Targeting FAK-NEDD9 protein-protein interaction to prevent focal adhesion assembly and vascular proliferation in pulmonary arterial hypertension

项目摘要/摘要 摘要肺动脉高压(PAH)是一种以肺源性疾病为特征的严重心肺疾病。 影响远端血管内皮细胞增殖、细胞外基质(ECM)扩张和间质纤维化 肺小动脉。尽管血管重构对PAH的发病率和早期死亡率很重要 患者，已批准的针对调节ECM扩张或 目前尚不存在血管增殖现象。焦点粘连是一种多聚体跨膜复合体， 内皮细胞与ECM整合素的接口。局灶性粘连导致细胞增殖和细胞外基质扩张 在一个由粘着斑激酶(FAK)启动的过程中。FAK的全局抑制可缓解血管病变 在体内重塑和改善PAH；然而，FAK抑制剂是为癌症量身定做的，并与OFF-1相关。 组织效应被认为是PAH患者的高危因素。因此，开发预防焦虑症的药物 选择性地在病变组织中进行黏附组装是治疗PAH的一种很有前途的方法。 本实验室鉴定神经前体细胞表达发育下调蛋白9 (NEDD9)作为调节PAH中血管纤维化的支架蛋白。NEDD9的SH3结构域是一个 FAK的主要结合靶点：FAK-NEDD9蛋白-蛋白复合体的形成调节灶性黏附 许多细胞系中的信号转导。在目前的提案中，我们首次提出了NEDD9-SH3 晶体结构。我们合成了几种FAK多肽模拟物(NEDD)，并证明了它与 使用多种化学方法将NEDD9转化为NEDD9。从蛋白质结构和新颖的实验 数据中，我们还在FAK结合槽附近的Cys18处发现了NEDD9所特有的活性硫醇残基。 我们进一步表明，肺内皮细胞NEDD9与ECM整合素ITGA5相互作用，并调节一个关键点 涉及血管增殖蛋白血管内皮生长因子的途径。这些观察结果确立了中心假设 当前项目摘要：在肺动脉内皮细胞中，NEDDide(或类似物)抑制FAK-NEDD9 损害焦点粘连组装的复杂结构。我们进一步假设NEDD9-SH3是一个 可修改的靶点，通过它来抑制局部黏附依赖的ECM扩张，血管内皮生长因子信号，从而， 肺动脉高压中的血管重塑。目标1：使用结构导向的方法进行合成和化学优化 用共价探针(S)修饰NEDDide(S)，利用Cys18选择性靶向NEDD9。我们将测试 基于荧光的完整质谱法测定NEDD(S)的结合亲和力、效力和选择性 通过体外和细胞内实验和其他方法证实NEDD(S)对焦点黏附的抑制作用。 目标2是：使用涉及突变的NEDD9结构和NEDDide(S)的获得和功能损失分析来显示 NEDD9-SH3在局灶性粘连中调节ITG5A-VEGF信号，是一个可修饰的靶点，通过它可以 在体外和体内抑制PAH-HPAECs和实验性PAH的ECM重塑和血管增殖。

Project Summary/Abstract Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by pulmonary endothelial proliferation, extracellular matrix (ECM) expansion, and interstitial fibrosis that affects distal pulmonary arterioles. Despite the importance of vascular remodeling to morbidity and early mortality in PAH patients, approved pharmacotherapies targeting the molecular events that regulate ECM expansion or angioproliferation do not exist currently. Focal adhesions are multimeric transmembrane complexes that interface endothelial cells with ECM integrins. Focal adhesions drive cellular proliferation and ECM expansion in a process that is initiated by focal adhesion kinase (FAK). Global inhibition of FAK mitigates vascular remodeling and improves PAH in vivo; however, FAK inhibitors are tailored for cancers and associated with off- tissue effects considered particularly high-risk for PAH patients. Thus, developing drugs that prevent focal adhesion assembly in affected tissue selectively is a promising approach to treat PAH. Our laboratory identified Neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9) as a scaffolding protein that regulates vascular fibrosis in PAH. The SH3 domain of NEDD9 is a principal binding target of FAK: formation of the FAK-NEDD9 protein-protein complex regulates focal adhesion signal transduction in numerous cell lines. In the current proposal, we present for the first time the NEDD9-SH3 crystal structure. We synthesized several FAK peptidomimetics (NEDDtides) and demonstrate binding of the NEDDtides to NEDD9 using numerous chemical methods. From the protein structure and novel experimental data, we also identified a reactive thiol residue at Cys18 near the FAK binding groove that is unique to NEDD9. We further show that pulmonary endothelial NEDD9 interacts with the ECM integrin ITGA5 and regulates a key pathway involving the angioproliferation protein VEGF. These observations establish the central hypothesis of the current project: In pulmonary artery endothelial cells, NEDDtide (or analog) inhibits FAK-NEDD9 complex formation to impair focal adhesion assembly. We postulate further that NEDD9-SH3 is a modifiable target by which to inhibit focal adhesion-dependent ECM expansion, VEGF signaling, and, thus, vascular remodeling in PAH. Aim 1 is: Use a structure-guided approach to synthesis and chemical optimization of NEDDtide(s) modified with covalent probe(s) to leverage Cys18 for targeting NEDD9 selectively. We will test binding affinity, potency, and selectivity of NEDDtide(s) using intact mass-spectrometry, fluorescent-based assays and other methods to demonstrate inhibition of focal adhesion by NEDDtide(s) in vitro and in cellulo. Aim 2 is: Use gain- and loss-of-function assays involving mutant NEDD9 constructs and NEDDtide(s) to show that NEDD9-SH3 modulates ITG5A-VEGF signaling at focal adhesions and is a modifiable target by which to inhibit ECM remodeling and angioproliferation in PAH-HPAECs in vitro and experimental PAH in vivo.