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高通量筛选，适用于鉴定对含有α 6亚基（α 6 nAChR）的烟碱乙酰胆碱受体具有选择性的化合物。为了实现这一目标，提出了两个具体目标：1。将建立一种HTS就绪测定法，并针对用α 6/β 2 β 3 nAChR稳定转染的现有高功能单克隆细胞系进行优化。膜电位和细胞内Ca2+染料方法均适用，第二个目标将采用可改进以提供最稳健和可靠结果的方法。2.将针对HTS条件配置优化的测定。提出了总体筛选工作流程。该工作流程结合了二次正交-效价-和选择性-计数器筛选方案，以识别和丢弃假阳性，并表征来自初始筛选的确认候选物。已经可用的检测适合正交和反筛选，使用不同的活动比的主要屏幕，进行了说明。第二个目标还包含一项建议，即从约2400种结构多样的供试化合物（包括高比例的已确定的CNS活性药物）的中试筛选中生成原理验证数据。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α 6 nAChR亚单位基因变异和α 6 nAChR功能与物质使用和依赖（特别是吸烟）以及帕金森病发展和治疗的重要特征联系起来。这些疾病都是一个重大的公共卫生问题。α 6 nAChRs影响这些现象的潜在分子机制仍然没有得到很好的理解。通过拟议的筛选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。这将通过新的科学见解和潜在的揭示新的治疗途径/靶点来产生影响。通过该筛选鉴定的化合物也可以用于相同条件的治疗。值得注意的是，鉴于α 6乙酰胆碱受体与多种物质的滥用倾向之间的既定关联，对药物依赖疗法的影响可能相当广泛。此外，α 6 nAChR选择性放射性示踪剂可以是用于帕金森病诊断和/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，目的是鉴定和表征对含有16个亚基的烟碱乙酰胆碱受体具有选择性的化合物。这些受体与以下主要的公共卫生问题有关：吸烟、酗酒和帕金森病。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.