DEVELOPING METHODS FOR CRYSTALLIZING FAMILY C GPCRS

开发 C 系列 GPCR 结晶方法

• 批准号: 7802803
• 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/7802803
• 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; Hormones; Housing; Human; Insecta; Laboratories; Lead; Licensing; Ligand Binding; Ligand Binding Domain; Ligands; Link; Membrane; Membrane Proteins; Mental Depression; Mental Health; 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; Psychiatric therapeutic procedure; 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）：该提案的目的是开发获得C g蛋白偶联受体（GPCR）的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来治疗精神病和神经系统疾病。 GPCR的特征在于存在七个跨膜1螺旋段，这些段通过交替的细胞内和细胞外环区分开。脊椎动物中的GPCR通常按顺序和结构相似性分为五个家庭，指定为Rhodopsin（family A），Secritin（Family B），谷氨酸盐（family C），粘合剂和卷曲/味觉2。 c gpcr家族在结构上与家庭A受体不同。尽管它们共享相同的7 TM拓扑，但与家族A受体没有序列同源性。家族C受体具有大型细胞外氨基末端配体结合位点，该结合位点由双子金星捕捞结构域组成。但是，可以通过小分子药物来调节几个家族C受体的活性，这些药物直接与7TM束结合并通过变构调节受体活性。 7TM束是一个理想的药物靶标，因为与位于金星蝇绑结构域中的天然激素结合位点观察到的密切相关的受体亚型之间的序列保守较少。尽管在过去的二十年中，尽管GPCR对GPCR的药物发现非常积极，但新的GPCR药物的数量令人失望。药物开发的主要瓶颈之一是缺乏有关GPCR的高分辨率结构信息，以识别和优化铅。 Confometrx联合创始人Brian Kobilka实验室开发了一个GPCRs的结晶技术的最新进步：生成GPCR-T4溶菌酶（GPCR-T4L）融合蛋白。该技术已应用于两个家族A 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（MGLURS）作为模型系统将该技术适应家庭C受体。 mglurs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标。在该SBIR的第一阶段，我们旨在确定使用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，我们将继续进行结晶试验和结构确定，并将使用此信息来启动新的MGLURS药物的开发。为生成MGLUR-T4L蛋白而开发的方法应容易适用于其他家族C GPCR。该建议是对NIH PA-06-375的回应，这是用于研究心理健康研究中脑衍生的GPCR的新型工具，以及有关膜蛋白结构生物学的路线图计划。 公共卫生相关性：我们建议开发方法来确定家族C G蛋白偶联受体（GPCR）的三维结构，这些结构在调节中枢神经系统中正常神经元功能方面起着至关重要的作用。它们的多样化生理作用使它们成为治疗几种精神病和神经系统疾病，包括焦虑症，精神分裂症，抑郁症，帕金森氏病和阿尔茨海默氏病的可行治疗靶标。家族C GPCR的高分辨率晶体结构将为开发更有效和选择性药物的开发提供宝贵的工具。