FOUR HELIX BUNDLE ANALOG OF A G PROTEIN COUPLED RECEPTOR

这项建议的目标是开发四股螺旋束类似物 G蛋白偶联受体(GPCRs)，加速基于结构的 这些重要蛋白质的药物开发。GPCR是完整的 介导细胞反应的未知结构的膜蛋白 对不同的刺激，并与许多人类疾病有关。一种可溶的 GPCR的四股螺旋束类似物将有助于结构 脂类或洗涤剂的测定和免除要求 目前阻碍了筛查检测。在包括Beta2在内的几个GPCR中 肾上腺素能受体(BETA2AR)，配体结合亲和力的决定因素 特异性集中在螺旋3-6。他们的定位 与可溶性四股螺旋束蛋白(4HB‘s)相似。 在第一阶段，BETA2AR-4HB将包括螺旋3-6和 来自BETA2AR的其他关键元素。它将进行优化，以实现稳定 在人细胞中的表达、膜的定位和特异性 激动剂和拮抗剂。二硫键可能会被引入以增加 稳定性和手掌部位增加，以确保膜锚定。 第二阶段将涉及BETA2AR-4HB的工程可溶变体， 与G蛋白偶联及膜结合蛋白和G蛋白结构测定 可溶解的形式。在第三阶段，结构信息将用于 确定先导化合物，以及开发的其他GPCRs的类似物。 建议的商业应用： 膜结合形式和可溶性形式的gpr模拟遗嘱的产生 极大地方便了结构确定，并允许基于解决方案 筛选激动剂和拮抗剂。这些进步将会加速 药物发现和基于结构的药物设计工作在整个 并将对GPCR超家族的发展产生重大影响 GPCR相关疾病的治疗药物。GPCR是 全球近30%的药物发现工作，超过60%的 可用药物与GPCR相互作用：估计为840亿美元 1995年的市场。

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.