Screening Method for GPCRs Related to Appetite

与食欲相关的GPCR的筛选方法

• 批准号: 7123080
• 负责人: MARIA INES MORANO
• 金额: $ 44.8万
• 依托单位: ORIGINUS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7123080
• 关键词: alleles; appetite; cell line; cell surface receptors; combinatorial chemistry; drug screening /evaluation; eating disorders; fluorescence microscopy; high throughput technology; obesity; receptor coupling; receptor expression; technology /technique development; transfection

DESCRIPTION (provided by applicant): The development of drugs aimed at reducing obesity and controlling eating disorders represents a major target of the pharmaceutical industry and has become one of the most exciting areas in drug discovery. The long term goal of this project is to develop tools for high throughput testing of drug compounds from combinatorial libraries that target G-Protein-Coupled Receptors (GPCR's) associated with appetite control and eating behavior. Novel targets for such drug development arise from the recent explosion of information on molecules and neural circuits involved in appetite regulation. This proposal focuses on developing testing platforms for GPCR families associated with feeding behavior. A distinguishing feature of the drug-discovery strategy we are pursuing is that is based on an efficient and highly reliable system that allows multiplexing of receptors in order to test numerous compounds against numerous targets. This strategy has been enabled by a novel transfection technology that is licensed by Originus, Inc. from the University of Michigan termed Surface Transfection and Expression Protocol (STEP), which has significant advantages over transient and even stable expression methods. Under Phase I funding, testing platforms for two GPCR families associated with feeding behavior-the Melanocortin Receptors (MCRs) and the Orexin Receptors (OXRs) were developed. STEP transfection of 384-we 11 microplates to test the expression and ligand activation of individually expressed MCRs and OXRs (Specific Aim 1) and multiplexing via co-expression of multiple MCRs (Specific Aim 2) were validated in two different cell lines. Under Phase II funding, we propose to build on the information from Phase I to validate platforms for other feeding-related GPCRs with different intracellular coupling, to extend the testing to additional cell lines, to develop new assays for HTS, to optimize allelic variants of the GPCRs, and to multiplex GPCRs of the same or different families in physiological relevant combinations. Finally we propose to initiate our own internal efforts for HTS of multiplexed GPCRs with single ligands per microplate well from a small library of known compounds and for ultraHTS (uHTS) of multiplexed GPCRs using pools of drugs from larger chemical libraries in each microplate well. The proposed experiments have been carefully designed based on interactions with many potential business partners and will allow Originus to demonstrate the efficacy of the STEP platforms for screening multiplexed feeding-related GPCRs and other GPCRs outside the immediate area of obesity drug screening. The proposed Phase II work will be the basis for the implementation of more broadly HTS and uHTS of libraries and testing of potential lead compound in collaboration with selected partners during Phase III.

描述（由申请人提供）：开发旨在减少肥胖和控制饮食失调的药物是制药行业的主要目标，并且已成为药物发现中最令人兴奋的领域之一。该项目的长期目标是开发用于高通量测试组合库中药物化合物的工具，这些组合库以与食欲控制和饮食行为相关的 G 蛋白偶联受体 (GPCR) 为目标。此类药物开发的新目标源于最近有关食欲调节的分子和神经回路信息的爆炸式增长。该提案的重点是为与进食行为相关的 GPCR 家族开发测试平台。我们所追求的药物发现策略的一个显着特征是基于高效且高度可靠的系统，该系统允许多重受体，以便针对众多靶点测试多种化合物。这一策略是通过密歇根大学 Originus, Inc. 授权的新型转染技术实现的，该技术称为表面转染和表达方案 (STEP)，该技术比瞬时甚至稳定表达方法具有显着优势。在第一阶段的资助下，开发了两个与进食行为相关的 GPCR 家族——黑皮质素受体 (MCR) 和食欲素受体 (OXR) 的测试平台。在两种不同的细胞系中验证了 384-we 11 微孔板的 STEP 转染，以测试单独表达的 MCR 和 OXR（具体目标 1）的表达和配体激活，以及通过多个 MCR 共表达进行复用（具体目标 2）。在第二阶段的资助下，我们建议以第一阶段的信息为基础，验证具有不同细胞内偶联的其他喂养相关 GPCR 的平台，将测试扩展到其他细胞系，开发新的 HTS 检测方法，优化GPCR，并以生理学相关组合复用相同或不同家族的 GPCR。最后，我们建议启动我们自己的内部工作，对来自已知化合物的小型库的每个微板孔使用单个配体的多重 GPCR 进行 HTS，以及使用来自每个微板孔中较大化学库的药物池进行多重 GPCR 的 ultraHTS (uHTS)。拟议的实验是根据与许多潜在商业合作伙伴的互动精心设计的，将使 Originus 能够证明 STEP 平台在筛选多重喂养相关 GPCR 和肥胖药物筛选直接领域之外的其他 GPCR 方面的功效。拟议的第二阶段工作将成为在第三阶段期间与选定的合作伙伴合作实施更广泛的 HTS 和 uHTS 库以及测试潜在先导化合物的基础。