High Throughput Screening for nAChRs: Cell Lines and Assay Development

描述（由申请人提供）：该研究项目对NIH/NIDA RFA-DA-11-007反应灵敏，目标是开发一种高通量筛选（HTS）方法，用于筛选和鉴定含有a3， a5， a6和/或b4亚基的神经元尼古丁乙酰胆碱受体（nAChRs）的选择性配体。nachr是配体门控离子通道，涉及广泛的生理功能、病理过程和药理作用。它们已被证明可以调节尼古丁的成瘾性，这是全球超过10亿人吸烟或使用其他烟草产品的主要原因。神经元nachr由a和b亚基组成，形成五聚体阳离子通道。在脊椎动物神经组织中已鉴定出9个a亚基（a2 - a10）和3个b亚基（b2 - b4）。虽然a7亚基形成功能性同质受体，但哺乳动物脑中的大多数nachr是含有两个或两个以上亚基的异质受体。多年来，a4b2 nAChR亚型（由a4， b2和其他亚基组成）一直是研究尼古丁成瘾和开发戒烟疗法的主要焦点，许多证据支持它们的参与；但自2007年以来，全基因组关联研究揭示了人类染色体15q24-25.1上的a5-a3-b4基因簇的标记与吸烟和吸烟相关疾病的显著关联。在这些标记中，最引人注目的变体是rs16969968，它在a5亚基的398位（D398N）将一个氨基酸从天冬氨酸变为天冬酰胺。这一发现以及最近的发现表明，除了a4和b2亚基外，a3、a5、a6和b4亚基在吸烟和吸烟相关疾病中也起着重要作用。鉴于这些新的信息，开发新的配体对含有a3， a5， a6和/或b4亚基的受体具有选择性是很重要的。因此，迫切需要一种能够在现有或新的化合物文库中可靠地识别选择性配体的HTS分析。我们提出了两个具体目标来实现这一目标：目标1：建立高功能的稳定转染细胞系，表达含有a3， a5， a6和/或b4亚基的人类nAChRs。目的2：利用IonFlux自动化、高通量膜片钳系统开发功能性HTS检测。

DESCRIPTION (provided by applicant): The goal of this research project, which is highly responsive to NIH/NIDA RFA-DA-11-007, is to develop a high throughput screening (HTS) assay for screening and identifying selective ligands for neuronal nicotinic acetylcholine receptors (nAChRs) containing a3, a5, a6 and/or b4 subunits. nAChRs are ligand-gated ion channels that are implicated in a wide range of physiological functions, pathological processes and pharmacological effects. They have been shown to mediate the addictive effects of nicotine, which are the main reason why more than a billion people worldwide smoke cigarettes or use other tobacco products. Neuronal nAChRs are composed of a and b subunits that form pentameric cation channels. Nine a subunits (a2 - a10) and three b subunits (b2 - b4) have been identified in vertebrate neuronal tissues. Although the a7 subunit forms functional homomeric receptors, most nAChRs in mammalian brain are heteromeric receptors that contain two or more subunits. For many years, a4b2 nAChR subtypes (composed of a4, b2 and perhaps other subunits) have been the primary focus for studying nicotine addiction and developing smoking cessation therapeutics, and much evidence supports their involvement; but since 2007 genome-wide association studies have revealed significant associations of markers in the human a5-a3-b4 gene cluster on chromosome 15q24-25.1 with smoking and smoking related diseases. Among these markers, the most compelling variant is rs16969968, which changes an amino acid from aspartate to asparagine at position 398 (D398N) in the a5 subunit. This and more recent discoveries suggest that in addition to a4 and b2 subunits, a3, a5, a6 and b4 subunits play important roles in smoking and smoking related diseases. In light of this new information, it is considered important to develop new ligands selective for receptors containing a3, a5, a6 and/or b4 subunits. Hence, the crucial need for a HTS assay that can reliably identify selective ligands in existing or new libraries of compounds. We propose two Specific Aims to accomplish this goal: Aim 1: To establish highly functional stably transfected cell lines that express human nAChRs containing a3, a5, a6 and/or b4 subunits. Aim 2: To develop a functional HTS assay using IonFlux automated, high throughput patch clamp system. PUBLIC HEALTH RELEVANCE: Cigarette smoking is the leading cause of preventable disease and premature death in the United States. Selective ligands of nicotinic receptors can be used in studying nicotine addiction and for developing new smoking cessation therapeutics. The goal of this project is to develop high throughput screening assay that will accelerate the development of selective ligands.