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）测定，其适合于鉴定对“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。描述了两个具体目标： 1. 将为一对现有的高功能单克隆细胞系建立和优化 HTS 就绪测定法。一种用 ¿3¿4- 稳定转染，另一种用 ¡4¿2- 烟碱乙酰胆碱受体 (nAChR) 稳定转染。膜电位和 Ca2 染料荧光方法均适用，目标 2 将采用可以改进以提供最稳健和最可靠结果的方法。 2. 优化的测定将针对 HTS 条件进行配置。提出了总体筛选工作流程，并将应用于每个优化的 ¿3¿4 和 ¿4¿2 HTS 就绪检测。该工作流程结合了二次正交效力和选择性反筛选协议，以识别和丢弃误报，并从初始筛选中表征已确认的候选者。描述了适用于正交筛选和反筛选的现有测定法，使用与初级筛选不同的活性。我们将使用约 2400 种结构多样的测试化合物的试点筛选，为每个完全配置的测定生成原理验证数据。该提案与 PAR-12-266 以及《家庭吸烟预防和烟草控制法案》(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 亚型的经过验证的检测方法。由此产生的筛选将通过加速 PAR-12-266 的两个既定目标的进展来产生影响：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 - 了解传统和新兴烟草产品的成分、成分、成分、添加剂和设计特征；……”。他们还将讨论 FDA 烟草产品中心的特定研究领域（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能受体...等）？）。该可用性一系列筛选还将推进 FSPTCA 确定的烟草产品成分的优先事项，这些成分将为后续研究提供有价值的线索，以了解它们与烟草使用和依赖的相关性及其精确的作用机制。这些研究将通过新的科学见解并可能通过揭示新的戒烟治疗途径/目标来提供影响。

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.