Screening Method for GPCRs Related to Appetite

描述（由申请人提供）：旨在减少肥胖和控制饮食失调的药物的开发是制药工业的主要目标，并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药物化合物的工具，这些药物化合物的目标是与食欲控制和饮食行为相关的g蛋白偶联受体（GPCR）。这类药物开发的新靶点来自于最近有关食欲调节的分子和神经回路的大量信息。本提案的重点是开发与摄食行为相关的GPCR家族的测试平台。我们正在追求的药物发现策略的一个显著特征是，它基于一个高效和高度可靠的系统，该系统允许多种受体的复用，以便针对多种靶点测试多种化合物。这一策略得益于密歇根大学Originus公司授权的一项名为表面转染和表达协议（STEP）的新型转染技术，该技术比瞬时甚至稳定的表达方法具有显著优势。在第一阶段的资助下，开发了两个与摄食行为相关的GPCR家族的测试平台——黑色素皮质素受体（mcr）和食欲素受体（oxr）。STEP转染384-we - 11微孔板，检测单独表达的mcr和oxr的表达和配体激活（Specific Aim 1），并通过多个mcr的共表达（Specific Aim 2）在两种不同细胞系中进行了验证。在第二阶段的资助下，我们建议在第一阶段的信息基础上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的等位基因变异，以及在生理相关组合中使用相同或不同家族的多重gpcr。最后，我们建议启动我们自己的内部工作，从一个小的已知化合物文库中对每个微孔中含有单个配体的多路gpcr进行HTS，并在每个微孔中使用来自较大化学文库的药物池对多路gpcr进行超HTS。根据与许多潜在商业伙伴的互动，我们精心设计了拟议的实验，并将使Originus能够证明STEP平台在筛选多路喂养相关的gpcr和其他gpcr方面的功效，这些gpcr不直接用于肥胖药物筛选。拟议的第二阶段工作将是在第三阶段与选定的合作伙伴合作，更广泛地实施库的HTS和uHTS以及测试潜在先导化合物的基础。

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.