High-Throughput Assay Development for Non-Nicotine Tobacco Components

描述（由申请人提供）：提出了一对高通量筛选（HTS）方法，适用于鉴定具有α 3beta4和α 4beta2尼古丁受体活性的非尼古丁烟草制品化合物。描述了两个具体目标：1。将针对一对现有的高功能单克隆细胞系建立并优化HTS-ready检测方法。一个稳定转染了alpha3beta4-，另一个稳定转染了alpha4beta2-烟碱乙酰胆碱受体（nAChRs）。膜电位和Ca2+染料荧光方法都是合适的，并且可以改进以提供最稳健和可靠的结果的方法将用于Aim 2。2. 优化后的测定方法将用于高温高温条件。提出了一个整体筛选工作流程，并将应用于每个优化的alpha3beta4和alpha4beta2 HTS-ready检测。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已确认的候选物的特征。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。我们将使用约2400种结构多样的测试化合物的先导筛选，为每个完全配置的分析生成原理验证数据。本提案与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，本提案将提供一套针对与烟草制品使用和依赖相关的nAChR亚型的验证分析。由此产生的屏幕将通过加速实现PAR-12-266的两个既定目标产生影响：1)“减少成瘾-理解……尼古丁以外影响可燃和不可燃烟草制品成瘾性的其他成分和成分。”2)“烟草制品的多样性——了解其成分、成分、成分、添加剂和设计特征；…传统的和新兴的烟草产品。”他们还将讨论FDA烟草制品中心的一个特定研究领域（http://www.fda.gov/downloads/TobaccoProducts/NewsEvents/UCM293998.pdf; #15；“什么高通量筛选可以开发和/或用于评估烟草制品和烟雾中可能影响成瘾的化合物（例如，作用于尼古丁或多巴胺能受体……等）？”）。这套屏幕的可用性也将促进FSPTCA的优先事项。通过这种筛选确定的烟草产品成分将成为后续研究的宝贵线索，以了解它们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和可能通过揭示新的戒烟治疗途径/目标产生影响。通过这些筛选确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 alpha3beta4 and alpha4beta2 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 alpha3beta4-, the other with alpha4beta2- 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 alpha3beta4 and alpha4beta2 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.