Characterization of Anandamide Transport in Brain

描述(申请人提供)：内源性大麻素及其G蛋白偶联受体是神经调节系统的组成部分，调节包括疼痛和奖励在内的多种大脑功能。内源性大麻酰胺是根据需要从神经元中释放出来的，并经历了一个快速的失活过程，被认为包括两个连续的步骤：载体介导的转运到细胞内，以及脂肪酸酰胺水解酶-1(FAAH-1)和FAAH-2在细胞内的水解。本申请的主要目的是解决目前仍未完全了解的ANANDAME失活的两个关键方面：(I)ANANDIAME转运的机制和意义；(Ii)FAAH介导的ANANDAME在中枢神经系统(CNS)外的水解作用。在之前的研究中，在这笔赠款的资助下，我们开发了第一个大脑不受欢迎的FAAH抑制剂，并表明这种O-芳基氨基甲酸酯衍生物(名为URB937)通过增强位于中枢神经系统外的CB1型大麻受体上的ANANDAME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的一个催化缺陷变异体(称为FAAH-like厌胺转运体，Flat)，它缺乏酰胺酶活性，但选择性地与厌氧核酰胺结合，促进这种化合物向细胞内的转运。最后，我们发现了一种配体(ARN272)，它可以抑制ANANDIAME与平板的结合，在体外阻断ANANDAME的转运，并在体内阻断ANANDAME的失活。目前的建议有两个主要目标。目的1：确定外周FAAH抑制的药效团分布，并确定新的脑不能抑制FAAH的药物。我们将合成URB937的类似物，以(I)确定O-芳基氨基甲酸酯化学型中外周分离的结构-活性关系；以及(Ii)识别新的脑不适用的FAAH抑制剂，这些抑制剂既可用于研究外周FAAH的功能，也可用作无中枢副作用的止痛药的原型。目的2：研究Flat在神经元脱氢表雄胺转运中的作用，寻找有效的、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，目的是探索底物(如ANANDAME)和抑制剂(如ARN272)与Flat相互作用的分子机制(S)；(B)我们将研究Flat介导ANANDAME转运的机制；以及(C)我们将利用ARN272的支架创建新型平板配体，作为研究Flat在ANANDAME转运中功能的工具。这些研究可能会产生新的分子工具，有助于阐明阿南达胺失活的机制，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。 与公共健康相关：人体产生类似大麻的分子，称为内源性大麻素，参与调节疼痛、情绪、记忆和成瘾。我们建议创造有效和选择性的化学探针，使研究人员能够调查脑细胞如何阻止内源性大麻素的活动。这些化学物质可能不仅作为实验工具有用，还可能帮助我们发现治疗药物滥用、疼痛和其他疾病的创新疗法。

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. PUBLIC HEALTH RELEVANCE: The human body produces marijuana-like molecules, called endocannabinoids, which are involved in the regulation of pain, mood, memory and addiction. We propose to create potent and selective chemical probes that will allow researchers to investigate how brain cells stop the actions of the endocannabinoids. These chemicals may not only be useful as experimental tools, but might also help us discover innovative therapies to treat drug abuse, pain and other diseases.