High-Throughput Assay Development for Non-Nicotine Tobacco Components

描述（由申请人提供）：提出了一对适用于鉴定对<$3 <$4和<$4 <$2烟碱受体具有活性的非尼古丁烟草产品化合物的高释放量筛选（HTS）测定。有两个具体的目标：1。将针对一对现有的高功能单克隆细胞系建立并优化HTS就绪测定。一个稳定转染有<$3 <$4-，另一个稳定转染有<$4 <$2-烟碱乙酰胆碱受体（nAChRs）。膜电位和Ca 2+染料荧光方法均适用，目标2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，并将其应用于优化的<$3 <$4和<$4 <$2 HTS就绪检测试剂盒。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。我们将使用约2400种结构多样的测试化合物的中试筛选为每个完全配置的检测生成原理验证数据。该提案与PAR-12-266和《家庭吸烟预防和烟草控制法》（FSPTCA）的相关性：如具体目标、意义和创新部分所述，该提案将提供一套针对与烟草产品使用和依赖相关的nAChR亚型的经验证的检测方法。由此产生的屏幕将通过加速PAR-12-266的两个既定目标的进展产生影响：1）“减少成瘾-理解.尼古丁以外影响可燃和不可燃烟草产品成瘾的其他成分和组分。“2）“烟草产品的多样性-了解烟草产品的组成、成分、成分、添加剂和设计特点;传统的和新兴的烟草产品。“他们还将讨论FDA烟草产品中心的一个特定研究领域（http：//www.fda.gov/downloads/TobaccoProducts/NewsEvents/UCM293998.pdf; #15;“可以开发和/或使用哪些高通量筛选来评估烟草产品和烟雾中可能影响成瘾的化合物（例如，作用于烟碱或多巴胺受体等）？）。这套屏幕的可用性也将推进FSPTCA的优先事项。通过这种筛选确定的烟草产品成分将是后续研究的宝贵线索，以了解其与烟草使用和依赖的相关性及其确切的作用机制。这些研究将通过新的科学见解产生影响，并可能揭示新的戒烟治疗途径/目标。通过这些筛选鉴定的化合物也可能是FSPTCA监管的候选物。

DESCRIPTION (provided by applicant): A pair of High Throughput Screening (HTS) assays suitable for identifying non-nicotine tobacco product compounds with activity at ¿3¿4 and ¿4¿2 nicotinic receptors is proposed. Two Specific Aims are described: 1. HTS-ready assays will be established and optimized for a pair of existing, highly-functional, monoclonal cell lines. One is stably transfected with ¿3¿4-, the other with ¿4¿2- nicotinic acetylcholine receptors (nAChRs). Both membrane-potential and Ca2+ dye fluorescence approaches are suitable and the approach which can be refined to provide the most robust and reliable results will be adopted for Aim 2. 2. The optimized assays will be configured for HTS conditions. An overall screening workflow is proposed, and will be applied to each of the optimized ¿3¿4 and ¿4¿2 HTS-ready assays. 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 screens. Already-available assays suitable for orthogonal- and counter-screening, using different activities than the primary screen, are described. We will generate proof-of-principle data for each fully-configured assay using a pilot screen of ~2400 structurally-diverse test compounds. Relevance of this proposal to PAR-12-266, and the Family Smoking Prevention and Tobacco Control Act (FSPTCA): As detailed in the Specific Aims, Significance and Innovation sections, this proposal will provide a set of validated assays targeting nAChR subtypes tied to use of, and dependence on, tobacco products. The resulting screens will produce impact by accelerating progress on two stated goals of PAR-12-266: 1) "Reducing Addiction - understanding ... other constituents and components beyond nicotine that affect addiction of combustible and non-combustible tobacco products." 2) "Diversity of Tobacco Products - understanding the constituents, components, ingredients, additives, and design features; ... of conventional and new and emerging tobacco products." They will also address a specific research area of the FDA Center for Tobacco Products (http://www.fda.gov/downloads/TobaccoProducts/NewsEvents/UCM293998.pdf; #15; "What high-throughput screens can be developed and/or used to evaluate compounds in tobacco products and smoke that may affect addiction (e.g., act on nicotinic or dopaminergic receptors...etc.)?). Availability of this suite of screens will also advance priorities of the FSPTCA. Tobacco product components identified by such screens will be valuable leads for follow-up studies to understand their relevance to tobacco use and dependence, and their precise mechanisms of action. These studies would provide impact through new scientific insights and potentially by revealing novel tobacco-cessation therapeutic avenues/targets. Compounds identified by these screens may also be candidates for regulation under the FSPTCA.