Structural Analysis of TF and Interacting Molecules

精确识别和表征相互作用的分子位点的能力， 凝血蛋白酶途径和与血栓形成相关的信号传导途径，将为可能有益地调节这些过程的化学试剂的计算和实验筛选打开大门。这些试剂的鉴定可以产生新类别的干预分子，以更详细地研究这些机制，并可能导致新的诊断和治疗方法来检测和治疗血栓性疾病和相关疾病。该项目的目标是产生组织因子（TF）与其功能相关的合作伙伴的相互作用的物理图像，并利用这些信息来帮助选择目标和设计可以在体外和体内调节TF功能的化学试剂。为了实现这一点，我们将确定和分析的组织因子的细胞内和细胞外分子相互作用的结构方面，通过利用和结合计算建模，NMR和其他生物物理方法。我们将使用这些结构来定义结合位点和相互作用的“热点”，计算筛选小分子库，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VIIa与因子IXa和Xa的三元复合物的改进模型;肽抑制剂与含有Xa的三元复合物的模型相互作用;开发起始复合物与蛋白酶激活受体PAR-1和PAR-2的细胞外相互作用的模型;并开发TF与相关整合素结构相互作用的模型。利用NMR和其他生物物理技术结合计算建模，我们将表征细胞质TF C-末端结构域（TFCD）在其磷酸化和非磷酸化状态下与信号传导伙伴的细胞内相互作用。从这些结构分析，我们将定义相互作用的目标和筛选小分子数据库的配体，可以调节这些相互作用。为了使这些研究，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.