Characterization of Anandamide Transport in Brain

Anandamide 在脑中转运的表征

• 批准号: 8916060
• 负责人: Daniele Piomelli
• 金额: $ 30.04万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-14 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8916060
• 关键词: ABCG2 gene; Acute Pain; Address; Adverse effects; Amidohydrolases; Analgesics; Animal Model; Arachidonic Acids; Binding; Blood - brain barrier anatomy; Brain; CNR1 gene; Carbamates; Cell membrane; Cells; Chemicals; Collaborations; Disease; Drug abuse; Employee Strikes; Endocannabinoids; Funding; G-Protein-Coupled Receptors; Genetic; Grant; Human body; Hydrolysis; In Vitro; Innovative Therapy; Lead; Ligands; Marijuana; Mediating; Membrane; Memory; Molecular; Moods; Movement; Neuraxis; Neurons; Organelles; Pain; Pain management; Peripheral; Play; Positioning Attribute; Process; Publications; Regulation; Research; Research Personnel; Rewards; Rodent; Role; Signal Transduction; Structure-Activity Relationship; Substance abuse problem; System; Tissues; Variant; Work; Xenobiotics; addiction; amidase; analog; anandamide; base; brain cell; cannabinoid receptor; carrier mediated transport; chronic pain; design; drug candidate; experience; fatty acid amide hydrolase; human disease; in vivo; inhibitor/antagonist; innovation; neuroregulation; novel; pharmacophore; prototype; research study; response; scaffold; segregation; small molecule; tool

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.

描述（由申请人提供）：内源性大麻素及其参加的G蛋白偶联受体是调节多种脑功能（包括疼痛和奖励）的神经调节系统的组成部分。内源性大麻素配胺根据需求从神经元释放，并经历快速失活过程，该过程被认为涉及两个顺序的步骤：载体介导的转运到细胞中的转运以及脂肪酸酰胺酰胺水解酶-1（FAAH-1）和FAAH-2的细胞内水解。本应用的主要目的是解决阿甘丹酰胺失活的两个关键方面，这些方面尚不完全理解：（i）anandamide运输的机制和意义； （ii）FAAH介导的中枢神经系统（CNS）之外介导的蜘蛛水解作用。在先前由这笔赠款资助的研究中，我们开发了第一个脑部覆盖法族抑制剂，并表明，这种O-芳基氨基甲酸酯衍生物（称为URB937）通过增强Anandamide在CB1型Cannabinoid受体的固有活性来抑制啮齿动物中与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的催化有缺陷的变体（称为FAAH样的Anandamide Transporter，FLAT），该变异缺乏amidase酶活性，但选择性地结合了Anandamide并促进该化合物进入细胞。最后，我们发现了一个配体（ARN272），该配体抑制了anandamide与平坦的结合，在体外阻断了anandamide的转运，并在体内中断Anandamide失活。本提案有两个主要目标。目的1：定义外围法族抑制剂的药效学特征并鉴定新的脑覆盖法族抑制剂。我们将综合urb937的类似物（i）确定O-芳基氨基甲酸酯化学型内周围隔离的结构活性关系； （ii）确定新的脑覆盖法族抑制剂，这些抑制剂既可以用来研究外围法族的功能，又可以用作缺乏中心副作用的镇痛药的原型。 AIM 2：表征平坦在神经元胺传输中的作用，并发现有效和选择性的平坦抑制剂。我们将进行三组研究：（a）我们将进行旨在探索底物（例如anandamide）和抑制剂（例如ARN272）的分子机制的计算和突变分析； （b）我们将研究平坦介导阿甘丹胺运输的机制； （c）我们将使用ARN272的脚手架来创建新型的扁平配体，这些配体可以用作研究Anandamide运输中Flat功能的工具。这些研究可能会产生新的分子工具，从而有助于阐明阿甘丹胺失活的机制，并可能导致发现新的候选药物疼痛，药物滥用和其他人类疾病。