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.

项目摘要/摘要 肺动脉高压（PAH）是一种以肺部为特征的严重心肺疾病 内皮增殖，细胞外基质（ECM）膨胀以及影响远端的间质性纤维化 肺动物。尽管血管重塑对PAH的发病和早期死亡的重要性很重要 患者，针对调节ECM扩展或 当前不存在血管增生。焦点粘连是多聚体跨膜复合物 界面内皮细胞与ECM整联蛋白。局灶性粘连驱动细胞增殖和ECM膨胀 在局灶性粘附激酶（FAK）引发的过程中。全球抑制FAK减轻血管 重塑并改善体内PAH；但是，FAK抑制剂是针对癌症量身定制的，并且与外部抑制剂有关 组织效应认为PAH患者特别高风险。因此，开发可防止焦点的药物 在受影响组织中的粘附组件选择性是治疗PAH的有前途的方法。 我们的实验室发现神经前体细胞表达了发育下调的蛋白质9 （NEDD9）作为调节PAH血管纤维化的脚手架蛋白。 NEDD9的SH3域是 FAK的主要结合靶标：FAK-NEDD9蛋白 - 蛋白质复合物的形成可调节局灶性粘附 众多细胞系中的信号转导。在当前的建议中，我们首次出席NEDD9-SH3 晶体结构。我们合成了几种FAK肽仪（NEDDTIDE），并证明了结合 使用多种化学方法使用NEDD9的NEDDIDE。来自蛋白质结构和新型实验 数据，我们还鉴定了NEDD9独有的FAK结合凹槽附近的Cys18的反应性硫醇残基。 我们进一步表明肺内皮NEDD9与ECM整合蛋白ITGA5相互作用并调节钥匙 涉及血管增生蛋白VEGF的途径。这些观察结果建立了中心假设 目前的项目：在肺动脉内皮细胞中，NEDDTIDE（或类似物）抑制FAK-NEDD9 复合形成以损害局灶性粘附组件。我们进一步假设NEDD9-SH3是 可修改的靶标可抑制局灶性粘附依赖性的ECM扩展，VEGF信号传导，从而抑制局部粘附的靶标 PAH中的血管重塑。目标1是：使用结构引导的方法进行合成和化学优化 用共价探针修饰的Neddtide（S）有选择地靶向NEDD9。我们将测试 使用完整的质谱，基于荧光的质谱法的结合亲和力，效力和选择性 测定和其他方法证明了在体外和纤维素中抑制Neddtide抑制局灶性粘附的方法。 AIM 2是：使用涉及突变NEDD9构建体和Neddtide的功能丧失和功能丧失测定 NEDD9-SH3在焦点粘附处调节ITG5A-VEGF信号传导，并且是一个可修改的靶标。 在体外抑制PAH-HPAECS中的ECM重塑和血管增生。

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

Clinical profile and underdiagnosis of pulmonary hypertension in US veteran patients.

• DOI: 10.1161/circheartfailure.112.000091
• 发表时间: 2013-09-01
• 期刊: Circulation. Heart failure
• 作者: Maron BA;Choudhary G;Khan UA;Jankowich MD;McChesney H;Ferrazzani SJ;Gaddam S;Sharma S;Opotowsky AR;Bhatt DL;Rocco TP;Aragam JR
• 通讯作者: Aragam JR

Diagnosis, Treatment, and Clinical Management of Pulmonary Arterial Hypertension in the Contemporary Era: A Review.

• DOI: 10.1001/jamacardio.2016.4471
• 发表时间: 2016-12-01
• 期刊: JAMA CARDIOLOGY
• 影响因子: 24
• 作者: Maron, Bradley A.;Galie, Nazzareno
• 通讯作者: Galie, Nazzareno