Methods for the Development of Arrestin2 Inhibitors.

描述(由申请人提供)：arrestin是一种胞浆蛋白，在长期接触配体时，通过调节G蛋白偶联受体(GPCRs)功能的下调来控制GPCRs。G蛋白偶联受体是许多最臭名昭著的导致成瘾的处方药的靶子，例如除了非法药物(如大麻)外，还有几种阿片类止痛剂(如维柯丁、奥施康定)。GPCR的这种下调是导致药物耐受性的一个因素，而药物耐受性是理解成瘾的关键问题。在被配体激活后，GPCRs在C末端被磷酸化，这允许与arrestin形成络合物。Arrestin是一种支架蛋白，然后可以招募其他蛋白结合伙伴，这会导致细胞内GPCR的脱敏和内化，这是耐受发生的基础过程。它还表明，在细胞内内化后，磷酸化模式决定了受体的最终命运。这包括导致受体降解、循环到细胞表面或与其他信使途径相互作用的途径。此外，有证据表明，arrestin的结合形式可能会因其结合伙伴的不同而不同。因此，生化证据导致了这样的假设，即arrestin与gpcr的交替复合体决定了受体的最终命运。因此，我们断言arrestin是一个有趣的药物靶点，其功能的调节提供了在受体被激活后控制其命运的可能性。该提案旨在开发arrestin-2作为药物靶标，建立基于核磁共振的针对arrestin-2的配体筛选技术，并根据针对化学多样性进行优化的化合物库来验证这些技术。这些特定的目标将在从低亲和力到高亲和力(10-3-10-8M)的一系列亲和力范围内实现对配体的有效采样。这提供了双重好处，允许对更大范围的化学多样性进行采样，并允许更广泛地绘制可能被用来调节arrestin-2的作用的潜在结合位点的图谱。靶向arrestin-2作为调节可能影响成瘾的生化途径的一种手段的概念是一个新的假说，实现这一点的配体的发展将为确定这些生物过程对成瘾的影响提供宝贵的工具。这种先导化合物的建立将进一步有助于设计和开发避免耐受的治疗剂，从而有可能治疗药物成瘾的症状。 公共卫生相关性：这一知识将通过建立筛选Arrestin-2配体的方法而有益于人类健康。Arrestin活性位点的特征和发现针对这些位点的配体的工具将在评估arrestin如何参与gpr信号转导中发挥重要作用。GPRC-arrestin相互作用介导了许多生物学事件，如脱敏、内化以及gpr与多个信使系统的相互作用。这些事件导致了许多生物学过程，包括药物耐受性和成瘾。这项工作将导致开发工具，以更好地了解这些过程，并可能有助于设计有助于治疗成瘾和避免治疗药物耐受的治疗剂。

DESCRIPTION (provided by applicant): Arrestin is a cytosolic protein that serves to control G-protein Coupled Receptors (GPCRs) by mediating downregulation of GPCR function upon prolonged exposure to a ligand. G-protein coupled receptors are the targets for many of the most notorious prescription pharmaceuticals that lead to addiction such as several of the opiod analgesics (e.g. Vicodin, Oxycontin) in addition to illicit drugs (e.g. cannabis). This downregulation of the GPCR is a factor leading to drug tolerance which is a key issue in understanding addiction. Following activation by a ligand, GPCRs are phosphorylated at the C-terminus which allows the formation of a complex with arrestin. Arrestin is a scaffold protein that can then recruit other protein binding partners and this leads to desensitization and internalization of the GPCR within the cell, processes that underlie the onset of tolerance. It has also been shown that after internalization within the cell, the phosphorylation pattern governs the eventual fate of the recptor. This includes pathways that lead to degradation of the receptor, recycling to the cell surface, or interfacing with other messenger pathways. Moreover, there is evidence that bound forms of arrestin may differ depending on its binding partner. The biochemical evidence thus leads to the hypothesis that alternate complexes of the arrestin with the GPCR governs the eventual fate of the receptor. Thus it is our assertion that arrestin is an intriguing drug target and the modulation of its function offers the potential to control the fate of the receptor after it has been activated. This proposal seeks to develop arrestin-2 as a drug target, establish techniques for Nuclear Magnetic Resonance Based Screening of ligands targeted to arrestin-2 and validate these techniques against a library of compounds optimized for chemical diversity. These specific aims will accomplish efficient sampling of ligands across a range of affinities from lower affinity to high affinity (10-3 - 10-8 M). This offers a double benefit in allowing the sampling of a greater range of chemical diversity and allowing a more extensive mapping of potential binding sites that may be exploited to modulate the action of arrestin-2. The notion-of targeting arrestin-2 as a means to modulate biochemical pathways that may affect addiction is a novel hypothesis and the development of ligands that accomplish this would offer an invaluable tool to determine the impact that these biological processes have on addiction. The establishment of such lead compounds would further serve the design and development of therapeutic agents that circumvent tolerance and hence potentially treat the symptoms of drug addiction. PUBLIC HEALTH RELEVANCE: This knowledge will benefit human health by establishing methods for screening of ligands against arrestin-2. The characterization of arrestin2 active sites and tools for the discovery of ligands targeted to these sites will be valuable in evaluating how arrestin contributes to GPCR signaling. The GPRC-arrestin interaction mediates many biological events such as desensitization, internalization and the interaction of GPCR's with multiple messenger systems. These events underly many biological processes including drug tolerance and addiction. This work will lead to the development of tools to better understand these processes and may contribute to the design of therapeutic agents that help to treat addiction and circumvent tolerance to therapeutic drugs.