Developing Methods for Crystallizing Class B GPCRs

开发 B 类 GPCR 结晶方法

• 批准号: 7748135
• 负责人: HO LEUNG NG
• 金额: $ 10万
• 依托单位: CONFOMETRX, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7748135
• 关键词: Abbreviations; Adrenergic Receptor; Affinity; Agonist; Amino Acids; Binding; Biochemical; Biological Models; C-terminal; Cell membrane; Cells; Characteristics; Chimeric Proteins; Computer Simulation; Crystallization; Crystallography; Data; Development; Drug Delivery Systems; Drug Design; Drug Industry; Endocrine; Family; Fees; Figs - dietary; Fostering; G-Protein-Coupled Receptors; Growth; Housing; Inflammatory; Insecta; Knowledge; Lead; Libraries; Licensing; Ligand Binding; Ligands; Location; Membrane Proteins; Metabolism; Methodology; Methods; Modification; Muramidase; N-terminal; Neurotransmitters; PACAPR-1 protein; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Physiological; Property; Proteins; Protocols documentation; Regulation; Resistance; Resolution; Rhodopsin; Rights; Screening procedure; Services; Small Business Innovation Research Grant; Source; Stream; Structure; Surface; Technology; Transmembrane Domain; United States National Institutes of Health; Universities; base; commercial application; commercialization; design; drug development; drug discovery; experience; extracellular; high throughput screening; insertion/deletion mutation; member; milligram; nervous system disorder; peptide hormone; pituitary adenylate cyclase activating polypeptide; programs; public health relevance; receptor; receptor structure function; scale up; small molecule; structural biology; success; therapeutic target; three dimensional structure; tool development

DESCRIPTION (provided by applicant): G-protein-coupled receptors (GPCRs) represent the largest group of targets for drug discovery. Drug development for these receptors has relied heavily on high-throughput screening of large libraries of compounds. This strategy has seen some successes for a number of receptors, particularly Class A (rhodopsin family) GPCRs for non-peptide hormones and neurotransmitters. However, this approach has been mostly unsuccessful for Class B GPCRs. In this subfamily, the endogenous ligands are all relatively large peptides ranging in size from 27 to 84 amino acids. Class B GPCRs are characterized by an extended ligand recognition surface that includes residues from the transmembrane domains, from the large N- terminal domain, and from the interhelical extracellular segments. This extended ligand recognition surface likely explains the difficulty in developing small molecule drugs selective for Class B GPCRs. Alternative or complementary approaches thus need to be developed that include high-resolution structural information, which can in turn be used to design drugs based on the receptor structure. At present, only a handful of GPCR structures have been solved, and none within the Class B GPCR subfamily. A recent advance in crystallization technology, generating GPCR-T4 Lysozyme (GPCR-T4L) fusion proteins, has led to high-resolution structures of two Class A GPCRs, opening new opportunities for structure-based design of drugs. ConfometRx has obtained the exclusive licensing rights for the commercial application of GPCR-T4L technology, which was developed in Brian Kobilka's lab at Stanford University. We propose here to adapt this technology to Class B receptors. In phase I of this SBIR, we aim to determine the feasibility of using the GPCR-T4L technology to express and purify a functional T4L fusion receptor for a selected Class B GPCR. In phase II, we will proceed with the crystallization trials and structure determination. In addition, once the methodology developed here is established, it will be applied to other Class B GPCRs. The structural information collected with this approach could become a critical component for the successful development of new small molecule drugs. PUBLIC HEALTH RELEVANCE: In this SBIR, we propose to determine the three-dimensional structure of a receptor that belongs to an important family of drug targets, the Class B family of G-protein-coupled receptors. They participate in the physiological regulation of metabolism and growth, and in immuno/neuro-modulations as well as endocrine/exocrine secretions, but drug development targeted at these receptors has been mostly unsuccessful. By solving the structure of such a receptor, we hope to provide a critical tool for the development of new drugs.

描述（由申请人提供）：G蛋白偶联受体（GPCR）代表了最大的药物发现靶标。这些受体的药物开发在很大程度上依赖于对大型化合物库的高通量筛选。该策略为许多受体提供了一些成功，尤其是A类（Rhodopsin家族）GPCR，用于非肽激素和神经递质。但是，对于B类GPCR，这种方法主要是不成功的。在此亚科中，内源配体的大小为27至84个氨基酸。 B类GPCR的特征是扩展的配体识别表面，其中包括来自跨膜结构域的残基，来自大的N末端结构域以及来自细胞间外段的残基。这种扩展的配体识别表面可能解释了为B类GPCR选择小分子药物的困难。因此，需要开发包括高分辨率结构信息的替代方法或互补方法，这些方法又可以根据受体结构来设计药物。目前，仅解决了少数GPCR结构，而B类GPCR亚家族中没有任何结构。结晶技术的最新进步，生成GPCR-T4溶菌酶（GPCR-T4L）融合蛋白，导致了两种A类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。 Confometrx已获得GPCR-T4L技术商业应用的独家许可权，该公司是在斯坦福大学Brian Kobilka实验室开发的。我们在这里建议将该技术适应B类受体。在该SBIR的第一阶段，我们旨在确定使用GPCR-T4L技术表达和纯化选定B类GPCR的功能性T4L融合受体的可行性。在第二阶段，我们将继续进行结晶试验和结构测定。此外，一旦建立了此处开发的方法，它将应用于其他B类GPCR。使用这种方法收集的结构信息可能成为成功开发新的小分子药物的关键组成部分。公共卫生相关性：在此SBIR中，我们建议确定属于重要药物靶标家族的受体的三维结构，即G蛋白偶联受体的B类家族。他们参与了代谢和生长的生理调节，免疫/神经调节以及内分泌/外分泌分泌物，但是针对这些受体的药物开发主要是没有成功的。通过解决这种受体的结构，我们希望为开发新药提供关键的工具。