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

描述（由申请人提供）：本申请涉及NIH开发和调整用于HTS的生物测定的努力。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-受体具有亲和力的小分子化合物的HTS检测方法。具体地说，我们试图确定在人类Y2受体的功能性拮抗剂的化合物。经过优化后，这种化合物可能在临床上对焦虑、抑郁和/或酒精中毒有用，这也是斯克里普斯制药行业经验丰富的个人所追求的。目前还没有临床可测试的化合物可以通过y2受体抑制NPY信号传导。NPY-Y2受体是我们之前首次描述的，最近在人类身上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。具体目的和目的：我们将使用HitHunter cAMP试验的改进版本。该检测具有高信号背景比，用于监测gpcr的细胞活化，特别是gi偶联受体。这是一种使用酶片段互补（EFC）从细胞裂解物中测量细胞cAMP的均匀微滴板测定法。我们的目标是初步实施DiscoveRx HitHunter cAMP XS技术，然后开发本文所述的进一步分析，适用于HTS（从384孔格式移动到1536孔格式），利用机器人技术筛选60万个化合物库，用于NPY-Y2受体拮抗剂。我们将开发和使用HA-Y2/CHO-K1细胞，并在10微米化合物中抑制50%的NPY，在单点测量将确定命中。命中和效力的确认将在10点剂量反应曲线中进行。NPY-Y1受体的反筛选将有助于评估选择性和分析命中。IC50的测定将允许选择化合物进行化学优化。一种放射性检测方法将用于消除“假阳性”。这种检测格式的成功开发和实施将为未来G.s和g.i.偶联受体HTS运动铺平道路。与公共卫生相关：来自人类（和动物）研究的许多证据表明，大脑NPY-Y2受体与情感性障碍（抑郁和焦虑）和酒精中毒有关。值得注意的是，以前只有少数零散的努力集中在开发用于精神病学目的的Y2受体药物上。我们的方法新颖有力，是成功开发这种药物以减轻人类痛苦的综合战略的一部分。

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.