High-throughput cell-based assays for identifying antagonists of multiple smoking-associated human nicotinic acetylcholine receptor subtypes.
High-throughput cell-based assays for identifying antagonists of multiple smoking-associated human nicotinic acetylcholine receptor subtypes.
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DOI：
10.1016/j.slasd.2021.10.001
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Whiteaker P
中科院分区：
文献类型：
--
作者：
Kassner M;Eaton JB;Tang N;Petit JL;Meurice N;Yin HH;Whiteaker P
有大量证据表明，除了尼古丁之外，烟草烟雾中发现的其他化合物也会显着影响吸烟行为。此外，近年来，提供尼古丁的非燃烧产品的供应呈爆炸式增长，例如电子烟和基本上不受监管的“自制”电子烟设备。仅烟草烟雾中就有数千种化合物，而且新产品不断引入新的化合物。要发现这些化合物中的哪些在影响烟草/尼古丁成瘾的烟碱乙酰胆碱受体 (nAChR) 的多种吸烟相关亚型中具有活性，需要采用高通量筛选 (HTS) 方法。因此，我们开发了一组 HTS 友好的基于细胞的检测，所有检测均在相同的细胞背景下进行，并使用相同的膜电位染料读数，以测量 α3β4-、α4β2- 和 α6β2-nAChR 亚型的功能。这些亚型均与人类吸烟行为显着且一致地相关。我们通过对 Prestwick FDA 批准的一组扩展药物库进行初步筛选来验证我们的检测方法。筛选显示出优异的性能参数，并且在识别拮抗剂时实现了中等命中率（所有三种测定的平均值为 1.2%）（选择拮抗剂是因为内源性拮抗剂对尼古丁/烟草产品消费的影响尚未得到充分研究）。使用正交测定（86Rb+ 流出）的验证率在三种测定中平均为 73%。由此产生的检测组代表了一个有价值的新平台，可用于筛选和鉴定 nAChR 亚型选择性化合物。这为识别燃烧和非燃烧烟草产品中与吸烟相关的化合物提供了资源，并在寻找其他戒烟疗法方面具有潜在的相关性。
There is substantial evidence that in addition to nicotine, other compounds found in tobacco smoke significantly influence smoking behavior. Further, recent years have seen an explosion in the availability of non-combusted products that deliver nicotine, such as e-cigarettes and “home-brew” vaping devices that are essentially unregulated. There are many thousands of compounds in tobacco smoke alone, and new products are constantly introducing new compounds. Uncovering which of these compounds are active, across multiple smoking-relevant subtypes of the nicotinic acetylcholine receptor (nAChR) that influence tobacco/nicotine addiction, requires a high-throughput screening (HTS) approach. Accordingly, we developed a panel of HTS-friendly cell-based assays, all performed in the same cellular background and using the same membrane potential dye readout, to measure the function of the α3β4-, α4β2-, and α6β2-nAChR subtypes. These subtypes have each been prominently and consistently associated with human smoking behavior. We validated our assays by performing pilot screening of an expanded set of the Prestwick FDA-approved drug library. The screens displayed excellent performance parameters, and moderate hit rates (mean of 1.2% across all three assays) were achieved when identifying antagonists (chosen since effects of endogenous antagonists on consumption of nicotine/tobacco products are under-studied). Validation rates using an orthogonal assay (86Rb+ efflux) averaged 73% across the three assays. The resulting panel of assays represents a valuable new platform with which to screen and identify nAChR subtype-selective compounds. This provides a resource for identifying smoking-related compounds in both combusted and non-combusted tobacco products, with potential relevance in the search for additional smoking-cessation therapies.
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影响因子：
1.7
作者：
Bertrand, S;DevillersThiery, A;Bertrand, D
通讯作者：
Bertrand, D
DOI：
10.1111/j.1601-183x.2009.00495.x
发表时间：
2009-08
期刊：
Genes, brain, and behavior
影响因子：
--
作者：
Hoft NR;Corley RP;McQueen MB;Huizinga D;Menard S;Ehringer MA
通讯作者：
Ehringer MA
影响因子：
33.9
作者：
Babb, Stephen;Malarcher, Arm;Jamal, Ahmed
通讯作者：
Jamal, Ahmed
影响因子：
4.7
作者：
Avelar, Alicia J.;Akers, Austin T.;Henderson, Brandon J.
通讯作者：
Henderson, Brandon J.
影响因子：
6
作者：
Benowitz, Neal L.;Nardone, Natalie;Jacob, Peyton, III
通讯作者：
Jacob, Peyton, III