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3？4和4？2烟碱受体活性的非尼古丁烟草产品化合物。描述了两个具体目标：1.将为现有的、高功能的克隆细胞系建立和优化HTS-Ready检测方法。一种是稳定地转染3？4-，另一种是？4？2-烟碱型乙酰胆碱受体(NAChRs)。膜电位法和钙染料荧光法都是合适的，AIM 2.2将采用可以改进以提供最可靠结果的方法。优化的分析方法将配置用于高温超导条件。提出了一个全面的筛选工作流程，并将应用于每一种优化的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协议，以从初始筛选中识别和丢弃假阳性，并确定已确认的候选人的特征。描述了适用于正交筛选和反筛选的现有分析方法，这些方法使用与主筛选不同的活性。我们将使用约2400种结构多样的测试化合物的中试筛选，为每个完全配置的分析生成原理证明数据。这项建议与PAR-12-266和《家庭吸烟预防和烟草控制法案》(FSPTCA)的相关性：正如在具体目标、意义和创新部分中详细描述的那样，这项建议将提供一套针对与烟草产品的使用和依赖相关的nAChR亚型的有效检测方法。由此产生的筛选结果将产生影响，加速实现PAR-12-266规定的两个目标：1)“减少成瘾--了解……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成分。”2)“烟草产品的多样性--了解传统和新出现的烟草产品的成分、成分、添加剂和设计特征。”他们还将讨论FDA烟草产品中心(http://www.fda.gov/downloads/TobaccoProducts/NewsEvents/UCM293998.pdf；#15的一个特定研究领域：“可以开发和/或使用哪些高通量筛查来评估烟草产品和烟雾中可能影响成瘾的化合物(例如作用于尼古丁或多巴胺能受体等？)。这套屏幕的提供也将推进FSPTCA的优先事项。这些筛查确定的烟草产品成分将是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产生影响，并可能通过揭示新的戒烟治疗途径/目标来提供影响。这些筛选确定的化合物也可能是FSPTCA监管的候选者。

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.