Molecular Imaging of G-Protein-Coupled Receptors for Drug Development

描述（由申请人提供）：g蛋白偶联受体（gpcr）是人类中最重要的蛋白质之一，因为它们是许多信号通路的“门户”，包括那些导致疾病的信号通路，并且它们的功能可通过药物干预。通过识别和结合特定的化学物质，gpcr将信号从细胞外部传递到细胞内部，在那里它们触发一系列生物事件。因此，gpcr是结构测定中最丰富的一类蛋白质。它们是最大的一类膜蛋白；人类基因组编码约800个gpcr，其中约一半是潜在的药物靶点。然而，目前仅60种受体用于小分子药物，25种受体用于基于天然配体的生物治疗；然而，与gpcr结合的药物约占所有治疗药物的三分之一。到2014年，全球每年与gpcr相互作用的药物市场预计将达到1180亿美元。另外还有80多种gpcr可能适用于抗体疗法，这是一种受到越来越多关注的新型攻击途径，我们将继续探索。从液晶磷脂双层中的大膜蛋白制备用于x射线晶体学和溶液核磁共振的样品的困难一直是基于结构的药物发现的主要障碍。为了克服这一障碍，我们同时开发了固态核磁共振光谱的新方法，并将其应用于膜蛋白的结构测定。我们的主要目标是趋化因子受体CXCR1，在该合同的前五年，我们在确定其结构方面取得了实质性进展。我们对白细胞介素-8 （IL-8）与CXCR1相互作用的研究为发现可能通过与CXCR1结合而影响炎症、癌症转移和其他疾病的药物提供了一个框架。为了加速设计和发现与CXCR1和其他gpcr相互作用的药物，我们正在开发一种通用的方法来确定生理条件下gpcr在其天然磷脂环境中的三维结构。这项研究是多学科的，涉及分子生物学、生物化学、结构生物学、核磁共振波谱学和计算。它在培养能够跨越化学和生物边界工作和互动的科学家方面非常有效。我们认识到，这需要现有的最高水平的技术；因此，它是由加州大学圣地亚哥分校（UCSD）和两家生物技术公司（马萨诸塞州安多弗的剑桥同位素实验室（CIL）和加利福尼亚州圣地亚哥的膜受体技术（MRT））组成的生物工程研究合作伙伴关系。公司的关键技术是合成同位素标记的氨基酸和前体，制造独特的同位素标记细菌生长培养基；MRT拥有表达、纯化和重折叠生物活性gpcr的技术，该技术最初是由M-Fold生物技术公司的汉斯·基弗博士开发的，他仍然参与这项研究。

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are among the most important proteins in humans because they are the 'gateway' for many signally pathways, including those that contribute to diseases, and their functions are amenable to intervention by drugs. By recognizing and binding specific chemicals, GPCRs transduce signals from the outside of cell to the inside where they trigger a cascade of biological events. Consequently, GPCRs are the most fecund class of proteins for structure determination. They are the largest class of membrane proteins; ~800 GPCRs are encoded in the human genome, about half of which are potential drug targets. However, only ~60 of them are currently used as receptors for small molecule drugs and ~25 of them for bio- therapeutics based on the natural ligand; nonetheless, drugs that bind to GPCRs account for about one-third of all therapeutic drugs. The annual worldwide market for drugs that interact with GPCRs is predicted to be $118 Billion by 2014. More than 80 additional GPCRs are potentially amenable to antibody therapeutics, a novel avenue of attack that is receiving increasing attention, and one that we will explore. The difficulty in preparing samples for X-ray crystallography and Solution NMR from large membrane proteins in liquid crystalline phospholipid bilayers has been the principal roadblock to structure-based drug discovery. In order to overcome this roadblock we have simultaneously developed new methods for solid-state NMR spectroscopy and applied to structure determination of membrane proteins. Our principal target is the chemokine receptor CXCR1, and we have made substantial progress towards determining its structure during the first five years of this award. Our studies of interleukin-8 (IL-8) interacting with CXCR1 provide a framework for discovery of drugs that may affect inflammation, cancer metastasis, and other diseases by binding to CXCR1. In order to accelerate the design and discovery of drugs that interact with CXCR1 and other GPCRs, we are developing a general method for determining the three-dimensional structures of GPCRs in their native phospholipid environment under physiological conditions. This research is multidisciplinary, involving molecular biology, biochemistry, structural biology, NMR spectroscopy, and computation. It is highly effective at training scientists who can work and interact across chemical and biological boundaries. We recognize that it requires the highest levels of technology available; as a result, it is organized as a Bioengineering Research Partnership among the University of California, San Diego (UCSD) and two biotechnology companies (Cambridge Isotope Laboratories (CIL), Andover, Massachusetts and Membrane Receptor Technologies (MRT), San Diego, California. CIL's key technology is the synthesis of isotopically labeled amino acids and precursors, and the manufacture of unique isotopically labeled bacterial growth media; their effort is led by Joel Bradley, Ph.D. MRT has the technology for the expression, purification, and refolding of biologically active GPCRs developed originally by Hans Kiefer, Ph.D., at M-Fold Biotech and he remains involved in the research. PUBLIC HEALTH RELEVANCE: Most diseases that afflict humans can be treated or cured with drugs. The majority of therapeutic drugs are chemicals targeted to protein receptors that reside in cell membranes, the largest class of which is G-protein coupled receptors (GPCRs). Determining the structures of GPCRs is a very high priority goal of biomedical research because it will accelerate the discovery of new drugs to treat a wide range of diseases. The research project described in this proposal will advance the method of NMR spectroscopy so that it can be used to determine the structures of GPCRs in their native environment of phospholipid bilayers under physiological conditions. This is a technically demanding project that requires the methods of chemistry, physics, and biology. This project is focused on the chemokine receptor CXCR1 and its interactions with its natural ligand interleukin-8 (IL-8) as an example that affects several diseases, including inflammatory disease and cancer metastasis. The methods demonstrated with this example should be applicable to many other GPCRs (and diseases) as well as other classes of membrane proteins.