Characterization of Anandamide Transport in Brain

描述（由申请人提供）：内源性大麻素及其相关的G蛋白偶联受体是调节多种脑功能的神经调节系统的组成部分，包括疼痛和奖励。内源性大麻素anandamide根据需要从神经元中释放出来，并经历一个快速的失活过程，该过程被认为涉及两个连续的步骤：载体介导的转运进入细胞和脂肪酸酰胺水解酶-1 （FAAH-1）和FAAH-2在细胞内水解。本应用程序的主要目的是解决阿南达明失活的两个关键方面，这仍然是不完全了解：(i)阿南达明转运的机制和意义；（ii） faah介导的anandamide水解在中枢神经系统（CNS）外的作用。在之前的研究中，由该基金资助，我们开发了第一个大脑无关的FAAH抑制剂，并表明这种o -芳基氨基甲酸酯衍生物（称为URB937）通过增强位于CNS外的cb1型大麻素受体的anandamide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催化缺陷变体（称为FAAH-Like Anandamide Transporter， FLAT），该变体缺乏酰胺酶活性，但选择性地结合Anandamide并促进该化合物运输到细胞中。最后，我们发现了一种配体（ARN272），它可以抑制anandamide与FLAT的结合，阻断anandamide在体外的转运，并阻断anandamide在体内的失活。目前的建议有两个主要目的。目的1：确定外周FAAH抑制的药效团谱，并鉴定新的脑外FAAH抑制剂。我们将合成URB937的类似物来(i)确定氨基甲酸酯o芳基化学型中外周分离的结构-活性关系；（ii）发现新的脑外FAAH抑制剂，可用于研究外周FAAH的功能，并作为无中枢副作用的止痛剂的原型。目的2：表征FLAT在神经元阿南达胺转运中的作用，并发现有效和选择性的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anandamide）和抑制剂（如ARN272）与FLAT相互作用的分子机制；(b)我们将研究FLAT介导anandamide转运的机制；(c)我们将使用ARN272的支架来创建新的FLAT配体，这些配体可以作为研究FLAT在anandamide运输中的功能的工具。这些研究可能会产生新的分子工具，有助于阐明anandamide失活的机制，并可能导致发现治疗疼痛、药物滥用和其他人类疾病的新候选药物。

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.