Assay Development Relating to the HTS of the Neuropeptide Y-Y2 Receptor

与神经肽 Y-Y2 受体 HTS 相关的检测方法开发

• 批准号: 7169427
• 负责人: Claes Robert Wahlestedt
• 金额: $ 17.31万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169427
• 关键词: NIH Roadmap Initiative tag; affinity labeling; chemical registry /resource; drug discovery /isolation; high throughput technology; inhibitor /antagonist; neuropeptides; radiotracer; receptor binding; robotics; technology /technique development

DESCRIPTION (provided by applicant): This application relates to the NIH efforts to develop and adapt biological assays for use in HTS. Our team has extensive experience in drug discovery and pharmaceutical screening campaigns. Our overall aim is to develop assays for HTS of small molecule compounds that display affinity to the neuropeptide Y (NPY) Y2- receptor. Specifically, we seek to identify compounds that are functional antagonists at the human Y2- receptor. Following optimization, also to be pursued at Scripps by individuals with much pharmaceutical industry experience, such a compound might be clinically useful in anxiety, depression and/or alcoholism. No clinically testable compounds that inhibit signaling of NPY through the Y2-receptor are in existence today. The NPY-Y2 receptor was first described by us previously and recent studies in human in particular have made us more convinced than ever that it is an important target in psychiatric disease and addiction. SPECIFIC OBJECTIVES AND AIMS: We will use a modified version of the HitHunter cAMP assay. This assay features high signal to background ratios for monitoring cellular activation of GPCRs, particularly G.i -coupled receptors. It is a homogeneous microtiter plate assay for measuring cellular cAMP from cell lysates using Enzyme Fragment Complementation (EFC). We aim to initially implement the DiscoveRx HitHunter cAMP XS technology and then develop further assays described herein, amenable to HTS (moving from 384- to 1536- well format) to screen a 600,000 compound library utilizing robotics for NPY-Y2 receptor antagonists. We will develop and use HA-Y2/CHO-K1 cells and 50% inhibition of NPY at 10 microM compound, measured at a single point will define hits. Confirmation of hits and potencies will be in 10 point dose response curves. Counter-screening of the NPY-Y1 receptor will help evaluate selectivity and parse down hits. IC50 determination will allow selection of compounds for chemical optimization. A radioactive detection method will be used to eliminate "false positives". Successful development and implementation of this assay format will pave the way for future G.s and G.i- coupled receptor HTS campaigns. RELEVANCE TO PUBLIC HEALTH: Many lines of evidence from studies on humans (and animals) suggest that the brain NPY-Y2 receptor is involved in affective disorders (depression as well as anxiety) and in alcoholism. Notably, only a few scattered efforts have previously focused on developing a Y2 receptor drug for psychiatric purposes. Our approach is novel, powerful and part of a comprehensive strategy to succeed in developing such a drug to alleviate human suffering.

描述（由申请人提供）：本申请涉及NIH为开发和调整HTS中使用的生物测定所做的努力。 我们的团队在药物发现和药物筛选活动方面拥有丰富的经验。 我们的总体目标是开发对神经肽Y（NPY）Y2受体显示亲和力的小分子化合物的HTS测定。 具体而言，我们寻求鉴定作为人Y2受体的功能性拮抗剂的化合物。 在优化之后，也将由具有许多制药行业经验的个人在Scripps进行，这样的化合物可能在临床上用于焦虑，抑郁和/或酒精中毒。 目前还不存在通过Y2受体抑制NPY信号传导的临床可测试化合物。 NPY-Y2受体是我们以前首次描述的，特别是最近在人类中的研究使我们比以往任何时候都更加确信它是精神疾病和成瘾的重要靶点。 特异性试剂盒和目的：我们将使用HitHunter cAMP检测试剂盒的改良版本。 该测定的特征在于用于监测GPCR（特别是GPCR偶联受体）的细胞活化的高信号背景比。 这是一种均相微量滴定板试验，用于使用酶片段互补（EFC）测量细胞裂解物中的细胞cAMP。 我们的目标是最初实施DiscoveRx HitHunter cAMP XS技术，然后开发本文所述的进一步测定，适用于HTS（从384孔形式移动到1536孔形式），以利用机器人技术筛选600，000个化合物文库用于NPY-Y2受体拮抗剂。 我们将开发和使用HA-Y2/CHO-K1细胞，并且在单个点测量的10 μ M化合物对NPY的50%抑制将定义命中。 将在10点剂量反应曲线中确认命中率和效价。 NPY-Y1受体的反筛选将有助于评估选择性和解析向下命中。 IC 50测定将允许选择用于化学优化的化合物。 将使用放射性检测方法来消除“假阳性”。 这种测定形式的成功开发和实施将为未来的G.s和G.i偶联受体HTS活动铺平道路。 与公共卫生的相关性：许多来自人类（和动物）研究的证据表明，大脑NPY-Y2受体与情感障碍（抑郁和焦虑）和酗酒有关。 值得注意的是，以前只有少数分散的努力集中在开发用于精神病目的的Y2受体药物上。 我们的方法是新颖的，强大的，是成功开发这种药物以减轻人类痛苦的全面战略的一部分。