Lang W, Qin C, Lin S, Khanolkar A D, Goutopoulos A, Fan P, Abouzid K, Meng Z, Biegel D, Makriyannis A
Department of Pharmaceutical Sciences, and Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269, USA.
J Med Chem. 1999 Mar 11;42(5):896-902. doi: 10.1021/jm980461j.
Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N, N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1'-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of hydrolysis of (R)-alpha-methanandamide (2.4%) (anandamide = 100%) was about 10-fold lower than that of its (S)-enantiomer (23%). In contrast, (R)-beta-methanandamide was 6-times more susceptible (121%) than the (S)-beta-enantiomer (21%). Interestingly, an inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH. Metabolic stability is also imparted to analogues with a short hydrocarbon headgroup as well as to those possessing 2-monomethyl or 2,2-dimethyl substituents. 2-Arachidonylglycerol and racemic 1-arachidonylglycerol were shown to be excellent AAH substrates. To identify AAH inhibitors, hydrolysis of anandamide was also studied in the presence of a select group of cannabimimetics. Of these, (-)-Delta8-THC and SR141716A, a biarylpyrazole CB1 antagonist, were found to inhibit enzymatic activity. These newly defined enzyme recognition parameters should provide a foundation for the rational development of stable, therapeutically useful anandamide analogues with high receptor affinity.
花生四烯酸乙醇胺水解酶（AAH）催化花生四烯酸乙醇胺（花生四烯酸酰胺）——一种内源性大麻素受体配体——的水解反应。为了阐明AAH底物的结构要求，我们使用在实验室开发的两种反相高效液相色谱（RP-HPLC）分析法，研究了大鼠脑微粒体AAH对一系列花生四烯酸酰胺类似物的水解作用。发现花生四烯酸酰胺（1）是最佳底物，其表观Km为2.34 mM，Vmax为2.89 nmol/分钟/毫克蛋白质。虽然花生四烯酸乙醇胺（2）具有相似的Km值，但其Vmax约为花生四烯酸酰胺的一半。N，N-双（2-羟乙基）花生四烯酸酰胺（3）未被水解，这表明该酶对未取代或单-N-取代的花生四烯酸酰胺具有特异性。在乙醇酰胺头部基团的1'-位带有甲基的类似物也被发现对酶促周转具有更大的抗性，因此代谢稳定性增强。该酶表现出高立体选择性，因为（R）-α-甲花生四烯酸乙醇胺的水解速率（2.4%）（花生四烯酸乙醇胺=100%）比其（S）-对映体（23%）低约10倍。相反，（R）-β-甲花生四烯酸乙醇胺比（S）-β-对映体（21%）更易被水解6倍（121%）。有趣的是，AAH立体选择性与脑大麻素受体亲和力之间呈现负相关，因为具有高受体亲和力的对映体对AAH水解的敏感性较低。短烃基头部基团的类似物以及具有2-单甲基或2,2-二甲基取代基的类似物也具有代谢稳定性。2-花生四烯酸甘油酯和外消旋1-花生四烯酸甘油酯被证明是优良的AAH底物。为了鉴定AAH抑制剂，还研究了在一组精选的大麻模拟物存在下花生四烯酸乙醇胺的水解情况。其中，（-）-Δ8-四氢大麻酚和SR141716A（一种联芳基吡唑CB1拮抗剂）被发现可抑制酶活性。这些新定义的酶识别参数应为合理开发具有高受体亲和力的稳定、治疗有用的花生四烯酸乙醇胺类似物提供基础。
