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

描述(由申请人提供)：本申请涉及美国国立卫生研究院开发和调整用于HTS的生物检测的努力。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分析。具体地说，我们试图确定在人类Y2受体上具有功能性拮抗剂的化合物。在斯克里普斯具有丰富制药行业经验的个人也将进行优化之后，这种化合物可能在临床上对焦虑、抑郁和/或酒精中毒有用。目前还没有通过Y2受体抑制NPY信号的临床可测化合物存在。NPY-Y2受体是我们以前首先描述的，最近对人类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。具体目标和目的：我们将使用HitHunter阵营试验的修改版本。该方法具有高信号与背景比的特点，可用于监测GPCRs的细胞激活，特别是G.I偶联受体。这是一种利用酶片段互补(EFC)从细胞裂解物中测定细胞cAMP的均一微量平板分析方法。我们的目标是最初实施DiscoveRx HitHunter Camp XS技术，然后开发本文描述的进一步分析方法，适用于HTS(从384孔格式移动到1536孔格式)，以利用机器人技术筛选60万个化合物文库，寻找NPY-Y2受体拮抗剂。我们将开发和使用HA-Y2/CHO-K1细胞，在10微米的化合物中对NPY的50%抑制将定义为HITS。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解析命中。IC50测定将允许选择化合物进行化学优化。将使用一种放射性检测方法来消除“假阳性”。这种分析形式的成功开发和实施将为未来G.S和G.I偶联受体HTS活动铺平道路。与公共健康相关：来自对人类(和动物)的研究的许多证据表明，大脑NPY-Y2受体与情感障碍(抑郁和焦虑)和酒精中毒有关。值得注意的是，之前只有几个零星的努力专注于开发一种用于精神疾病的Y2受体药物。我们的方法新颖、有效，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.