM之间，活性最强的化合物是AA-5-HT。该物质对大鼠嗜碱性白血病（RBL-2H3）细胞的FAAH也有活性（IC50 = 5.6μM），并且抑制N18TG2和RBL-2H3完整细胞对[14C]花生四烯酸乙醇胺的水解，而不影响[14C]花生四烯酸乙醇胺的摄取。虽然AEG表现为竞争性抑制剂并被FAAH制剂水解为花生四烯酸（AA），但AA-5-HT对FAAH催化的水解具有抗性，表现为紧密结合的非共价混合抑制剂。AA-5-HT不干扰cPLA2介导的、离子霉素或抗原诱导的[3H]AA从RBL-2H3细胞的释放，在无细胞实验中也不干扰cPLA2活性。最后，AA-5-HT不激活CB1大麻素受体，因为它在体外结合试验中作为非常弱的配体起作用，并且在10-15mg/kg体重时，在小鼠进行的“旷场”、“热板”和直肠体温过低试验中无活性。相反，AEG在这些试验以及AA-5-HT也有活性的“环形”不动试验中表现为大麻模拟物质。AA-5-HT是迄今为止报道的第一种对cPLA2和CB1受体均无活性的FAAH抑制剂，而AEG代表一种新型的大麻素受体激动剂。
