In Vitro Screening Strategies

项目2的目标是G蛋白依赖的基于细胞的功能的改进和应用 食欲素1受体(0X1R)活性监测方法，适用于测定食欲素1受体的活性及其作用机制 0X1R拮抗剂的作用及其作为治疗新疗法的潜力 尼古丁依赖症。此外，一系列不依赖G蛋白的基于细胞的功能分析将 开发和优化以进一步表征0X1R配体并允许鉴定化合物 表现出“功能选择性”。项目1中合成的化合物和项目1中出现的化合物 分子文库探针生产中心的OXIR高通量筛选(HTS)活动 (MLPCN)样本收集将以上述检测为特征 高通量重组0X1R。用于评估高优先级有效的选择性的反筛选 还将开发调节剂，并将使用初级化合物确认感兴趣的化合物的活性 神经细胞，以帮助弥合体外和体内药理学之间的差距。这种多重化验 方法与在项目3中生成的药代动力学数据一起被设计为驱动迭代 药物化学计划(项目1)，旨在确定有效的、选择性的、细胞渗透剂0X1R 将推进尼古丁依赖动物模型体内疗效研究的拮抗剂(项目4)。

The objectives of Project 2 are the refinement and application of G protein-dependent cell-based functional assays for monitoring Orexin 1 receptor (0X1R) activity, suitable to determine potency and mechanism of action of 0X1R antagonists that have the potential to be developed as novel therapeutics for the treatment of nicotine dependence. In addition, a series of G protein-independent cell-based functional assays will be developed and optimized to further characterize 0X1R ligands and allow for identification of compounds exhibiting 'functional selectivity'. Compounds synthesized in Project 1 and compounds emerging from the OXIR high throughput screening (HTS) campaign of the 'Molecular Libraries Probe Production Centers Network' (MLPCN) sample collection will be characterized in the aforementioned assays against recombinant 0X1R in high throughput fashion. Counterscreens to assess selectivity of high priority potent modulators will also be developed and activity of interesting compounds will be confirmed using primary neuronal cells to help bridge the gap between in vitro and in vivo pharmacology. This multiple assay approach together with pharmacokinetic data generated in Project 3 is designed to drive an iterative medicinal chemistry program (Project 1) aimed at identifying potent, selective, cell penetrant 0X1R antagonists that will advance to in vivo efficacy studies in animal models of nicotine dependence (Project 4).