Proteomic Studies of Dendrimer-based Nanomedicines

描述（由申请人提供）：尽管有许多关于新型纳米药物（如树突-药物偶联物）的设计和递送的报道，但只有相对较少的研究在分子水平上检查了它们的治疗潜力。特别是，目前还没有方便和快速的方法来识别候选药物的细胞内靶点的特定亚群。这项建议的长期目标是应用蛋白质组学方法来识别细胞内药物靶点，并开发基于纳米颗粒的新型治疗剂。本申请的目的是使用树突状蛋白酪氨酸磷酸酶（PTP）抑制剂偶联物作为模型系统，建立一个蛋白质组学平台，有效地、可重复地表征这些纳米药物的特异性和活性。我们的基本原理是，用PTP抑制剂功能化的树状大分子不仅可以有效地在细胞内递送这些小的候选药物，而且还可以提供一个强大的蛋白质组学工具来探测它们在活细胞中的治疗靶点。拟议的研究将利用我们在树状大分子表面创建多功能基团的能力，使它们能够实现针对特定目标的有效细胞内递送，然后可以很容易地分离用于随后的蛋白质组学分析。在强有力的初步数据的指导下，这项急需的技术将通过追求三个具体目标来实现：1)合成含有固定化PTP抑制剂的多功能树状大分子；2)鉴定癌细胞匀浆中的PTP靶点；3)在培养的完整癌细胞中鉴定PTP蛋白靶点。在目标1下，已经证明的合成路线将应用于制备多功能树突状物试剂。在目标2和目标3中，将分别在体外和活细胞中检测这些树突状物质的活性和特异性。这个项目是创新的，因为拟议的研究将是第一个结合使用树状大分子作为药物载体（纳米药物）和潜在的治疗药物与蛋白质组学来确定治疗靶点的研究。它将利用一种强大的技术直接识别活细胞中的治疗靶点。一旦这一策略的实用性得到确立，很有可能将其扩展到大量基于树状大分子和其他纳米粒子的纳米药物的表征。完全开发的工具将在分子水平上精确地提供纳米药物如何与细胞内靶标反应和相互作用的详细知识。这项研究的意义在于它扩展了蛋白质组学的重要作用及其对一个新领域的贡献，并为药物发现提供了一个强有力的工具，使我们能够在分子水平上评估纳米药物的作用。此外，功能化的树状大分子和其他相关纳米颗粒将为新型蛋白质组学试剂提供独特的材料。

DESCRIPTION (provided by applicant): Despite numerous reports of the design and delivery of novel nanomedicines such as dendrimer-drug conjugates, only relatively few studies have examined their therapeutic potential at the molecular level. In particular, there are currently no convenient and quick approaches to identify specific subsets of intracellular targets of drug candidates. The long-term goals of this proposal are to apply proteomic approaches to identify intracellular drug targets and to develop novel therapeutic agents based on nanoparticles. The objective of this application is to use dendrimer-protein tyrosine phosphatase (PTP) inhibitor conjugates as a model system to establish a proteomic platform that efficiently and reproducibly characterizes the specificity and activity of these nanomedicines. Our rationale is that dendrimers functionalized with PTP inhibitors will not only efficiently deliver these small drug candidates intracellularly, but will also provide a powerful proteomic tool to probe their therapeutic targets in living cells. The proposed research will capitalize on our ability to create multi-functional groups on the surface of dendrimers such that they can achieve efficient intracellular delivery for specific targeting and then can be readily isolated for subsequent proteomic analyses. Guided by strong preliminary data, this much needed technology will be achieved by pursuing three specific aims: 1) to synthesize multi-functionalized dendrimers containing an immobilized PTP inhibitor; 2) to identify PTP targets in cancer cell homogenates; and 3) to identify PTP protein targets in intact cancer cells in culture. Under aim 1, an already proven synthetic route will be applied to the preparation of multifunctionalized dendrimer agents. Under aims 2 and 3, the activity and specificity of these dendrimer agents will be examined in vitro and in living cells, respectively. This project is innovative as the proposed studies will be among the first to combine the use of dendrimers as drug carriers (nanomedicines) and potential therapeutic agents with proteomics to identify therapeutic targets. It will capitalize on a powerful technique to directly identify therapeutic targets in living cells. Once the utility of this strategy is established, it is highly likely that it can be expanded to the characterization of a large number of nanomedicines based on dendrimers and other nanoparticles. The fully developed tool will provide detailed knowledge at the molecular level on precisely how nanomedicines react and interact with their intracellular targets. The research will be of significance because it expands the important role of proteomics and its contribution to a new field and provides a powerful tool needed for drug discovery that will allow us to evaluate the actions of nanomedicines at the molecular level. In addition, functionalized dendrimers and other related nanoparticles will provide unique materials for use as novel proteomics reagents. PUBLIC HEALTH RELEVANCE: This application is proposing noteworthy studies that have the potential applicability to identifying therapeutic targets in living systems in high throughput, as well as to developing new generation medicine, in particular nanomedicine. The proposed research has strong relevance to general public health, since the technology can be potentially expanded to any disease and drug discovery area.