van der Stelt Mario, van Kuik J Albert, Bari Monica, van Zadelhoff Guus, Leeflang Bas R, Veldink Gerrit A, Finazzi-Agrò Alessandro, Vliegenthart Johannes F G, Maccarrone Mauro
Department of Bio-organic Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
J Med Chem. 2002 Aug 15;45(17):3709-20. doi: 10.1021/jm020818q.
This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.
本研究旨在探寻内源性大麻素酰基链与大麻素受体、膜转运蛋白以及脂肪酸酰胺水解酶（FAAH）相互作用的结构要求。为此，通过使用不同的脂氧合酶在花生四烯酸乙醇胺（N-花生四烯酰乙醇胺，AEA）和2-花生四烯酸甘油酯（2-AG）的不同位置引入1-羟基-2Z,4E-戊二烯系统，来限制酰基链的灵活性。这带来了选择性并减弱了AEA和2-AG的结合效力。尽管置换常数适中，但发现15(S)-羟基-二十碳-5Z,8Z,11Z,13E-四烯酰-N-(2-羟乙基)胺可选择性地结合至CB(1)受体，而其1-花生四烯酰基-sn-甘油类似物和13(S)-羟基-十八碳-9Z,11E-二烯酰-N-(2-羟乙基)胺可选择性地结合至CB(2)受体。11(S)-羟基-二十碳-5Z,8Z,12E,14Z-四烯酰-N-(2-羟乙基)胺不与任何一种受体结合，而12(S)-羟基-二十碳-5Z,8Z,10E,14Z-四烯酰-N-(2-羟乙基)胺确实以与AEA相似的亲和力结合至两种CB受体。所有氧化型花生四烯酸乙醇胺衍生物都是FAAH的良好抑制剂（低微摩尔K(i)），但对AEA转运体无效。2-AG迅速异构化为1(3)-花生四烯酰基-sn-甘油。1-和3-花生四烯酰基-sn-甘油均不与任何一种CB受体结合，也不干扰AEA转运。因此，异构化后，2-AG失活，从而降低了2-AG的有效浓度。(1)H NMR光谱分析表明，与水相比，氯仿在15(S)-羟基-二十碳-5Z,8Z,11Z,13E-四烯酸的酰基链中并未诱导出明显不同的构象。在存在明确水分子的情况下对AEA及其类似物进行的分子动力学（MD）模拟表明，酰基链紧密折叠的构象并非CB(1)结合的唯一要求。C(2)-C(15)环的结构细节，如11位的sp(2)碳，对于受体结合是必要的。MD模拟可能表明，AEA和12(S)-羟基-二十碳-5Z,8Z,10E,14Z-四烯酰-N-(2-羟乙基)胺的戊基尾部的平均取向与低亲和力、无活性配体的不同。
