HTS Assay Development for alpha6/3beta2beta3 Subtype Nicotinic Receptors

alpha6/3beta2beta3 亚型烟碱受体的 HTS 检测开发

• 批准号: 8212661
• 负责人: PAUL WHITEAKER
• 金额: $ 25.4万
• 依托单位: ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212661
• 关键词: Adopted; Agonist; Alcohol abuse; Alcohol dependence; Alcohol or Other Drugs use; Award; Biological Assay; Cell Line; Collection; Data; Dependence; Development; Diagnosis; Drosophila acetylcholine receptor alpha-subunit; Drug Addiction; Dyes; Eligibility Determination; Ensure; Etiology; Genetic Variation; Health; Housing; Human; Laboratories; Libraries; Ligand Binding; Ligands; Link; Mediating; Membrane Potentials; Molecular; Monitor; N-terminal; Nicotine; Nicotine Dependence; Nicotinic Receptors; Parkinson Disease; Patients; Pharmaceutical Preparations; Pharmacology; Play; Positron-Emission Tomography; Psychological reinforcement; Public Health; Publishing; Research; Resources; Rewards; Role; Screening procedure; Smoking; Staging; Substance Addiction; Techniques; Testing; Tobacco smoking; United States National Institutes of Health; Writing; addiction; assay development; design; disease diagnosis; genetic association; high throughput screening; insight; meetings; neurochemistry; novel therapeutics; radiotracer; receptor; response; single photon emission computed tomography; small molecule libraries; success; therapy development; tool

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.

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，适用于鉴定含有alpha6亚基（alpha6 nAChRs）的尼古丁乙酰胆碱受体的选择性化合物。为实现这一目标，提出了两个具体目标：将针对稳定转染alpha6/Beta2Beta3 nAChRs的现有高功能单克隆细胞系建立并优化HTS-ready检测方法。膜电位和细胞内Ca2+染色方法都是合适的，并且可以改进以提供最稳健和可靠的结果的方法将被用于第二个目标。优化后的分析将配置为高温条件。提出了一种整体筛选工作流程。该工作流程结合了二次正交效价和选择性反筛选方案，以识别和丢弃假阳性，并从初始筛选中确定确定的候选物的特征。描述了适用于正交筛选和反筛选的现有测定法，使用不同于主筛选的活性。第二个目标还包含一个从大约2400种结构多样的测试化合物（包括高比例的已建立的中枢神经系统活性药物）的试点筛选中生成原理证明数据的建议。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亚基基因变异和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。每一种情况都是一个重大的公共卫生问题。alpha6 nachr影响这些现象的潜在分子机制尚不清楚。通过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 nachr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/靶点产生影响。通过这种筛选确定的化合物也可以用于治疗相同的疾病。值得注意的是，考虑到alpha6 nachr与多种物质滥用倾向的既定关联，对药物依赖治疗的影响可能相当广泛。此外，α 6 nAChR选择性放射性示踪剂可能是一种有价值的PET/SPECT配体，用于帕金森病的诊断和/或治疗成功的监测。