Towards a novel platelet inhibitor: Targeting the C1 domain of CalDAG-GEFI to inhibit Rap1b

新型血小板抑制剂：靶向 CalDAG-GEFI 的 C1 结构域抑制 Rap1b

• 批准号: 9099516
• 负责人: Mrinalini Ramanan
• 金额: $ 3.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099516
• 关键词: Affect; Affinity; Aspirin; Binding; Binding Sites; Biochemical; Biological Assay; Blood Platelets; Calcium; Calcium Binding; Cause of Death; Cell Line; Cells; Confocal Microscopy; DAG/PE-Binding Domain; Data; Dependence; Diglycerides; Drug Design; Environment; Fibrinogen; Fluorescein-5-isothiocyanate; Foundations; Future; GTP Phosphohydrolase Activators; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Heart Diseases; Hemorrhage; In Vitro; Integrins; Label; Lead; Lipid Binding; Lipids; Maps; Marketing; Mediating; Membrane; Molecular Models; Monitor; Monomeric GTP-Binding Proteins; Mus; Myocardial Infarction; NMR Spectroscopy; Nucleotides; Operative Surgical Procedures; Patients; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Physiological; Platelet Activation; Platelet Inhibitors; Plavix; Play; Proteins; Reading; Regulation; Rest; Risk; Role; Sedimentation process; Signal Transduction; Site; Specificity; System; Testing; Time; United States; Variant; Work; abstracting; biophysical analysis; improved; inhibitor/antagonist; molecular modeling; mutant; novel; novel strategies; prevent; public health relevance

 DESCRIPTION (provided by applicant): Towards a novel platelet inhibitor: Targeting the C1 domain of CalDAG-GEFI to inhibit Rap1b Abstract Several anti-platelet drugs on the market such as Plavix protect patients against heart attacks, but also cause excessive bleeding in patients undergoing surgery, due to irreversible platelet activation. CalDAG-GEFI however, a regulator of the small GTPase Rap1b, reversibly activates platelets and prevents excessive bleeding. Biochemical and biophysical studies of Rap1b and CalDAG-GEFI interactions lay the foundation for future anti-platelet drug design that can takes advantage of the unique anti-thrombotic potential of CalDAG- GEFI. This project aims to build a fundamental molecular model of C1-Rap1b interactions for future drug design efforts. Hypothesis: C1 domain of CalDAG-GEFI critically regulates Rap1b activation through (1) CalDAG-GEFI membrane localization and, (2) initial Rap1b binding for subsequent GEF activation. In Aim 1, we investigate the role of Rap1b-C1 binding in CalDAG-GEF activation. Absence of C1 domain has been shown to reduce Rap1b activation by CalDAG-GEFI. We propose initial recruitment of Rap1b by C1 promotes GEF binding and activation of Rap1b. We will delineate this mechanism by determining nucleotide dependence of C1-Rap1b interactions, mapping sites of interaction using protein NMR, and generating defective C1 mutants to characterize structural changes within each protein that ultimately promote Rap1b binding to GEF domain. We have been able to generate HSQCs for both C1 domain and Rap1b, establishing an NMR-tractable system for studies proposed. In parallel, we investigate the role of lipid binding to the C1 domain for CalDAG-GEFI membrane association and GEF regulation in Aim 2. We propose IPL signaling to C1 is needed for membrane localization of CalDAG-GEFI. We have preliminary data for acidic PhosphatidylInositolPhosphates (PIP) lipids binding to the C1 domain. We will probe the specificity of PIP to C1 domain through in vitro lipid co-sedimentation assays that mimic platelet membrane composition. We will confirm in vitro data regarding Rap1b activation through C1 binding and CalDAG-GEFI localization in a physiological environment. We propose both C1 initial binding to Rap1b and CalDAG-GEFI membrane localization are physiologically relevant phenomena. We will test the functionality and localization of generated CalDAG-GEFI mutants in a platelet-like, megakaryocytic, cell line. Fibrinogen binding and αIIbβ3 binding assays will e used to test functionality of CalDAG-GEFI mutants, and confocal microscopy to monitor CalDAG-GEFI localization.

 描述（由申请人提供）：朝向新型血小板抑制剂：靶向CalDAG-GEFI的C1结构域以抑制Rap1b摘要市场上的几种抗血小板药物如Plastine可保护患者免受心脏病发作，但由于不可逆的血小板活化，也会导致接受手术的患者过度出血。然而，CalDAG-GEFI，一种小的GT3 Rap1b的调节剂，可逆地激活血小板并防止过度出血。Rap1b和CalDAG-GEFI相互作用的生物化学和生物物理研究为未来的抗血小板药物设计奠定了基础，这些药物可以利用CalDAG-GEFI独特的抗血栓形成潜力。该项目旨在为未来的药物设计工作建立C1-Rap 1b相互作用的基本分子模型。假设：CalDAG-GEFI的C1结构域通过（1）CalDAG-GEFI膜定位和（2）初始Rap1b结合随后的GEF激活来关键性地调节Rap1b激活。在目的1中，我们研究Rap1b-C1结合在CalDAG-GEF激活中的作用。C1结构域的缺失已被证明可以减少CalDAG-GEFI对Rap1b的激活。我们建议由C1的Rap1b的初始招聘促进GEF结合和Rap1b的激活。我们将通过确定C1-Rap1b相互作用的核苷酸依赖性，使用蛋白质NMR绘制相互作用位点，并产生有缺陷的C1突变体来表征最终促进Rap1b与GEF结构域结合的每个蛋白质内的结构变化来描述这种机制。我们已经能够生成C1结构域和Rap1b的HSQCs，为拟议的研究建立了一个NMR易于处理的系统。同时，我们研究了脂质结合C1结构域的CalDAG-GEFI膜协会和GEF调节目标2的作用。我们建议IPL信号传导到C1是CalDAG-GEFI膜定位所需的。我们有酸性磷脂酰肌醇磷酸（PIP）脂质结合C1结构域的初步数据。我们将通过体外脂质共沉降试验模拟血小板膜成分来探测PIP对C1结构域的特异性。我们将通过C1结合和生理环境中的CalDAG-GEFI定位确认有关Rap1b激活的体外数据。我们建议C1初始结合Rap1b和CalDAG-GEFI膜定位生理相关的现象。我们将在血小板样巨核细胞系中测试产生的CalDAG-GEFI突变体的功能和定位。将使用纤维蛋白原结合和α IIb β 3结合测定来测试CalDAG-GEFI突变体的功能性，并使用共聚焦显微镜来监测CalDAG-GEFI定位。