Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

Abstract

Src homology 3 (SH3) proteins regulate numerous fibroproliferative pathophenotypes including pulmonary arterial hypertension (PAH) but are challenging to target therapeutically. We innovated a peptidomimetic that occupies the canonical focal adhesion kinase (FAK) binding site on the SH3 domain of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) protein, a pro-PAH regulator. Peptidomimetic derivatization with a bromoacetamide group alkylated a NEDD9 cysteine positioned uniquely among SH3 domains (Cys18), which stabilized the RT loop, prevented FAK binding, and inhibited human pulmonary artery endothelial cell (HPAEC) migration. When linked to a thalidomide moiety, the peptide showed degrader activity of NEDD9 protein and, therefore, we next investigated therapeutic application of NEDD9 inhibition. In HPAECs, si-NEDD9 downregulated sulfatase-1, which increased podosome rosette formation and cell migration via 6-O-desulfation of glycocalyx-forming heparan sulfate proteoglycans, and reversed vascular remodeling and PAH in vivo. Whereas sulfatase-1 overexpression decreased pulmonary endothelial podosome formation, cell migration, and tube formation and increased collagen III synthesis, sulfatase-1 knockdown prevented fibroproliferative remodeling and pulmonary hypertension in PAH in vivo. These data leverage cysteinyl thiol reactivity to establish an SH3 domain-targeting structure-validated covalent peptide and identify two convergent mechanisms through NEDD9 that control endothelial phenotype, including reverse remodeling via sulfatase-1 transcriptional control. Overall, this study advances an SH3-specific therapeutic approach with relevance to PAH and other fibroproliferative pathophenotypes.