Biosensors of GPCR Activation & Inhibition for Drug Discovery

描述（由申请人提供）：配体对G蛋白偶联受体（gpcr）的亲和力，效力和功效的测量是药理学和药物发现的基础。测量GPCR第二信使（如cAMP和肌醇磷酸）产生的细胞功能测定是常规使用的，并且自引入以来在概念上保持不变。然而，就其性质而言，第二信使被高度放大，并在配体-受体相互作用引起的生物物理事件的下游进行反调控反应。此外，剂量反应曲线忽略了受体配体结合的潜在动力学，因此可能在激动剂和拮抗剂的效力测量中给出误导性的结果。随着坎地沙坦（candesartan）、马拉维洛克（maraviroc）等成功疗法的描述，确定GPCR靶向药物改善临床效用的动力学特性的潜力正得到越来越广泛的认可。然而，由于缺乏适当的方法，在药物发现环境中测量结合动力学和内在功效是罕见的。我们假设，在大多数化合物的早期药物化学分析级联中使用这些特性，而不是回顾性地使用一些高级化合物，将提高药物化学工作的整体效率。最近，GPCR生物传感器已经被开发出来，通过荧光共振能量转移作为受体构象的函数的变化来测量结合动力学和内在功效，独立于其他细胞成分。我们建议将这些生物传感器用于药物发现环境的常规使用，并测试这种方法对几种潜在治疗兴趣的其他GPCR的可转移性。该项目将确定生物传感器开发和验证药物发现应用的途径，并将开发特定传感器以实现多种药物发现项目。由于这是最大的经过验证的药物靶点家族，因此加强药物化学工作以创造有效疗法的一般工具可能对公共卫生产生重大的长期影响。

DESCRIPTION (provided by applicant): Measurements of the affinity, potency and efficacy of ligands to G protein-coupled receptors (GPCRs) are fundamental to pharmacology and drug discovery. Cellular functional assays measuring the production of GPCR second messengers such as cAMP and inositol phosphates, are used routinely and remain conceptually unchanged since their introduction. However, by their nature 2nd messengers are highly amplified and counter-regulated responses well downstream of the biophysical events resulting from ligand-receptor interactions. Further, dose response curves neglect the underlying kinetics of receptor ligand binding and can therefore give misleading results for both agonist and antagonist potency measurements. The potential for kinetic properties to determine improved clinical utility of GPCR targeted drugs is becoming more broadly appreciated with the characterization of successful therapeutics such as candesartan, maraviroc and others. However, the measurement binding kinetics and intrinsic efficacy in a drug discovery setting is rare due to the lack of appropriate methods. We hypothesize that availability of these properties early in the medicinal chemistry assay cascade for most compounds, rather than retrospectively for a few advanced compounds, will improve the overall efficiency of medicinal chemistry efforts. Recently, GPCR biosensors have been developed to measure binding kinetics and intrinsic efficacy by changes in Fluorescence Resonance Energy Transfer as a function of receptor conformation, independent of other cellular components. We propose to adapt these biosensors for routine use in a drug discovery environment and test the transferability this approache to several additional GPCR's of potential therapeutic interest. This project will define a path by which biosensors can be developed and validated for drug discovery applications and specific sensors will be developed to enable multiple drug discovery programs. Since this is the largest family of validated drug targets, general tools which enhance medicinal chemistry efforts to create efficacious therapeutics are likely to have a significant long term impact on public health. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop GPCR biosensors for use in drug discovery in order to provide a method for routine measurement of ligand binding kinetics and intrinsic efficacy. If successful, this will improve the ability of medicinal chemists to design drugs which act at GPCR targets that may be useful for a wide range of human diseases.