Novel Cell-based Real Time Platform for GPCR Drug Discovery

摘要 用于GPCR药物发现的高通量筛选测定受到复合物的阻碍 与当前技术相关的仪器，以及假阳性和关闭的高风险， 目标效应。大多数目前可用的GPCR药物筛选测定是终点检测。 依赖于信号传导发生后GPCR信号传导事件的二级报告子的测定。 这些测定进一步受到测定所需外源试剂的限制 读数。在SBIR提案的第一阶段，我们建议开发一种新的无试剂，真实的- GPCR信号的时间平台技术。所提出的技术的基础是使用 我们的新型荧光蛋白LucY（Lucigen Yellow）作为蛋白质相互作用报告基因。我们 建议按照以下原则开发分裂荧光重组测定法： 双分子荧光互补（BiFC）。LucY对其他分裂有利- 荧光团系统，如基于GFP的荧光团系统，因为其可逆性质， 荧光允许“信号开启”和“信号关闭”筛选。我们提出的分析需要 既不使用辅助试剂，也不使用复杂的仪器，从而最大限度地减少了 假阳性在药物筛选中使用这项技术将需要最少的投资， 仪器和试剂，这很少是这类目标的选择。

Abstract High throughput screening assays for GPCR drug discovery are hampered by the complex instrumentation associated with current technologies, and the high risk of false-positives and off- target effects. The majority of currently available GPCR drug screening assays are end-point assays that rely on secondary reporters of GPCR signaling events after signaling has occurred. These assays are further constrained by the requirement of an exogenous reagent for the assay readout. In the Phase I of this SBIR proposal, we propose to develop a novel reagent-free, real- time platform technology for GPCR signaling. The basis for the proposed technology is the use of our novel fluorescent protein, LucY (for Lucigen Yellow), as a protein interaction reporter. We propose to develop a split-fluorescence reassembly assay following the principles of Bimolecular Fluorescence Complementation (BiFC). LucY is advantageous to other split- fluorophore systems, like those based on GFP, because the reversible nature of its fluorescence allows for both 'signal on' and 'signal off' screening. Our proposed assay requires neither secondary reagents nor complex instrumentation, and thus minimizes the occurrence of false positives. Use of this technology in drug screening will require minimal investments in instrumentation and reagents, which is rarely an option for this class of targets.