Parallel Multimodal High-throughput screening to identify activators of the orexin receptors

项目总结 GPCR超家族的配体调节受体已被证明是一个丰富的靶点来源 人类无数疾病的治疗方法的发展。食欲素1和食欲素2受体为A类 GPCRs在中枢神经系统中有差异表达。食欲素1受体是最丰富的 在蓝斑中表达，被认为控制情绪、奖励和自主神经的各个方面 神经系统，而食欲素2受体在控制觉醒的区域表达，如 结节乳头状核，是调节睡眠/觉醒的重要部位。近20年后，他们的 初步发现，第一个有效的双重增食欲素1/2受体拮抗剂已被带来治疗 失眠市场(BelSomera®、Suvorexant、Merck)。尽管做出了巨大的努力来确定 食欲素受体，几乎没有小分子激动剂的报道出现在初级或专利文献中。雅致 食欲素基因敲除动物的基因消融实验和脑室内给药实验 食欲素A或食欲素B多肽的研究表明食欲素受体激动剂或增强剂对许多潜在的 症状包括：1)抑郁；2)学习和记忆(认知)；3)注意力缺陷多动障碍 (ADHD)；4)结肠癌的治疗；5)包括发作性睡病在内的睡眠障碍。而食欲素多肽是 两种食欲素受体的有效激动剂，它们都不是选择性的，也不能很好地穿越血脑屏障， 使它们成为体内药理学研究的不良探针底物。这提供了识别小的 分子，脑部增食欲素受体的穿透性激活剂，以询问受体的功能 疾病状态。我们已经优化了一种基于细胞的高通量筛选相容的初步检测方法 具体测量食欲素1和食欲素2的功能活性。初步的10k中试筛选 导致了这些受体的一些小分子激动剂的鉴定 该检测方法可靠，重现性好。斯克里普斯机构药物的HTS全面筛查活动 发现文库(~640k化合物)将导致多种增食欲素受体激动剂的鉴定 以及进一步发展的增效剂。在迭代过程中，这些经过验证的命中将被表征 通过一系列基于细胞的体外分析来确定效力、选择性和作用机制。 初步的药物化学先导优化将确定早期的构效关系和体外 将对药物代谢进行评估，以确认早期线索的可伸缩性。这些努力将提供一流的 用于进一步探讨食欲素受体在疾病动物模型中的功能的化学工具。

PROJECT SUMMARY The GPCR superfamily of ligand regulated receptors has proven to be a rich source of targets for development of therapeutics for a myriad of human diseases. The orexin 1 and orexin 2 receptors are class A GPCR’s differentially expressed in the central nervous system. The orexin 1 receptor is most abundantly expressed in the locus coeruleus and is thought to control aspects of emotion, reward, and the autonomic nervous system, whereas the orexin 2 receptor is expressed in regions controlling arousal such as the tubermammillary nucleus, an important site for the regulation of sleep/wakefulness. Almost 20 years after their initial discovery, the first potent dual orexin 1 / orexin 2 receptor antagonist therapeutic has been brought to market for insomnia (Belsomera ®, suvorexant, Merck). Despite a massive effort to identify antagonists of the the orexin receptor, almost no reports of small molecule agonists appear in the primary or patent literature. Elegant genetic ablation experiments using orexin knock-out animals and experiments with intracerebroventricular dosing of orexin A or orexin B peptides suggests a role for orexin receptor agonists or potentiators for a number of potential indications including: 1) depression; 2) learning and memory (cognition); 3) attention deficit hyperactivity disorder (ADHD); 4) treatment for colon cancer; and 5) sleep disorders including narcolepsy. While the orexin peptides are potent agonists of both orexin receptors, they are not selective nor do they cross the blood brain barrier well, making them poor probe substrates for in vivo pharmacology studies. This provides the impetus to identify small molecule, brain penetrant activators of the orexin receptors to interrogate the receptors function in the context of disease states. We have optimized a cell-based high-throughput screening compatible primary assay that specifically measures the functional activity of orexin 1 and orexin 2. A preliminary 10k pilot screen was performed leading to the identification of a number of small molecule agonists of these receptors demonstrating this assay to be robust and reproducible. A full HTS screening campaign of the Scripps Institutional Drug Discovery Library (~640k compounds) will lead to the identification of multiple classes of orexin receptor agonists and potentiators for further development. In an iterative process, these validated hits will be characterized through a cascade of in vitro cell-based assays to determine potency, selectivity and mechanism of action. Preliminary medicinal chemistry lead optimization will identify early structure activity relationships and in vitro drug metabolism will be assessed to confirm tractibility of early leads. These efforts will provide first in-class chemical tools to be used to further probe orexin receptor function in animal models of disease.