Using Dendrimers to Design Multivalent Therapeutic Agents

描述（由申请人提供）：本研究项目的总体目标是开发合成的多价框架，用于识别和操纵在细胞间相互作用中重要的生物识别过程。多价在许多生物相互作用中起着关键作用，开发合成多价系统来系统地探索这些过程对于推进多价治疗的合理发展至关重要。由于生物学中典型的个体受体/配体相互作用通常很弱，因此使用多价性来增强这些相互作用可以增强功能的亲和性。此外，多价相互作用可以创建可用于以新方式调节过程的配体模式，而这些结构结构不能由传统的小分子疗法形成。拟议的研究描述了树状大分子作为工具的发展，以促进对旨在介导多价癌细胞识别过程的多价框架要求的理解。我们的假设是，碳水化合物功能化的树状大分子可以用来形成聚集素簇，这些聚集素/糖状树状大分子簇将有效地决定癌细胞的细胞间识别事件。我们选择糖树状大分子作为多价框架，因为它们的大小（生成）和端基功能化易于操作。我们选择凝集素作为我们的靶蛋白是因为它们在癌症过程中的已知功能。三个具体目标如下。1)合成新的碳水化合物功能化树状大分子。2)表征糖树状聚合物/凝集素结合相互作用。3)用糖树突分子进行细胞和动物模型（秀丽隐杆线虫）的测定。针对特定的目标1，提出了化学酶合成形成n-乙酰乳胺和Thomsen-Friedenriech抗原功能化的树状大分子。本文还提出了载肽底物的树状大分子的合成方法。针对特定的目的2，提出了利用荧光寿命波形、荧光各向异性、尺寸排除色谱-多角度光散射、表面等离子体共振和ELISA实验表征糖树状聚合物/凝集素聚集体的方法。由于半乳糖凝集素-1和-3对癌细胞聚集过程的影响，我们的目标是表征糖树状聚合物/半乳糖凝集素相互作用的结合亲和力，以及在糖树状聚合物存在时向细胞显示的半乳糖凝集素的模式。针对特定目标3，我们将与韦恩州立大学Karmanos癌症研究所的Avraham Raz博士合作进行血管生成和细胞粘附试验。我们还建议进行同型细胞聚集试验、趋化性和迁移试验、机械弹性研究、前药模拟物释放的动力学研究以及秀丽隐杆线虫的研究。

DESCRIPTION (provided by applicant): The overarching goal of this research program is to develop synthetic multivalent frameworks for the discernment and manipulation of biological recognition processes that are important in intercellular interactions. Multivalency plays a critical role in many biological interactions, and the development of synthetic multivalent systems to systematically probe these processes is critical for advancement of the rational development of multivalent therapeutics. Because typical individual receptor/ligand interactions in biology are often weak, augmentation of these interactions using multivalency can enhance functional avidity. Moreover, multivalent interactions can create patterns of ligands that can be used to modulate processes in novel ways, and these architectures of structure cannot be formed by traditional small-molecule therapeutics. The proposed research describes the development of dendrimers as tools to advance the understanding of the requirements for multivalent frameworks that are designed to mediate multivalent cancer cellular recognition processes. Our hypothesis is that carbohydrate functionalized dendrimers can be used to form clusters of galectins and that these galectin/glycodendrimer clusters will effectively arbitrate the intercellular recognition events of cancer cells. We have chosen glycodendrimers as the multivalent frameworks for the proposed studies because of the ease of manipulation of their size (generation) and of their end group functionalization. We have chosen galectins as our target proteins because of their known functions in cancer processes. The three specific aims are as follows. 1) Synthesize new carbohydrate-functionalized dendrimers. 2) Characterize the glycodendrimer/galectin binding interactions. 3) Perform cell based and animal model (C. elegans) assays with glycodendrimers. For specific aim 1, chemoenzymatic syntheses to form N-acetyllactosamine and Thomsen-Friedenriech antigen functionalized dendrimers are proposed. The synthesis of dendrimers bearing peptide substrates for matrix metalloproteases is also proposed. For specific aim 2, characterization of glycodendrimer/galectin aggregates is proposed using fluorescence lifetime waveform, fluorescence anisotropy, size exclusion chromatography-multiangle light scattering, surface plasmon resonance and ELISA experiments. Because of the effect of galectins -1 and -3 on cancer cellular aggregation processes, our goal is to characterize the binding affinity of the glycodendrimer/galectin interactions and the pattern of galectin that is displayed to the cells in the presence of glycodendrimers. For specific aim 3, we will perform angiogenesis and cellular adhesion assays in collaboration with Dr. Avraham Raz of the Karmanos Cancer Institute at Wayne State University. We also propose to perform homotypic cellular aggregation assays, chemotaxis and migration assays, mechanoelastic studies, kinetic studies on release of prodrug mimics, and studies with C. elegans.