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疾病的有效和高选择性变构药物分子的发展。gpcr的特点是存在七个跨膜的α -螺旋片段，由细胞内和细胞外环区交替分隔。根据序列和结构相似性，脊椎动物中的gpcr通常分为5个家族，分别是Rhodopsin （Family A）、Secretin （Family B）、Glutamate （Family C）、Adhesion和Frizzled/Taste2。C家族gpcr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位点，由双叶捕蝇草结构域组成。然而，已经有可能通过直接结合7TM束的小分子药物来调节几种C家族受体的活性，并以变构的方式调节受体活性。7TM蛋白束是一个理想的药物靶点，因为与位于捕蝇草结构域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低。尽管在过去的二十年里，GPCR药物的学术和工业研究非常活跃，但新的GPCR药物的数量却令人失望。药物开发的主要瓶颈之一是缺乏用于识别和优化先导物的高分辨率gpcr结构信息。conometrx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-T4L）融合蛋白。该技术已应用于六种A族gpcr的高分辨率结构，为基于结构的药物设计开辟了新的机会。我们建议将该技术应用于C家族受体，以代谢性谷氨酸受体（mGluRs）为模型系统。mGluRs主要在中枢神经系统中表达，是治疗几种神经精神疾病的潜在治疗靶点。在该SBIR的I期研究中，我们证明了使用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确定，并将利用这些信息开始开发一类新的mGluRs药物。用于生成mGluR-T4L蛋白的方法应该很容易适用于其他C家族gpcr。该提案是为了响应NIH PA-10-081，精神健康研究中研究脑源性gpcr的新工具，以及膜蛋白结构生物学的路线图倡议。

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.