DEVELOPING METHODS FOR CRYSTALLIZING FAMILY C GPCRS

描述(申请人提供)：本提案的目标是开发获得C家族G蛋白偶联受体(GPCRs)高分辨率晶体结构的方法。这些结构将极大地促进有效和高度选择性的变构药物分子的开发，用于治疗精神和神经疾病。GPCRs的特征是存在七个跨膜的α-螺旋片段，由交替的细胞内和细胞外环区分隔。脊椎动物中的GPCRs通常根据序列和结构的相似性分为五个家族，分别是视紫红质(A家族)、分泌素(B家族)、谷氨酸(C家族)、粘附性和FrizzledTaste2。C家族GPCRs在结构上不同于A家族受体。虽然它们具有相同的7TM拓扑结构，但与A家族受体没有序列同源性。C家族受体有一个大的胞外氨基末端配体结合部位，由一个双叶捕蝇器结构域组成。然而，通过直接与7TM束结合的小分子药物来调节几个C家族受体的活性，并通过变构来调节受体的活性是可能的。7TM束是一个理想的药物靶点，因为与位于捕蝇器结构域中的天然激素结合部位相比，密切相关的受体亚型之间的序列保守性较低。尽管在过去20年里，学术界和工业界在GPCRs的药物发现方面进行了非常积极的研究，但GPCRs新药的数量一直令人失望。药物开发的主要瓶颈之一是缺乏关于GPCRs的高分辨率结构信息，以确定和优化线索。ConfometRx联合创始人Brian Kobilka的实验室开发了A家族GPCRs结晶技术的最新进展：产生GPCR-T4溶菌酶(GPCR-T4L)融合蛋白。这项技术已应用于6个A族GPCR的高分辨结构，为基于结构的药物设计开辟了新的机遇。我们建议将这项技术应用于C家族受体，使用代谢性谷氨酸受体(MGluRs)作为模型系统。MGluRs主要在中枢神经系统表达，是治疗几种神经精神疾病的潜在靶点。在这项SBIR的第一阶段，我们论证了利用GPCR-T4L技术表达和纯化功能性mGluR-T4L融合蛋白的可行性。在第二阶段，我们将继续进行结晶试验和结构测定，并将利用这些信息来启动新类别mGluRs药物的开发。为产生mGluR-T4L蛋白而开发的方法学应该很容易适用于其他C家族GPCR。这项建议是对NIH PA-10-081的响应，这是在精神健康研究中研究脑源性GPCRs的新工具，以及膜蛋白结构生物学的路线图倡议。 公共卫生相关性：我们建议开发方法来确定C家族G蛋白偶联受体(GPCRs)的三维结构，已知GPCRs在调节中枢神经系统的正常神经功能中发挥重要作用。它们不同的生理作用使其成为治疗几种精神和神经疾病的可行靶点，包括焦虑症、精神分裂症、抑郁症、帕金森氏病和阿尔茨海默病等。C家族GPCRs的高分辨晶体结构将为开发更有效和更具选择性的药物提供宝贵的工具。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop methods to obtain high-resolution crystal structures of Family C G- protein-coupled receptors (GPCRs). These structures would greatly facilitate the development of effective and highly selective allosteric drug molecules for the treatment of psychiatric and neurological disorders. GPCRs are characterized by the presence of seven membrane-spanning alpha-helical segments separated by alternating intracellular and extracellular loop regions. GPCRs in vertebrates are commonly divided into five families by sequence and structural similarity, specified as Rhodopsin (Family A), Secretin (Family B), Glutamate (Family C), Adhesion, and Frizzled/Taste2. Family C GPCRs are structurally distinct from Family A receptors. While they share the same 7 TM topology, there is no sequence homology with Family A receptors. Family C receptors have a large extracellular, amino terminal ligand binding site consisting of a bilobed venus flytrap domain. However, it has been possible to modulate the activity of several Family C receptors by small molecule drugs that bind directly to the 7TM bundle and regulate receptor activity allosterically. The 7TM bundle is an ideal drug target because there is less sequence conservation between closely related receptor subtypes than observed for the native hormone binding site located in the Venus flytrap domain. Despite very active academic and industrial research efforts on drug discovery for GPCRs over the past two decades, the number of new GPCR drugs has been disappointing. One of the major bottlenecks in drug development has been the lack of high-resolution structural information on GPCRs for both identifying and optimizing leads. A recent advance in crystallization technology for family A GPCRs was developed in the laboratory of ConfometRx co-founder Brian Kobilka: generating GPCR-T4 Lysozyme (GPCR-T4L) fusion proteins. This technology has been applied to the high-resolution structures of six Family A GPCRs, opening new opportunities for structure-based design of drugs. We propose to adapt this technology to Family C receptors, using the metabotropic glutamate receptors (mGluRs) as a model system. The mGluRs are expressed primarily in the central nervous system and are potential therapeutic targets for the treatment of several neuropsychiatric disorders. In Phase I of this SBIR, we demonstrated the feasibility of using the GPCR-T4L technology to express and purify functional mGluR-T4L fusion proteins. In Phase II, we will proceed with the crystallization trials and structure determination and will use this information to initiate the development of a new class of mGluRs drugs. The methodology developed for generating mGluR-T4L proteins should be readily applicable to other Family C GPCRs. This proposal is in response to the NIH PA-10-081, Novel Tools for Investigating Brain-derived GPCRs in Mental Health Research, as well as Roadmap initiatives on the Structural Biology of Membrane Proteins. PUBLIC HEALTH RELEVANCE: We propose to develop methods to determine the three-dimensional structures of Family C G-protein- coupled receptors (GPCRs), which are known to play essential roles in regulating normal neuronal function in the central nervous system. Their diverse physiologic roles make them viable therapeutic targets for the treatment of several psychiatric and neurological disorders including anxiety disorders, schizophrenia, depression, Parkinson's disease, and Alzheimer's disease among others. High-resolution crystal structures of Family C GPCRs will provide valuable tools for the development of more effective and selective drugs.