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 remodelin...