Methods for the Development of Arrestin2 Inhibitors.

描述（由申请人提供）：抑制蛋白是一种胞质蛋白，通过在长期暴露于配体时介导G蛋白偶联受体（GPCR）功能下调来控制GPCR。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点，除了非法药物（例如大麻）之外，还包括几种阿片类镇痛药（例如维柯丁，奥施康定）。GPCR的这种下调是导致药物耐受性的一个因素，这是理解成瘾的一个关键问题。 在被配体激活后，GPCR在C末端磷酸化，这允许与抑制蛋白形成复合物。Arrestin是一种支架蛋白，然后可以招募其他蛋白质结合伴侣，这导致细胞内GPCR的脱敏和内化，这是耐受发生的基础过程。也已经表明，在细胞内内化后，磷酸化模式控制受体的最终命运。这包括导致受体降解，再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，结合形式的抑制蛋白可能会有所不同，这取决于其结合伴侣。因此，生物化学证据导致了这样的假设，即抑制蛋白与GPCR的交替复合物控制受体的最终命运。因此，我们认为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，建立针对arrestin-2的配体的基于核磁共振的筛选技术，并针对针对化学多样性优化的化合物库验证这些技术。这些特定的目标将在从低亲和力到高亲和力（10-3 - 10-8 M）的亲和力范围内实现配体的有效取样。这提供了双重益处，允许对更大范围的化学多样性进行采样，并允许对可能被利用来调节抑制蛋白-2的作用的潜在结合位点进行更广泛的映射。将抑制蛋白-2作为调节可能影响成瘾的生化途径的手段的概念是一个新的假设，实现这一点的配体的发展将提供一个宝贵的工具来确定这些生物过程对成瘾的影响。这种先导化合物的建立将进一步有助于设计和开发规避耐受性的治疗剂，从而潜在地治疗药物成瘾的症状。 公共卫生相关性：这些知识将通过建立筛选抑制蛋白-2配体的方法而有益于人类健康。arrestin 2活性位点的表征和用于发现靶向这些位点的配体的工具将在评估arrestin如何促进GPCR信号传导方面具有价值。GPRC-arrestin相互作用介导许多生物学事件，例如脱敏、内化和GPCR与多个信使系统的相互作用。这些事件是许多生物过程的基础，包括药物耐受和成瘾。这项工作将导致工具的开发，以更好地了解这些过程，并可能有助于设计治疗药物，帮助治疗成瘾和规避耐受性的治疗药物。

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.