Screening Method for GPCRs Related to Appetite

描述(申请人提供)：旨在减少肥胖和控制饮食失调的药物的开发代表了制药行业的主要目标，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从组合库中高通量测试药物化合物的工具，这些药物化合物针对与食欲控制和饮食行为相关的G蛋白偶联受体(GPCRs)。最近涉及食欲调节的分子和神经回路的信息爆炸，产生了此类药物开发的新靶点。这项提案的重点是为GPCR家庭开发与进食行为相关的测试平台。我们正在追求的药物发现战略的一个显著特征是，它基于一个高效和高度可靠的系统，该系统允许对受体进行多路复用，以便针对许多靶点测试许多化合物。这一策略是由密歇根大学Originus，Inc.授权的一种名为表面转染和表达协议(STEP)的新型转染技术实现的，该技术比瞬时甚至稳定的表达方法具有显著的优势。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--黑素皮质素受体(MCR)和食欲素受体(OXR)。用384-WE11微板分步转染法检测MCR和OXR的单独表达和配体活化(特异性目标1)和多个MCR的多重表达(特异性目标2)。在第二阶段拨款中，我们建议以第一阶段的资料为基础，验证其他与摄食有关的不同细胞内偶联的GPCR的平台，将测试扩展至更多细胞系，开发新的HTS检测方法，优化GPCR的等位基因变体，并在生理上相关的组合中多重使用相同或不同家庭的GPCR。最后，我们建议启动我们自己的内部努力，从一个已知化合物的小库中对每个微板井中具有单配体的多路GPCR进行HTS，并使用每个微板井中较大的化学库中的药物池来进行多路GPCR的超高温超导(UHTS)。拟议的实验是根据与许多潜在业务合作伙伴的互动而精心设计的，将使Originus能够展示STEP平台在筛选与喂养相关的多路GPCR和其他GPCR方面的有效性，这些GPCR不属于肥胖药物筛选的直接领域。拟议的第二阶段工作将成为在第三阶段与选定的合作伙伴合作，更广泛地实施图书馆的高温超导和超高温超导以及测试潜在的铅化合物的基础。

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.