Structural Analysis of TF and Interacting Molecules

精确识别和表征相互作用的分子位置的能力，对于 凝血酶途径和与血栓形成相关的信号通路的研究，将为计算和实验筛选可能有益地调节这些过程的化学制剂打开大门。这类药物的鉴定可能会产生新的介入性分子来更详细地研究这些机制，并可能导致检测和治疗血栓形成及相关疾病的新的诊断和治疗方法。该项目的目标是制作组织因子(TF)与其功能相关伙伴相互作用的物理图像，并利用这些信息帮助选择靶点和设计能够在体外和体内调节组织因子功能的化学试剂。为了实现这一点，我们将利用并结合计算模型、核磁共振和其他生物物理方法来确定和分析组织因子的细胞内和细胞外分子相互作用的结构方面。我们将使用这些结构来定义结合部位和相互作用的“热点”，通过计算筛选小分子文库，并设计特定的配体来调节特定的相互作用。具体地说，我们将利用计算对接和分子力学来开发组织因子/因子V11a与因子IXa和Xa的临时化合物的改进模型；多肽抑制剂与含有Xa的三元化合物的相互作用模型；起始化合物与蛋白酶激活的受体PAR-1和PAR-2的细胞外相互作用模型；以及Tf与相关整合素结构的相互作用模型。利用核磁共振和其他生物物理技术，结合计算模型，我们将表征细胞质Tf C末端结构域(TFCD)在磷酸化和非磷酸化状态下与信号伙伴的细胞内相互作用。从这些结构分析中，我们将定义相互作用目标，并筛选能够调节这些相互作用的配体的小分子数据库。为了能够进行这些研究，我们将扩展和增强我们的计算对接代码，以处理这些多组分、灵活的分子系统的复杂性。

The ability to precisely identify and characterize the molecular sites of interaction, both for the coagulation protease pathway and for the signaling pathways related to thrombogenesis, will open the door for computational and experimental screening of chemical agents that may beneficially modulate these processes. The identification of such agents may generate new classes of interventional molecules to study these mechanisms in greater detail and may lead to new diagnostic and therapeutic approaches to the detection and treatment of the thrombotic and related diseases. The goal of this project is to produce a physical picture of the interactions of Tissue Factor (TF) with its functionally relevant partners, and to utilize this information to help select targets and design chemical agents that can modulate TF function in vitro and in vivo. To accomplish this we will determine and analyze the structural aspects of the intracellular and extracellular molecular interactions of Tissue Factor by utilizing and combining computational modeling, NMR and other biophysical methods. We will use these structures to define binding sites and interaction "hot spots", computationally screen small molecule libraries, and design specific ligands to modulate particular interactions. Specifically we will utilize computational docking, and molecular mechanics to develop improved models of the temary complexes of tissue factor/factor Vlla with factors IXa and Xa; model interactions of peptide inhibitors with the ternary complex containing Xa; develop models for the extracellular interactions of the initiation complex with the protease activated receptors PAR-1 and PAR-2; and develop models of the interaction of TF with relevant integrin structures. Utilizing NMR, and other biophysical techniques in conjunction with computational modeling, we will characterize the intracellular interactions of the cytoplasmic TF C-terminal domain (TFCD) in both its phosphorylated and unphosphorylated states with signaling partners. From these structural analyses we will define interaction targets and screen small molecule databases for ligands that can modulate these interactions. To enable these studies we will extend and enhance our computational docking codes to deal with the complexity of these multi-component, flexible molecular systems.