DEVELOPMENT OF HIGH DENSITY DRUG SCREENING OF NEURAL GPCR's USING STEP ARRAYS

描述（由申请人提供）：基因组学和蛋白质组学的最新进展使药物发现发生了快速变化，通过对分离的蛋白质靶点进行简单的分析，提高了高通量筛选（HTS）方法的速度，但牺牲了信息的深度，并且难以决定在管道中追求哪些化合物。因此，人们认识到需要更智能的筛选平台，特别是强调细胞系统生物学的平台。更复杂的筛选策略对于发现靶向g蛋白偶联受体（gpcr）的新分子尤为重要。越来越多的体外和体内研究表明，GPCRs之间的相互作用可以改变它们的药理特征和运输，甚至可以将它们的偶联转换为不同的细胞内信号通路。此外，在神经科学领域，考虑细胞的细胞背景（即神经元类型）是高度相关的，因为神经元和神经胶质中发生了独特的药物反应。Originus公司可以通过使用密歇根大学独家许可的称为表面转染和表达协议（STEP）的专有固相细胞转染技术，为解决上述所有问题的更复杂的脑gpcr筛选平台的开发做出贡献。提出的新高密度STEP平台将提高屏幕的效率和经济性，同时通过比较小分子同时在神经元背景下对多个脑gpcr的影响来增强信息的深度。此外，这个新的平台可以同时监测每个个体或多重脑gpcr的几个信号通路的激活。大多数在大脑中表达的gpcr在健康的心理功能和精神障碍（包括精神分裂症、焦虑、抑郁和进食相关障碍）的药物治疗中具有潜在的意义。我们将在本提案的第一阶段关注一些神经相关靶点，包括黑素皮质素系统，食欲素系统，大麻素系统和多巴胺能系统。高密度STEP平台将优化为96孔微孔板格式，但每孔包含36个单独的转染点，每个板有3,456个单独的数据点。每个转染点将表达个体或“生理相关”的脑gpcr组合。第一个原型将使用钙通量读数，而第二个原型将允许同时筛选四种不同的报告基因分析。我们计划研究不同gpcr在两种不同细胞背景下的激活反应，并比较该化合物在HTS常用细胞系和神经元细胞系中的药理特征。在第二阶段，我们计划测试更多的GPCR组合，基于它们在神经回路中的共表达，表征在人群中发现的高频等位基因变异，通过RNAi功能丧失分析特定的细胞内通路，并通过高含量分析转向亚细胞成像。尽管目前正在研发中的新药为降低精神障碍的人力成本和经济成本带来了希望，但最近批准的具有治疗脑功能障碍的新作用机制的药物非常有限。该项目的最终目标是开发“智能”药物发现平台，以便更好地了解分子途径、细胞活动和体内神经元功能之间的复杂相互关系，从而使治疗合理地针对复杂的精神疾病。

DESCRIPTION (provided by applicant): Recent advances in genomics and proteomics have produced rapid changes in drug discovery, increasing the speed of the High Throughput Screening (HTS) methods by using simple assays on isolated protein targets but sacrificing depth of information and making decisions difficult as to which compounds to pursue in the pipeline. Thus, the need for smarter screening platforms is being recognized, especially platforms that emphasize Cellular Systems Biology. More sophisticated screening strategies are particularly important for discovering new molecules that target G-protein Coupled Receptors (GPCRs). A growing number of studies in vitro and in vivo have shown interactions between GPCRs that modify their pharmacological profile and trafficking, or even switch their coupling to different intracellular signaling pathways. Moreover, in the field of Neuroscience it is highly relevant to consider the cellular background of the cells (i.e. neuronal type) because of unique drug responses that occur in neurons and glia. Originus Inc. can contribute to the development of more sophisticated screening platforms for brain GPCRs that address all of the above considerations, by using a proprietary solid phase cell transfection technology termed Surface Transfection and Expression Protocol (STEP) exclusively licensed from the University of Michigan. The proposed new High-Density STEP platform will increase the efficiency and economy of the screens while enhancing the depth of information by comparing the effects of small molecules on multiple brain GPCRs simultaneously and in a neuronal background. Moreover, this new platform can be developed to monitor the activation of several signaling pathways for each individual or multiplexed brain GPCRs at the same time. Most GPCRs expressed in brain have potential significance in healthy mental function and in pharmaceutical treatment of mental dysfunction, including schizophrenia, anxiety, depression and feeding- related disorders. We will focus during Phase I of this proposal on some of the neurally relevant targets, including the melanocortin system, the orexin system, the cannabinoid system, and the dopaminergic system. The High-Density STEP platforms will be optimized for a 96-well microplate format but containing 36 individual transfection spots per well, resulting in 3,456 individual data-points per plate. Each transfection spot will express individual or "physiological relevant" combinations of brain GPCRs. The first prototype will use calcium flux readout, while the second prototype will allow the simultaneous screening of four different reporter gene assays. We plan to study the activation responses of the different GPCRs in two different cellular backgrounds and compare the compound's pharmacological profiles in cell lines commonly used in HTS and neuronal cell lines. During Phase II we plan to test more GPCR's combinations based on their co-expression in neural circuits, to characterize allelic variants found in high frequency in populations, to analyze specific intracellular pathways by RNAi loss-of-function, and to move to subcellular imaging via High Content Analysis. PUBLIC HEALTH RELEVANCE Although new medicines today in the research and development pipeline offer hope of reducing the human and economic costs of mental disorders, approvals of drugs with novel mechanisms of action for brain dysfunction have been recently very limited. The ultimate goal of this project is the development of "smart" drug discovery platforms that allow a better understanding of the complex interrelationships of molecular pathways, cellular activities and ultimately in vivo neuronal function to enable therapies rationally aimed at complex mental diseases.