DEVELOPING METHODS FOR CRYSTALLIZING FAMILY C GPCRS

开发 C 系列 GPCR 结晶方法

• 批准号: 8022838
• 负责人: Dan Feng
• 金额: $ 25万
• 依托单位: CONFOMETRX, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-08 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8022838
• 关键词: Abbreviations; Adenosine; Adhesions; Adrenergic Receptor; Affinity; Agonist; Alzheimer' s Disease; Anxiety Disorders; Binding; Binding Sites; Biological Models; Birds; Brain; Cells; Chimeric Proteins; Computer Simulation; Conserved Sequence; Couples; Crystallization; Crystallography; Cysteine-Rich Domain; Data; Development; Disease; Drug Delivery Systems; Drug Design; Drug Industry; Family; Family Planning; Fees; Fostering; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutamates; Goals; Grouping; Homo; Hormones; Housing; Human; Insecta; Laboratories; Lead; Licensing; Ligand Binding; Ligand Binding Domain; Ligands; Link; Membrane; Membrane Proteins; Mental Depression; Mental Health; Mental disorders; Metabotropic Glutamate Receptors; Methodology; Methods; Molecular Sieve Chromatography; Muramidase; Neuraxis; Neurons; Parkinson Disease; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phylogenetic Analysis; Physiological; Play; Proteins; Protocols documentation; Purinergic P1 Receptors; Research; Resistance; Resolution; Rhodopsin; Rights; Role; Schizophrenia; Screening procedure; Secretin; Sequence Homology; Services; Small Business Innovation Research Grant; Source; Specific qualifier value; Stream; Structure; Technology; Transmembrane Domain; United States National Institutes of Health; Universities; Venus Flytrap; Vertebrates; base; commercial application; commercialization; design; dimer; drug development; drug discovery; experience; extracellular; family structure; large scale production; member; monomer; nervous system disorder; neuropsychiatry; novel; programs; public health relevance; receptor; receptor binding; response; scale up; small molecule; structural biology; therapeutic target; three dimensional structure; tool; tool development

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 1-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 two Family A GPCRs (the 22AR and Adenosine A2a receptor), 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 aim to determine 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-06-375, 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.

描述（由申请人提供）：本提案的目标是开发获得 C 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有效且高度选择性的变构药物分子的开发，用于治疗精神和神经系统疾病。 GPCR 的特征是存在七个跨膜 1 螺旋片段，这些片段由交替的细胞内和细胞外环区域分隔开。脊椎动物中的 GPCR 通常根据序列和结构相似性分为 5 个家族，具体为视紫红质（A 家族）、促胰液素（B 家族）、谷氨酸（C 家族）、粘附和卷曲/味觉 2。 C 家族 GPCR 在结构上与 A 家族受体不同。虽然它们具有相同的 7 TM 拓扑结构，但与 A 族受体没有序列同源性。 C 家族受体具有一个大的细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，通过直接与 7TM 束结合并变构调节受体活性的小分子药物，可以调节几种 C 族受体的活性。 7TM 束是理想的药物靶点，因为与位于维纳斯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较少。尽管过去二十年学术界和工业界对 GPCR 药物发现的研究非常活跃，但新 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化合物的 GPCR 高分辨率结构信息。 ConfometRx 联合创始人 Brian Kobilka 的实验室开发了 A 族 GPCR 结晶技术的最新进展：生成 GPCR-T4 溶菌酶 (GPCR-T4L) 融合蛋白。该技术已应用于两个 A 族 GPCR（22AR 和腺苷 A2a 受体）的高分辨率结构，为基于结构的药物设计开辟了新的机会。我们建议使用代谢型谷氨酸受体 (mGluR) 作为模型系统，将该技术应用于 C 族受体。 mGluR 主要在中枢神经系统中表达，是治疗多种神经精神疾病的潜在治疗靶点。在该 SBIR 的第一阶段，我们的目标是确定使用 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，我们将进行结晶试验和结构测定，并将利用这些信息来启动新型 mGluRs 药物的开发。为生成 mGluR-T4L 蛋白而开发的方法应该很容易适用于其他 C 族 GPCR。该提案是对 NIH PA-06-375、心理健康研究中脑源性 GPCR 调查新工具以及膜蛋白结构生物学路线图倡议的回应。 公共健康相关性：我们建议开发确定 C 族 G 蛋白偶联受体 (GPCR) 三维结构的方法，已知 GPCR 在调节中枢神经系统正常神经元功能中发挥重要作用。它们多样化的生理作用使它们成为治疗多种精神和神经疾病的可行治疗靶点，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。 C 家族 GPCR 的高分辨率晶体结构将为开发更有效和更具选择性的药物提供有价值的工具。