DEVELOPING METHODS FOR CRYSTALLIZING FAMILY C GPCRS

描述（由申请人提供）：本提案的目标是开发获得C家族G蛋白偶联受体（GPCR）高分辨率晶体结构的方法。这些结构将极大地促进用于治疗精神和神经障碍的有效且高选择性的变构药物分子的开发。GPCR的特征在于存在由交替的细胞内和细胞外环区分隔的七个跨膜α-螺旋片段。脊椎动物中的GPCR通常根据序列和结构相似性分为五个家族，具体为视紫红质（家族A）、分泌素（家族B）、谷氨酸（家族C）、粘附和卷曲/味觉2。家族C GPCR在结构上不同于家族A受体。虽然它们共享相同的7 TM拓扑结构，但与家族A受体没有序列同源性。家族C受体具有由双叶捕蝇草结构域组成的大的胞外氨基末端配体结合位点。然而，已经可以通过直接结合7 TM束并变构地调节受体活性的小分子药物来调节几种家族C受体的活性。7 TM束是理想的药物靶标，因为与位于捕蝇草结构域中的天然激素结合位点相比，密切相关的受体亚型之间的序列保守性更小。尽管在过去的二十年里，学术界和工业界对GPCR药物的发现进行了非常积极的研究，但新的GPCR药物的数量一直令人失望。药物开发的主要瓶颈之一是缺乏用于识别和优化线索的GPCR的高分辨率结构信息。ConfometRx联合创始人Brian Kobilka的实验室开发了A家族GPCR结晶技术的最新进展：生成GPCR-T4溶菌酶（GPCR-T4 L）融合蛋白。该技术已应用于六个A家族GPCR的高分辨率结构，为基于结构的药物设计提供了新的机会。我们建议将这种技术应用于C家族受体，使用代谢型谷氨酸受体（mGluRs）作为模型系统。mGluRs主要在中枢神经系统中表达，并且是用于治疗几种神经精神障碍的潜在治疗靶标。在SBIR的I期，我们证明了使用GPCR-T4 L技术表达和纯化功能性mGluR-T4 L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，并将使用这些信息来启动一类新的mGluRs药物的开发。为产生mGluR-T4 L蛋白而开发的方法应容易地适用于其它C家族GPCR。该提案是对NIH PA-10-081，在精神健康研究中调查脑源性GPCR的新工具以及膜蛋白结构生物学路线图倡议的回应。 公共卫生相关性：我们建议开发的方法来确定的三维结构的家族C G蛋白偶联受体（GPCRs），这是已知的发挥重要作用，在调节正常的中枢神经系统的神经功能。它们的不同生理作用使它们成为治疗几种精神和神经障碍的可行治疗靶标，包括焦虑症、精神分裂症、抑郁症、帕金森病和阿尔茨海默病等。家族C GPCR的高分辨率晶体结构将为开发更有效和选择性的药物提供有价值的工具。

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.