Characterization of Anandamide Transport in Brain

描述（由申请人提供）：内源性大麻素及其相关G蛋白偶联受体是调节多种大脑功能（包括疼痛和奖赏）的神经调节系统的组成部分。内源性大麻素anandamide根据需要从神经元释放，并经历快速失活过程，该过程被认为涉及两个连续步骤：载体介导的转运到细胞中以及脂肪酸酰胺水解酶-1（FAAH-1）和FAAH-2的细胞内水解。本申请的主要目的是解决大麻素失活的两个关键方面，这两个方面仍然没有完全理解：（i）大麻素转运的机制和意义;以及（ii）FAAH介导的大麻素水解在中枢神经系统（CNS）外的作用。在以前的研究中，由该基金资助，我们开发了第一个脑不渗透FAAH抑制剂，并表明这种O-芳基氨基甲酸酯衍生物（称为URB 937）通过增强大麻素对位于CNS外的CB 1型大麻素受体的内在活性来抑制啮齿动物的急性和慢性疼痛相关反应。此外，我们分子克隆了FAAH-1的催化缺陷变体（称为FAAH样大麻素转运蛋白，FLAT），其缺乏酰胺酶活性，但选择性地结合大麻素并促进该化合物转运到细胞中。最后，我们发现了一个配体（ARN 272），抑制大麻素结合FLAT，阻断大麻素在体外转运和中断大麻素在体内失活。本建议有两个主要目的。目的1：确定外周FAAH抑制的药效团谱，并鉴定新的脑不通透FAAH抑制剂。我们将合成URB 937的类似物，以（i）确定O-芳基氨基甲酸酯化学型内外周分离的结构-活性关系;和（ii）鉴定新的脑不渗透FAAH抑制剂，其可用于研究外周FAAH的功能和作为没有中枢副作用的镇痛剂的原型。目的2：研究FLAT在神经元内源性花生四烯酸转运中的作用，寻找有效的选择性FLAT抑制剂。我们将进行三组研究：（a）我们将进行计算和突变分析，旨在探索底物（如花生四烯酸）和抑制剂（B）我们将研究FLAT介导大麻素转运的机制;以及（c）我们将使用ARN 272的支架来创建新的FLAT配体，其可以用作研究FLAT在花生四烯酸转运中的功能的工具。这些研究可能会产生新的分子工具，这将有助于阐明花生四烯酸失活的机制，并可能导致发现新的药物候选人的疼痛，物质滥用和其他人类疾病。

DESCRIPTION (provided by applicant): The endogenous cannabinoids and their attending G protein-coupled receptors are components of a neuromodulatory system that regulates multiple brain functions, including pain and reward. The endocannabinoid anandamide is released from neurons upon demand and undergoes a rapid deactivation process that is thought to involve two sequential steps: carrier-mediated transport into cells and intracellular hydrolysis by fatty acid amide hydrolase-1 (FAAH-1) and FAAH-2. The main objective of the present application is to address two key aspects of anandamide deactivation, which remain incompletely understood: (i) the mechanism and significance of anandamide transport; and (ii) the roles of FAAH-mediated anandamide hydrolysis outside the central nervous system (CNS). In previous studies, funded by this grant, we developed the first brain-impermeant FAAH inhibitor and showed that this O-aryl carbamate derivative (termed URB937) suppresses acute and chronic pain-related responses in rodents by enhancing the intrinsic activity of anandamide at CB1-type cannabinoid receptors located outside the CNS. Furthermore, we molecularly cloned a catalytically defective variant of FAAH-1 (termed FAAH-Like Anandamide Transporter, FLAT) that lacks amidase activity, but selectively binds to anandamide and facilitates the transport of this compound into cells. Finally, we discovered a ligand (ARN272) that inhibits anandamide binding to FLAT, blocks anandamide transport in vitro and interrupts anandamide deactivation in vivo. The present proposal has two primary aims. Aim 1: To define pharmacophore profiles for peripheral FAAH inhibition and identify new brain-impermeant FAAH inhibitors. We will synthesize analogs of URB937 to (i) determine structure-activity relationships for peripheral segregation within the O-aryl carbamate chemotype; and (ii) identify new brain-impermeant FAAH inhibitors, which may be utilized both to investigate the functions of peripheral FAAH and as prototypes for analgesic agents devoid of central side effects. Aim 2: To characterize the role of FLAT in neuronal anandamide transport and discover potent and selective FLAT inhibitors. We will perform three sets of studies: (a) we will conduct computational and mutational analyses aimed at exploring the molecular mechanism(s) through which substrates (such as anandamide) and inhibitors (such as ARN272) interact with FLAT; (b) we will investigate the mechanism through which FLAT mediates anandamide transport; and (c) we will use the scaffold of ARN272 to create novel FLAT ligands that may serve as tools to investigate the functions of FLAT in anandamide transport. These studies are likely to generate new molecular tools that will help elucidate the mechanism of anandamide deactivation and may lead to the discovery of new drug candidates for pain, substance abuse and other human diseases.