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

靶向FAK-NEDD9蛋白-蛋白相互作用预防肺动脉高压中的粘着斑组装和血管增殖

• 批准号: 10656686
• 负责人: Sirano Dhe-Paganon
• 金额: $ 63.68万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656686
• 关键词: 3-Dimensional; Ablation; Affect; Affinity; Animal Model; Arterial Disorder; Arteries; Attenuated; Binding; Biological Assay; Blood Vessels; Cancer cell line; Cell Line; Cell Proliferation; Cells; Cessation of life; Chemicals; Clinical; Complex; Connective Tissue; Data; Development; Disease; Distal; Endothelial Cells; Endothelium; Event; Experimental Models; Extracellular Matrix; Fibrillar Collagen; Fibronectins; Fibrosis; Fluorescence Recovery After Photobleaching; Focal Adhesion Kinase 1; Focal Adhesions; Genetic; Heart Diseases; Heart failure; Hematological Disease; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human; ITGA5 gene; Impairment; In Vitro; Integrins; Laboratories; Link; Lung; Lung diseases; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Morbidity - disease rate; Neural Groove; Neural Inhibition; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Positioning Attribute; Process; Proliferating; Proteins; Proteomics; Pulmonary Heart Disease; Pulmonary Vascular Resistance; Pulmonary arterial remodeling; SH3 Domains; Scaffolding Protein; Signal Transduction; Small Interfering RNA; Specificity; Structure; Sulfhydryl Compounds; Testing; Time; Tissues; Vascular Endothelial Growth Factors; Vascular Proliferation; Vascular remodeling; X-Ray Crystallography; adverse outcome; analog; angiogenesis; arteriole; body system; cell type; design; effective therapy; experimental study; gain of function; high risk; improved; in vivo; innovation; interstitial; kinase inhibitor; liquid chromatography mass spectrometry; loss of function; matrigel; molecular drug target; mortality; mutant; nerve stem cell; novel; overexpression; peptidomimetics; prevent; protein complex; protein protein interaction; protein structure; pulmonary arterial hypertension; pulmonary artery endothelial cell; pulmonary vascular remodeling; right ventricular failure; small molecule; targeted treatment

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.

项目概要/摘要 肺动脉高压（PAH）是一种严重的心肺疾病，其特征是肺动脉高压 内皮增殖、细胞外基质 (ECM) 扩张和影响远端的间质纤维化 肺小动脉。尽管血管重塑对 PAH 的发病率和早期死亡率很重要 患者，针对调节 ECM 扩张的分子事件的批准药物疗法或 目前不存在血管增殖。粘着斑是多聚跨膜复合物， 内皮细胞与 ECM 整合素的界面。粘着斑驱动细胞增殖和 ECM 扩张 由粘着斑激酶 (FAK) 启动的过程。 FAK 的整体抑制可减轻血管 重塑并改善体内PAH；然而，FAK 抑制剂是针对癌症而设计的，并且与非 组织效应被认为对 PAH 患者来说风险特别高。因此，开发预防局灶性药物 选择性地在受影响的组织中进行粘附组装是治疗 PAH 的一种有前途的方法。 我们的实验室鉴定出神经前体细胞表达发育下调蛋白 9 (NEDD9) 作为调节 PAH 血管纤维化的支架蛋白。 NEDD9 的 SH3 结构域是 FAK 的主要结合靶点：FAK-NEDD9 蛋白-蛋白复合物的形成调节粘着斑 许多细胞系中的信号转导。在当前的提案中，我们首次提出了 NEDD9-SH3 晶体结构。我们合成了几种 FAK 肽模拟物 (NEDDtides) 并证明了 使用多种化学方法将 NEDD 推向 NEDD9。从蛋白质结构和新颖的实验 根据数据，我们还在 FAK 结合槽附近的 Cys18 处鉴定了 NEDD9 特有的反应性硫醇残基。 我们进一步表明肺内皮 NEDD9 与 ECM 整合素 ITGA5 相互作用并调节关键 涉及血管增殖蛋白 VEGF 的通路。这些观察结果建立了中心假设 当前项目的内容：在肺动脉内皮细胞中，NEDDtide（或类似物）抑制 FAK-NEDD9 复杂的形成会损害粘着斑组装。我们进一步假设 NEDD9-SH3 是 可修改的目标，通过其抑制粘着斑依赖性 ECM 扩张、VEGF 信号传导，从而 PAH 中的血管重塑。目标 1 是：使用结构引导方法进行合成和化学优化 用共价探针修饰的 NEDDtide，利用 Cys18 选择性靶向 NEDD9。我们将测试 使用完整的基于荧光的质谱法测定 NEDDtide 的结合亲和力、效力和选择性 测定和其他方法来证明 NEDDtide 在体外和细胞内抑制粘着斑。 目标 2 是：使用涉及突变 NEDD9 构建体和 NEDDtide 的功能获得和丧失检测来显示 NEDD9-SH3 调节粘着斑处的 ITG5A-VEGF 信号传导，并且是一个可修改的靶标 抑制体外 PAH-HPAEC 和体内实验性 PAH 的 ECM 重塑和血管增殖。

项目成果

Systems biology: An emerging strategy for discovering novel pathogenetic mechanisms that promote cardiovascular disease.

• DOI: 10.21542/gcsp.2016.27
• 发表时间: 2016-09-30
• 期刊: Global cardiology science & practice
• 作者: Maron BA;Leopold JA
• 通讯作者: Leopold JA

Mechanism of Progressive Heart Failure and Significance of Pulmonary Hypertension in Obstructive Hypertrophic Cardiomyopathy.

• DOI: 10.1161/circheartfailure.116.003689
• 发表时间: 2017-04
• 期刊: Circulation. Heart failure
• 作者: Covella M;Rowin EJ;Hill NS;Preston IR;Milan A;Opotowsky AR;Maron BJ;Maron MS;Maron BA
• 通讯作者: Maron BA

Towards widespread noninvasive assessment of pulmonary vascular resistance in clinical practice.

在临床实践中走向广泛的肺血管阻力无创评估。

• DOI: 10.1016/j.echo.2013.10.010
• 发表时间: 2014
• 期刊: Journal of the American Society of Echocardiography : official publication of the American Society of Echocardiography
• 作者: Opotowsky,AlexanderR;Santos,Mário;Maron,BradleyA;Afilalo,Jonathan;Waxman,AaronB;Landzberg,MichaelJ;Forfia,PaulR
• 通讯作者: Forfia,PaulR

Independence Day: Separating Right Ventricular Function From Pulmonary Arterial Hypertension in Systemic Sclerosis.

独立日：将系统性硬化症中的右心室功能与肺动脉高压分开。

• DOI: 10.1161/circulationaha.116.023237
• 发表时间: 2016
• 期刊: Circulation
• 影响因子: 37.8
• 作者: Maron,BradleyA

TORward a Molecular Convergence Point in Pulmonary Arterial Hypertension With mTOR.

TORward 通过 mTOR 实现肺动脉高压的分子收敛点。

• DOI: 10.1016/j.jacbts.2018.08.003
• 发表时间: 2018
• 期刊: JACC. Basic to translational science
• 作者: Maron,BradleyA;Wilkins,MartinR
• 通讯作者: Wilkins,MartinR