HTS Assay Development for alpha6/3beta2beta3 Subtype Nicotinic Receptors

描述(由申请人提供)：总体目标是开发和实施一种高通量筛选，适用于鉴定对含有α6亚单位(α6 nAChRs)的烟碱型乙酰胆碱受体具有选择性的化合物。为了实现这一目标，提出了两个特定的目标：1.建立和优化HTS-Ready检测方法，用于稳定转染Alpha6/Beta2Beta3 nAChRs的现有高功能单抗细胞系。膜电位法和细胞内钙染料法都是合适的，第二个目标将采用可以改进以提供最稳健和可靠结果的方法。2.优化后的分析将针对HTS条件进行配置。提出了一个总体筛选工作流程。该工作流程结合了二次正交化效价和选择性反筛选方案，以便从初始筛选中识别和丢弃假阳性，并确定已确认的候选对象的特征。描述了适用于正交筛选和反筛选的现有分析方法，这些方法使用与主筛选不同的活性。第二个目标还包括一项提案，即从约2400种结构多样化的测试化合物(包括高比例的已建立的中枢神经系统活性药物)的试点筛查中生成原理证明数据。该项目的健康相关性源于遗传关联和神经化学研究，这些研究将alpha6 nAChR亚单位基因变异和alpa6 nAChR功能与物质使用和依赖(特别是与吸烟有关)联系起来，并与帕金森氏病的发展和治疗的重要特征联系起来。每一种情况都是一个重大的公共卫生问题。α6 nAChRs影响这些现象的潜在分子机制仍不清楚。拟议筛查确定的化合物可能成为有价值的研究工具，以更好地了解与Alpha6 nAChRs相关的主要公共卫生问题的病因学。这将通过新的科学见解产生影响，并可能通过揭示新的治疗途径/目标来产生影响。通过这一筛选确定的化合物也可以用于相同条件下的有用治疗。值得注意的是，鉴于字母6非乙酰胆碱受体与各种物质的滥用责任的既定联系，对药物依赖治疗的影响可能是相当广泛的。此外，α6 nAChR选择性放射性示踪剂可能是用于帕金森氏病诊断和/或监测治疗成功的有价值的PET/SPECT配体。 公共卫生相关性：该项目旨在开发和实施一种快速测试大型化合物库的技术，目的是识别和表征对包含16个亚基的烟碱型乙酰胆碱受体具有选择性的化合物。这些受体与以下重大公共健康问题有关：吸烟、酗酒和帕金森氏症。该项目确定的化合物将有助于了解这些疾病的根本原因，并可作为开发用于治疗和/或诊断的化合物的线索。

DESCRIPTION (provided by applicant): The overall objective is to develop and implement a High Throughput Screen suitable for identifying compounds with selectivity for nicotinic acetylcholine receptors which contain the alpha6 subunit (alpha6 nAChRs). To meet this objective, two Specific Aims are proposed: 1. An HTS-ready assay will be established and optimized for an existing, highly-functional, monoclonal cell line stably transfected with alpha6/Beta2Beta3 nAChRs. Both membrane-potential and intracellular Ca2+ dye approaches are suitable, and the approach which can be refined to provide the most robust and reliable results will be adopted for the second Aim. 2. The optimized assay will be configured for HTS conditions. An overall screening workflow is proposed. This workflow incorporates secondary orthogonal- potency-, and selectivity-counter-screening protocols to identify and discard false positives, and to characterize confirmed candidates, from the initial screen. Already-available assays suitable for orthogonal- and counter-screening, using different activities than the primary screen, are described. The second Aim also contains a proposal to generate proof-of-principle data from a pilot screen of ~2400 structurally-diverse test compounds (including a high proportion of established CNS-active drugs). The health relevance of this project arises from genetic association and neurochemical studies which link alpha6 nAChR subunit gene variants and alpa6 nAChR function to substance use and dependence (particularly to tobacco smoking), and to important features of Parkinson's Disease development and treatment. Each of these conditions is a major public-health issue. The underlying molecular mechanisms by which alpha6 nAChRs influence these phenomena are still not well understood. Compounds identified by the proposed screen could become valuable research tools to understand better the etiology of the major public health issues associated with alpha6 nAChRs. This would provide impact through new scientific insights and potentially by revealing novel therapeutic avenues / targets. Compounds identified by this screen could also be useful treatments for the same conditions. Notably, the impact on drug dependence therapies could be quite broad, given the established association of alpha6 nAChRs with abuse liability for diverse substances. Further, an alpha6 nAChR- selective radiotracer could be a valuable PET/SPECT ligand for Parkinson's Disease diagnosis and/or monitoring of treatment success. PUBLIC HEALTH RELEVANCE: This project is intended to develop and implement a technique for rapidly testing a large library of compounds, with the objective of identifying and characterizing compounds selective for nicotinic acetylcholine receptors which contain the 16 subunit. These receptors have been associated with the following major public- health issues: smoking, alcohol abuse, and Parkinson's Disease. Compounds identified by this project would be useful in understanding the underlying causes of these conditions, and may serve as leads for the development of compounds used in their treatment and / or diagnosis.