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高通量测试来自组合库的药物化合物的工具，该组合库靶向与食欲控制和进食行为相关的G蛋白偶联受体（GPCR）。这种药物开发的新靶点来自于最近有关食欲调节分子和神经回路的信息爆炸。该提案的重点是为与喂养行为相关的GPCR家庭开发测试平台。我们所追求的药物发现策略的一个显著特征是基于一个高效且高度可靠的系统，该系统允许受体的多重化，以便针对众多靶标测试众多化合物。该策略已通过由Originus，Inc.许可的新型转染技术实现。来自密歇根大学的称为表面转染和表达方案（STEP）的方法，其相对于瞬时甚至稳定表达方法具有显著的优点。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-黑皮质素受体（MCRs）和食欲素受体（OXRs）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（特异性目标1）的表达和配体活化，以及通过多种MCR（特异性目标2）的共表达的多重化。在第二阶段的资助下，我们建议建立在第一阶段的信息基础上，以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的等位基因变体，并在生理相关组合中多重检测相同或不同家族的GPCR。最后，我们建议启动我们自己的内部努力，HTS的多重GPCR与单一配体每个微孔板孔从一个小的图书馆的已知化合物和ultraHTS（uHTS）的多重GPCR使用池的药物从更大的化学库在每个微孔板孔。根据与许多潜在商业合作伙伴的互动，精心设计了拟议的实验，并将使Originus能够证明STEP平台筛选多重喂养相关GPCR和肥胖药物筛选直接领域以外的其他GPCR的有效性。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.