Reporter Imaging of Protein-Protein Interactions

描述(由申请人提供)： 随着分子成像在生物学和临床医学的许多领域的应用，分子成像本身就继续演变为一门学科。这项首次竞争性更新申请的长期目标是开发专注于报告基因的技术，用于PET、光学和其他新兴模式，应用于基因治疗、癌症生物学、发育生物学以及许多其他领域。一般的假设是，报告基因技术可以被开发和利用来研究活着的受试者的基本分子/细胞事件。在过去四年的筹资期间，在这方面取得了很好的进展。我们通过开发和验证基于酶的(单纯疱疹病毒1型胸苷激酶(HSVI-tk))和基于受体的(多巴胺2型受体(D2R))报告基因分析，帮助推动了这一领域的发展。我们已经将这些报告基因主要应用于成像基因治疗领域。目前更新的具体目标是通过对活体受试者的细胞内网络成像提供工具，将开发的方法带到下一个水平。蛋白质之间的相互作用是许多生物过程的核心，包括正常组织和疾病组织。在目标1中，我们改进了最近开发的一种策略，通过可诱导酵母双杂交(IY2H)策略来成像发生在细胞核中的蛋白质-蛋白质相互作用。在目标2中，我们改进了一种称为可诱导分裂报告(ISR)策略的替代方法，该策略能够测量细胞内许多位置的蛋白质-蛋白质相互作用。在目标3中，我们测试了在目标1和目标2中开发的成像策略，使用在活体小鼠的肿瘤治疗模型中使用格尔达那霉素类似物来靶向热休克蛋白90(Hsp90)和p23的相互作用的新方法。最后，在目标4中，我们建立了转基因模型，以进一步测试活体小鼠深层组织中蛋白质-蛋白质相互作用的成像方法。在所有目标中，应注意开发可推广的方法，这些方法应适用于使用多模式报告基因方法的其他研究。这项拟议工作的意义在于，它将有助于开发和验证专注于成像细胞内通信网络的新技术。这应该会导致许多应用，包括成像旨在抑制活体受试者蛋白质-蛋白质相互作用的药物相互作用的方法。可能会出现更快速的方法来验证癌症治疗的临床前模型并将其转化为临床。

DESCRIPTION (provided by applicant): Molecular imaging continues to evolve into a discipline in its own right with applications to many areas of biology and clinical medicine. The long-term goal of this first-time competitive renewal application is to develop technologies focused on reporter genes for use with PET, optical, and other emerging modalities for applications to gene therapy, cancer biology, developmental biology, as well as many other areas. The general hypothesis is that reporter gene technologies can be developed and utilized to study fundamental molecular/cellular events in living subjects. Excellent progress has been made in this regard over the last 4 year funding period. We have helped to push the field forward by developing and validating enzyme based (Herpes-simplex Virus Type 1 thymidine kinase (HSVI-tk)) and receptor based (Dopamine Type 2 receptor (D2R)) reporter gene assays. We have applied these reporter genes primarily in the area of imaging gene therapy. The specific aims of the current renewal are to take the developed approaches to the next level by providing tools "systems imaging" by imaging intracellular networks in living subjects. Protein-protein interactions are at the heart of many biological process including normal and diseased tissue. In Aim 1 we refine a recently developed strategy to image protein-protein interactions that occur in the cell nucleus through an inducible yeast two-hybrid (IY2H) strategy. In Aim 2 we refine an alternate approach referred to as an inducible split reporter (ISR) strategy which is capable of measuring protein-protein interactions in many locations within a cell. In Aim 3 we test the imaging strategies developed in Aims 1 and 2 using new approaches being used to target the interaction of heat shock protein 90 (Hsp90) with p23 using geldanamycin analogs in tumor therapy models in living mice. Finally, in Aim 4 we develop transgenic models to further test methods for imaging protein-protein interactions in deep tissues of living mice. In all aims, care is taken to develop generalizable approaches that should be applicable to other studies using multimodality reporter gene approaches. The significance of the proposed work is it will help to develop and validate new technologies focused on imaging intracellular communication networks. This should lead to many applications including methods to image the interaction of drugs designed to inhibit protein-protein interaction in living subjects. More rapid methods to validate and translate pre-clinical models for cancer therapeutics into the clinic will likely result.