FOUR HELIX BUNDLE ANALOG OF A G PROTEIN COUPLED RECEPTOR

G 蛋白偶联受体的四螺旋束类似物

• 批准号: 2785459
• 负责人: Clifford Robinson
• 金额: $ 9.85万
• 依托单位: THREE-DIMENSIONAL PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2785459
• 关键词: G protein; beta adrenergic receptor; cell line; chemical stability; fluorescence microscopy; gene expression; membrane activity; molecular cloning; polymerase chain reaction; protein engineering; protein localization; protein structure; receptor binding; receptor coupling; transfection

The objective of this proposal is to develop four-helix bundle analogs of G-Protein-Coupled Receptors (GPCRs), to accelerate structure-based drug development for these important proteins. GPCRs are integral membrane proteins of unknown structure that mediate cellular responses to diverse stimuli and are linked to many human diseases. A soluble four-helix bundle analog of a GPCR would facilitate structure determination and obviate requirements for lipids or detergents that currently hamper screening assays. In several GPCRs including the BETA2 adrenergic receptor (BETA2AR), determinants of ligand binding affinity and specificity arc concentrated in helices 3-6. Their orientation resembles that of soluble four-helix bundle proteins (4HB's). In Phase I BETA2AR-4HB will be constructed incorporating helices 3-6 and other key elements from BETA2AR. It will be optimized for stable expression in human cells, membrane localization, and specificity for agonists and antagonists. Disulfide bonds may be introduced to increase stability and a palmitation site added to ensure membrane anchoring. Phase II will involve engineering soluble variants of BETA2AR-4HB which couple to G-Proteins, and structure determination of membrane-bound and soluble forms. In Phase III, structural information will be used to identify lead compounds, and analogs of other GPCRs developed. PROPOSED COMMERCIAL APPLICATION: Generation of membrane-bound and soluble forms of the GPCR analog will greatly facilitate structure determination, and allow solution-based screens for agonists and antagonists. These advancements will accelerate drug discovery and structure based drug design efforts for the entire GPCR superfamily and will have a major impact on development of therapeutic agents for GPCR related diseases. GPCRs are targets for nearly 30% of drug discovery efforts worldwide, and over 60% of available drugs interact with a GPCR: estimated to be a $84 billion market in 1995.

本提案的目的是开发四螺旋束类似物 G蛋白偶联受体（GPCR），以加速基于结构的 针对这些重要蛋白质的药物开发。GPCR不可或缺 介导细胞反应的未知结构的膜蛋白 与多种刺激有关，并与许多人类疾病有关。可溶性 GPCR的四螺旋束类似物将促进结构 油脂或洗涤剂的测定和检验要求， 目前妨碍筛选测定。在几个GPCR中，包括BETA 2 肾上腺素能受体（BETA2 AR），配体结合亲和力的决定因素 和特异性集中在螺旋3-6中。它们的取向 类似于可溶性四螺旋束蛋白（4 HB）。 在第一阶段，将构建包含螺旋3-6和 BETA2 AR的其他关键元素。它将被优化为稳定 在人细胞中的表达，膜定位，和特异性 激动剂和拮抗剂。可以引入二硫键以增加 稳定性和添加的棕榈化位点以确保膜锚定。 第二阶段将涉及BETA2 AR-4 HB的工程可溶性变体， 偶联到G蛋白，以及膜结合和 可溶形式。在第三阶段，结构信息将用于 识别先导化合物以及开发的其他GPCR类似物。 拟定商业应用： GPCR类似物的膜结合和可溶形式的产生将 极大地方便了结构确定，并允许基于溶液 筛选激动剂和拮抗剂。这些进步将加速 药物发现和基于结构的药物设计工作， GPCR超家族，并将产生重大影响的发展 用于GPCR相关疾病的治疗剂。GPCR是 全球近30%的药物发现工作，超过60%的 现有药物与GPCR相互作用：估计为840亿美元 1995年市场。