Compositions comprising neural precursor cell expressed, developmentally down-regulated 9 (NEDD9)-binding peptides and methods of use thereof

Abstract

Described herein are compositions comprising NEDDtide peptides and methods of use thereof, e.g., to treat diseases associated with focal adhesion kinase (FAK) and Neural Precursor Cell Expressed, Developmentally Down-Regulated 9 (NEDD9) protein-protein interactions; such diseases include pulmonary arterial hypertension, other pulmonary vascular diseases, and solid tumor cancers, e.g., by inhibiting focal adhesion assembly.

Description

[0001] Compositions Comprising Neural Precursor Cell Expressed, Developmentally Down-Regulated 9 (NEDD9)-Binding Peptides and Methods of Use Thereof

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Applications Serial Nos. 63 / 524,589, filed on June 30, 2023, and 63 / 603.177, filed on November 28, 2023. The entire contents of the foregoing are hereby incorporated by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant No. HL153502 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named “29618-0438WOl_ST26_SL.XML.” The XML file, created on July 1, 2024, is 9,747 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety7.

[0008] TECHNICAL FIELD

[0009] Described herein are compositions comprising NEDDtide peptides and methods of use thereof, e.g., to treat diseases associated with focal adhesion kinase (FAK) and Neural Precursor Cell Expressed, Developmentally Down-Regulated 9 (NEDD9) protein-protein interactions; such diseases include pulmonary arterial hypertension, other pulmonary vascular diseases, and solid tumor cancers, e.g., by inhibiting focal adhesion assembly.

[0010] BACKGROUND

[0011] Focal adhesions are multimeric protein complexes that link intracellular actin bundles with ECM anchoring proteins (Morgan et al. Nat Rev Mol Cell Biol 2007:8:957-69). Focal adhesions assert dynamic regulation over vascular endothelial cell proliferation and ECM expansion via organization of tensile proteins that contribute to interstitial fibrosis and angiogenesis; focal adhesions play a role in solid tumor cell proliferation and metastasis, and in extracellular matrix expansion in pulmonary vascular diseases including pulmonary arterial hypertension (PAH).

[0012] SUMMARY

[0013] The interaction between focal adhesion kinase (FAK) and Neural Precursor Cell Expressed, Developmentally Down-Regulated 9 (NEDD9) is a critical and ratelimiting step for the focal adhesion assembly. In turn, focal adhesions are important in solid tumor cell proliferation and metastasis, and extracellular matrix expansion in pulmonary vascular disease. Described herein are peptides collectively referred to as NEDDtides that inhibit FAK-NEDD9 protein-protein interactions.

[0014] Provided herein are peptides comprising or consisting of a sequence shown in Table 1, or a peptidomimetic thereof, optionally wherein the peptide comprises a sequence PX1KPX2R, where Xi is P, Q or K, and X2 is S or P, optionally PPKPSR (SEQ ID NO: 1), PPKPSRP (SEQ ID NO:2), PPKPSRPG (SEQ ID NO:3), PPKPSRPGY (SEQ ID NO:4), PPKPSRPGYP (SEQ ID NO:5), PKKPPR (SEQ ID NO:6), or DEAPPKPSRPGYPS (SEQ ID NOY).

[0015] In some embodiments, the peptide comprises or consists of 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or has a length in a range having endpoints in any of the preceding values (e.g., 6-14 or 6-20 amino acids).

[0016] In some embodiments, the peptide comprises an electrophilic arginine analog as described herein, e.g.. as shown in FIGs. 10A-H, e.g., 10F-H, e.g., an analog comprising the structure:

[0017] In some embodiments, the peptide is modified, e.g., by conjugation with phthalimide or 4-nitrophthalimide optionally on the N or C terminus, by biotinylation of the amino terminus and / or amidation of the carboxy terminus, or conjugation to a linear or branched-chain monomethoxv poly-ethylene glycol. In some embodiments, the peptide is modified by thalidomide on the N-terminal end and / or by C-terminal amidation.

[0018] Also provided are compositions comprising a peptide described herein, including pharmaceutical compositions comprising the peptides and a pharmaceutically acceptable carrier. In some embodiments, the peptide the carrier comprises liposomes.

[0019] Additionally, provided herein are methods for treating a subject who has a disorder associated with pulmonary arterial hypertension (PAH). Further, provided herein are the peptides, compositions, and pharmaceutical compositions as described herein for use in a method of treating a subject who has a disorder associated with pulmonary arterial hypertension (PAH), the method comprising administering to the subject a therapeutically effective amount of the peptide The methods comprise administering to the subject a therapeutically effective amount of a peptide, composition, or pharmaceutical composition as described herein.

[0020] Further, provided herein are methods for treating a subject who has a solid tumor. Also provided herein are the peptides, compositions, and pharmaceutical compositions as described herein for use in a method of treating a subject who has a solid tumor, the method comprising administering to the subject a therapeutically effective amount of the composition. The methods comprise administering to the subject a therapeutically effective amount of a peptide, composition, or pharmaceutical composition as described herein. In some embodiments, the solid tumor is breast cancer, gastric cancer, or pancreatic cancer. In some embodiments, administration of the therapeutically effective amount results in a reduction in risk of metastasis.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0022] DESCRIPTION OF DRAWINGS

[0023] FIGs. 1A-D. FAK-NEDD9 interactions and focal adhesion assembly. (A) Upregulation of focal adhesion kinase (FAK) by intermediates already established in the pathogenesis of pulmonary arterial hypertension (PAH) (e.g., oxidant stress, others) results in (B) phosphorylation at Y397 / Y576 and recruitment of NEDD9 to focal adhesion assembly site (C). The FAK-NEDD9 protein-protein complex enables phosphorylation of NEDD9 by c-Src and subsequent formation of a multimeric complex that links actin fibrils to membrane-spanning integrins, particularly integrin ITGA5. (D) Assembly of the focal adhesion results in HPAEC proliferation, ECM expansion, and interstitial fibrosis, all of which are pathogenic and promote PAH. HPAEC, human pulmonary artery endothelial cell; ECM extracellular matrix; FGF, fibroblast growth factor.

[0024] FIGs. 2A-C. Schematic illustration. (A) In pulmonary arterial hypertension (PAH), upregulation of FAK by oxidant stress, FGF -2 or other intermediaries recruits NEDD9 to focal adhesion assembly site. The poly-proline amino acid sequence of FAK (711-719) targets the FAK binding grove of NEDD9 at the SH3 domain, which is a critical step required for focal adhesion assembly. (B) A peptideometic modeled after AA 711-719 (NEDDtide) competes with FAK for NEDD9 to prevent FAK- NEDD9 complex formation and NEDD9-dependent focal adhesion assembly. (C) Predicted functional effects on focal adhesion signaling by modulating SH3 with NEDDtide, a mutant NEDD9 lacking the SH3 domain (AN9-SH3), or mutant N9- C18Athat includes a substitution of the cysteine at position 18 with alanine (which is impervious to oxidant stress at Cysl8). The SH3 domain is a modifiable target by which to inhibit pulmonary vascular remodeling in human pulmonary artery endothelial cells and, thereby, improve PAH in vivo. AN9-SD. mutant NEDD9 lacking the substrate domain; FGF, fibroblast growth factor; FAK, focal adhesion kinase; ECM, extracellular matrix; 4HB, 4 helical bundle segment; Ct, C-terminal domain.

[0025] FIGs. 3A-B. NEDD9 regulates pulmonary endothelial collagen and global vascular remodeling in PAH. (A) Compared to donor control human pulmonary artery endothelial cells (HPAECs), HPAECs isolated from PAH patients (PAH- HPAEC) showed increased NEDD9 positivity and collagen III expression. *P<0.01 vs. Control. (B) Paraffin-embedded lung tissue from discarded donor lung transplant specimens (controls), explanted idiopathic pulmonary fibrosis (IPF) patients, margin- free lung cancer biopsies, and explanted PAH patients was analyzed using hematoxylin and eosin (H / E), elastin stain, trichrome stain for collagen, and anti- NEDD9 immunohistochemistry. The relationship between NEDD9 expression is shown in luminosity arbitrary units and collagen amount in pulmonary arterioles. N=3-9 patients / condition, *P<0.05 by ANCOVA. Scale bar=50 pm. See ref. 1.

[0026] FIGs. 4A-B. NEDD9 inhibition prevents fibrotic vascular remodeling and PAH in vivo. (A) C57BL (WT) and NEDD9 / _mice were injected with Sugen-5416 (20 mg / kg) every 7 days during a 3-week period of hypoxia treatment (O2 10%).) Representative images of trichrome staining and von Willebrand Factor (vWF)- collagen III colocalization of pulmonary arterioles (20-50 pm in diameter). Scale bar=50 pm. N9, NEDD9; Col III, collagen III. N=6-9 mice / condition. (B) Male Sprague-Dawley rats were administered monocrotaline (MCT) (50 mg / kg), and the effect of NEDD9 siRNA (si-N9) on distal pulmonary arterial fibrosis was assessed by picrosirius red stain. N9, NEDD9. Bar=50 pm. *P<0.05 vs. control; **P<0.05 vs. PAH control. V, vehicle.

[0027] FIGs. 5A-D. NEDD9 regulates collagen net formation and matrix remodeling proteins under conditions of oxidant stress in HPAECs. Human pulmonary artery endothelial cells (HPAECs) were treated with vehicle control or aldosterone (ALDO) (10‘7mol / L), which is increased in PAH and induces vascular reactive oxygen species accumulation, for 24 hr. Cells were transfected with siRNA against scrambled (negative) control or NEDD9. Inhibition of NEDD9 decreased oxidant stress-induced (A) collagen III net formation, (B) cell stiffness analyzed by atomic force microscopy, (C) 3-dimensional matrigel contraction (data expressed as change in gel diameter from time point 0 - 25 hr), and (D) MMP-2 proteolytic activity assessed by zymography. (N=3 *P<0.01 vs. V; **P<0.03 vs. Scr or si-NEDD9; ***P<0.05 vs. V+ALDO). See ref. 1

[0028] FIGs. 6A-D. FAK-NEDD9-ITGA5 interactions in human pulmonary artery endothelial cells (HPAECs). (A) Anti-FAK and anti-NEDD9 immunofluorescence performed on HPAECs isolated from patients with pulmonary arterial hypertension demonstrates FAK expression and FAK-NEDD9 colocalization (N=3). Scale bar. 8 pm. (B,C) Anti-NEDD9 immunoprecipitation liquid chromatography-mass spectrometry on HPAECs identified FAK (retention time, 35.44 min; mass / charge ratio [m / z] 664.87533) and ITGA5 (45.10 min; m / z, 991.97843) (N=3). (D) Schematic summarizing from the published literature the interaction between focal adhesion-ITG5A and heparan sulfate. Sulfatase-1 (SULF1) desulfonates heparan sulfate proteoglycan (6-OS removal), which upregulates VEGF- mediated angioproliferation and ECM remodeling.

[0029] FIGs. 7A-D. NEDD9 regulates SULF1 signaling in HPAECs and PAH in situ. (A) Compared to HPAECs transfected with si-Scr (negative control), NEDD9 siRNA for 24 hr decreased Sulfatase-1 (SULF1) significantly by Western. (B) Lung sections isolated from SU-5416 / Hypoxia-PAH and monocrotaline (MCT)-PAH rats were analysed immunohistochemically using an antibody against the endothelial cell marker CD31, SULF1, and Heparan sulfate with 6-0-sulfate groups. Increased SULF1 in remodeled pulmonary arterioles associated with decreased 6-O-S Heparan sulfate (blue, DAPI). (C) Compared to control, SULF1 is increased in pulmonary endothelial cells of PAH patients compared to donor controls (blue. DAPI). (N=3 / condition for A-C). B,C Scale bar=30 / rm. (D) HPAECs and human pulmonary artery smooth muscle cells were co-cultured for 24 hr, immunostained with gold- labeled antibodies against Neuropilin- 1 (NRP-1) and 6-O-S Heparan sulfate, and analysed by transmission electron microscopy. Complexes involving Heparan sulfate and NLP-1, which is a co-receptor for VEGF, are seen at cell junctions (N=3).

[0030] FIGs. 8A-E. Biophysical characterization of interactions between NEDD9 and FAK-peptides. (A) Sequence alignment includes Site-1 and Site-2 of human isoforms FAK1 and FAK2 centered on FAK1 Pro715 and Pro876, respectively, highlighting cross-paralog and cross-site consensus motifs (PX1KPX2R, where Xi is P, Q or K, and X2 is S or P). All synthetic peptides were N- and C-terminally capped. Isothermal Titration Calorimetry (ITC) of short peptides revealed affinity7constants in the low micromolar range. (B) Isothermal calorimetry (ITC) (C) Fluorescence Polarization (FP) and (D) Flow Induced Dispersion Analysis (FIDA) used FITCylated pep 10 and pep 11 as chemical probes revealing activity by each of these two shorter peptides. The FIDA analysis was performed in premix mode with UV detector relying on NEDD9's autofluorescence by titrating peplO with and without NEDD9, revealing low-micromolar affinities in both radius of hydration (Rh) and fluorescence calculations (E). Top, Ribbon diagram of the SH3 domain with Cysl8 in magenta. Middle, stick format demonstrating that Cysl8 is within hydrogen binding distances to the backbone amides of Ala20 and Glu21 as well as the side chain of the latter. Bottom, one NEDDtide sequence (PPKPSR) was docked in silico to produce multiple poses, the highest ranked of which is shown with a surface representation of the NEDD9 SH3 domain in light-blue and Cysl8 in yellow.

[0031] FIGs. 9A-C. TMP ligands NEDD9-Cysl8, and successful intracellular NEDDtide transfection. (A) Biotin-tagged NEDDtide 1 (0.25 pg) was mixed into 500 ng of Chariot Transfection ReagentTM, which served as vehicle (V) control (left blot). Separately, the NEDDtide (0.25-4 pg) Chariot mixture was transfected into cultured human pulmonary artery endothelial cells for 1 hr, and anti-biotin immunoblotting was performed, presented here by chemiluminescence imaging. (B) Nucleophile substitution reaction used to predict TMP modification at NEDD9- Cysl8. (C) The MS2 spectra for recombinant NEDD9 (1 pg) treated with DTT (1 M) for 1 hr followed by TMP (100 pM) for 3 hr, confirming detection of the NEDD9- Cysl8-TMP peptide (N=3).

[0032] FIGs. 10A-H. Probe development flowchart and chemistry rationale. (A) Docked structure shows the FAK1 peptide in the NEDD9 SH3 domain, highlighting the proximity7of the position +5 Arginine guanidinium group to Cysl8. (B) Sequence alignment of human FAK1 / 2 kinases shows consensus sequence for binding to SH3 domains, from which three peptides were designed and synthesized replacing the +5 Arginine with an alternative residue containing an electrophilic bromo-acetamide warhead. (C) Mass-spec shows that after Ihr at RT, a 10-fold excess of peptides reacted completely with the SH3 domain. (D) Fluorescence polarization assay showed that the electrophilic peptides efficiently displaced a reversible fluorescent FAK1 peptidyl probe. (E) FAKlp31 counterscreened using MS against six randomly-chosen available proteins (TIRR, NSP10, ORFN, BRD4, KRAS, SENP1) (F) First- generation covalent NEDD9 peptidomimetics starting from a NEDDtide showing variations in the terminal arginine residue (modeled from data in Fig. 8). Compound optimization guided by experimental and calculated structures. (G) Synthesis using multi-step processes generating acrylamide amines that are then coupled to protected pentapdeptdies followed by deprotection. (H) Exemplary electrophilic arginine analogs. FIGs. 11A-E. NEDDtides bind specifically to NEDD9. (A) High quality electron density of the first 7 residues of the peptide ligand in the FAK binding groove of BCAR-SH3. The structure of the SH3 domain of human BCAR1 (92% homology with NEDD9-SH3) was determined in complex with a peptide containing the first KPXR motif of human FAK1. Cry stals formed in 2M NH4SO4 and 0. IM BisTris pH 6.5 generating datasets with excellent statistics. The surface representation shows the complex structure of the FAK peptide bound to the BCAR1 SH3 domain revealing the proximity' of Arg to Ser64; this electrostatic surface representation shows the complementarity7of the charges between the positively charged basic peptide ligand and the negatively charge acidic BCAR1 binding pocket. Atomic closeup of the interaction between the ionic side chains and the SH3 domain showing salt bonds. (B) HPAEC lysates were transfected with aNEDDtide conjugated with phthalimide at - COOH or H2N, or non-conjugated control for 1 hr and changes to NEDD9 expression were measured by anti-NEDD9 immunoblot. These findings show degradation of NEDD9 in the phthalimide-transfected conditions compared to control, provided important supporting evidence for NEDD9 targeting by the NEDDtide. (C) The Pl peptide sequence failed to degrade another SH3 domain protein, GBR2, which (based on predictive models) lacks a binding pocket that would recognize the proline repeat binding motif of the peptide. Pl: Thalidomyl-(Ahx)-PPKPSRP-NH2; Un: Untreated.

[0033] (D) The degrading effect of Pl at the maximum concentration (600 ng) for 10 min.

[0034] (E) Cultured human pulmonary artery endothelial cells transfected with NEDDtide (DEAPPKPSRPGYPS (SEQ ID NO:7)) with an N-terminal modification of Biotin- Ahx (1 ng / ul) for 2 hr and then anti-NEDD9 and anti-Biotin immunofluorescence was performed. These images of HPAECs show subcellular colocalization of NEDDtide and NEDD9 in areas that are structurally consistent with focal adhesions.

[0035] DETAILED DESCRIPTION

[0036] Described herein are peptides and peptidomimetics that bind to NEDD9 with high affinity (10 uM or below) and inhibit binding of NEDD9 to FAK. These peptides and peptidomimetics are useful, e.g., in inhibiting focal adhesion assembly. In turn, focal adhesions are important in solid tumor cell proliferation and metastasis, and extracellular matrix expansion in pulmonary' vascular disease. Thus the peptides and peptidomimetics can be used to reduce solid tumor cell proliferation and metastasis and to reduce fibrosis and treat pulmonary vascular disease including pulmonary arterial hypertension (PAH).

[0037] Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary' disease characterized by an obliterative vasculopathy affecting distal pulmonary arterioles, which promotes right heart failure, increased morbidity, and premature death.4The 3- year mortality rate for PAH is -40% in longitudinal studies,5and all-cause healthcare costs approach $10,000 / patient month.6Approved pharmacotherapies focus on vasoconstriction signaling pathways; however, abnormal vasoreactivity is a central feature in a small minority7of PAH patients.7By contrast, adverse vascular remodeling driven largely by ECM expansion (including fibrosis) and cellular proliferation increases pulmonary vascular resistance, which is a major determinant of poor clinical outcome in PAH patients.8

[0038] In PAH, accumulation of pulmonary7endothelial reactive oxygen species promotes the organization of transmembrane-spanning heterodimeric integrin proteins.9Integrin ITGA5 interacts with extracellular heparan sulfate glycosaminoglycans to increase (i) vascular endothelial growth factor (VEGF) signaling that stimulates pathogenic angiogenesis and (ii) fibrillar collagen (e.g., collagen III) deposition that increases ECM stiffness (reviewed in ref. 10). In PAH, pulmonary vascular ITGA5 is increased significantly compared to control, whereas ITGA5 inhibition limits ECM expansion and improves PAH in vivo.11-13In PAH, conversion of the ECM to a pathogenic connective tissue elevates pulmonary vascular resistance, which increases mortality7events.8

[0039] Focal adhesions are multimeric protein complexes that link intracellular actin bundles with ECM anchoring proteins.11Focal adhesions assert dynamic regulation over vascular endothelial cell proliferation, ECM expansion via organization of tensile proteins that contribute to interstitial fibrosis, and angiogenesis. Focal adhesion kinase (FAK) is a non-receptor ty rosine kinase that has both kinase phosphory lation and scaffolding functions. Several molecular intermediaries already established in the pathogenesis of PAH upregulate FAK including reactive oxygen species accumulation,1 14'16paxillin,17fibroblast growth factor (FGF)-2,18and other mechanosensing processes.19,20Bioactivation of FAK is modulated via (auto)phosphorylation at Y397, or Y576 by c-Src.21 At focal adhesions, FAK recruits various docking proteins to form proteinprotein interactions, which is a key event required for focal adhesion signal transduction. Converging lines of evidence show that FAK-dependent signaling is important and potentially modifiable in PAH.14,22For example, administration of FAK-siRNA or the FAK inhibitor PF-573-228 (targeting phosphorylation at FAK- 397) decreased vascular smooth muscle cell proliferation and migration in vitro to improve PAH in one experimental model in vivo. However, these earlier reports did not: establish the molecular mechanisms regulating focal adhesion assembly in PAH, target FAK-regulated pulmonary' endothelial ECM remodeling, or develop a sitespecific therapeutic deliverable.

[0040] Redox regulation of NEDD9 in HPAECs promotes fibrotic remodeling of pulmonary arterioles.1A post-transcriptional oxidative modification of the cysteine residue at position 18 (Cysl8) within the SH3 domain of NEDD9 induced by its reaction with oxidants such as hydrogen peroxide (H2O2) stabilizes NEDD9 and increases NEDD9-dependent transcriptional regulation of COL3A1 , which codes for fibrillar collagen III (an important ECM collagen isoform). HPAEC-derived exosomes increase NEDD9 expression in heterologous pro-vascular cell types1in vitro, underscoring the importance of pulmonary' endothelial control of vascular fibrosis globally, through paracrine signaling pathways between cell types. Fibrotic pulmonary arterioles from PAH patients overexpress NEDD9, whereas genetic ablation or molecular inhibition of NEDD9 with siRNA prevented vascular fibrosis and decreased pulmonary hypertension in two experimental animal models of PAH in vivo. Furthermore, we showed that plasma NEDD9 is diagnostic for PAH and prognosticates lung transplant-free survival.3Taken together, these findings establish the importance of NEDD9 to ECM pathobiology and demonstrate the translational clinical relevance of NEDD9 to PAH per se.

[0041] Post-translational oxidative modification of Cyst 8 to -SO3H stabilizes NEDD9 and induces NEDD9-dependent upregulation of COL3A1 in human pulmonary artery endothelial cells (HPAECs). which is a novel pathogenetic molecular mechanism underpinning pulmonary arterial hypertension (PAH).1,2Plasma NEDD9 is diagnostic for PAH and highly prognostic for lung transplant-free survival.3 NEDD9 is a docking protein that in cancer cell lines modulates pathogenic effects of focal adhesions.23The sequence of molecular events that enable NEDD9 signaling at focal adhesions are: (i) FAK recruits NEDD9 to focal adhesions and interacts directly with the NEDD9 SH3 domain, (ii) the resulting FAK-NEDD9 complex permits phosphory lation of a ty rosine motif (DYDY) in the NEDD9 C- terminal domain by FAK, (iii) phosphorylation at NEDD9-DYDY creates a Src binding site that is required for modification of the NEDD9 substrate (SH2) domain by Src and, ultimately, FAK-dependent signal transduction (reviewed in ref. 24). Indeed, NEDD9" ' tumor cells show decreased FAK functionality7and an attenuated metastatic phenotype,25which aligns with numerous other reports demonstrating that the FAK-NEDD9 protein-protein interaction is a critical step that controls focal adhesion pathogenicity7(FIGs. 1A-D).2627Thus, NEDD9 is (i) an established molecular target of FAK and (ii) profibrotic in PAH. Despite the implications of these events to PAH, NEDD9-specific therapeutics do not exist.

[0042] As shown herein, surface structure imaging using the crystal structure of NEDD9 showed a putative FAK binding groove near a reactive and functionally essential Cysl8. We constructed a series of synthetic peptides modeled after the polyproline sequence of FAK (NEDDtide), which we demonstrate binds tightly to NEDD9 by isothermal titration calorimetry (ITC). These collective findings establish that NEDDtide (or peptidomimetic analogs thereof) target the FAK binding groove of NEDD9-SH3 to inhibit the FAK-NEDD9 protein-protein interaction. NEDD9-SH3 is a modifiable target by which to inhibit focal adhesion-dependent ECM expansion, angioproliferative signaling, and, thereby, vascular remodeling in PAH (FIGs. 2A-C), as well as provide therapeutic effect in chronic thromboembolic pulmonary hypertension (CTEPH). NEDD9 is implicated in the pathogenesis of various solid tumor cancers.94In breast adenocarcinoma, NEDD9 targets TGF-P,95also implicated in PAH,96to alter the phenoty pe of cells and permit blood-bome metastasis.97NEDD9 is observed from peripheral blood samples in patients with gastric adenocarcinoma98and other cancers,99and inhibition of NEDD9 inhibits pancreatic cancer cell proliferation and invasion.94 NEDDtides and peptidomimetics

[0043] Described herein are peptides having or comprising a sequence comprising

[0044] PX1KPX2R, where Xi is P, Q or K, and X2 is S or P, e.g., as shown in Table 1, as well as peptidomimetics thereof.

[0045] TABLE 1 - EXEMPLARY PEPTIDES

[0046] Preferably the peptides comprise or consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or have a length in a range having endpoints in any of the preceding values (e.g.. 6-14 or 6-20).

[0047] In some embodiments, the peptide includes an electrophilic arginine analog as described herein, e g., as shown in FIGs. 10A-H, optionally at position 2 or 5 of the sequence PXKPXR.

[0048] The peptides can also be modified, e.g., by biotinylation of the amino terminus and / or amidation of the carboxy terminus, conjugation with phthalimide or 4- nitrophthalimide (e.g., to induce destabilization of target NEDD9 proteins) optionally on the C terminus (Winter et al., Science, Volume 348, Issue 6241, p. 1376-81 (2015)), or conjugation to a linear or branched-chain monomethoxy poly-ethylene glycol (PEG, i.e., PEGylation). The modification can be added, e.g., via an aminohexanoic acid (Ahx) linker. Other modifications can include fusion proteins, e.g.. Fc fusion proteins, fusion to human serum albumin, fusion to carboxy-terminal peptide, and other polypeptide fusion approaches to make drugs with more desirable pharmacokinetic profiles. See, e.g., Werle and Bemkop-Schniirch, Amino Acids. 2006 Jun;30(4):351-67; Strohl, BioDrugs. 2015; 29(4): 215-239; Wang et al., Signal Transduction and Targeted Therapy 7: 48 (2022). Peptides disclosed herein can be modified according to the methods known in the art for producing peptidomimetics. See, e.g., Qvit et al., Drug Discov Today. 2017 Feb: 22(2): 454-462; Farhadi and Hashemian, Drug Des Devel Ther. 2018; 12: 1239- 1254; Avan et al., Chem. Soc. Rev., 2014,43, 3575-3594; Pathak, et al., Indo American Journal of Pharmaceutical Research, 2015. 8; Kazmierski, W.M., ed., Peptidomimetics Protocols, Human Press (Totow NJ 1998); Goodman et al., eds., Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, Thiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746 (2003). In some cases, these modified peptidomimetic versions of the peptides and fragments disclosed herein exhibit enhanced stability- in vivo, relative to the non-peptidomimetic peptides.

[0049] Methods for creating a peptidomimetic include substituting one or more, e.g., all, of the amino acids in a peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as “retro” sequences. In another method, the N- terminal to C-terminal order of the amino acid residues is reversed, such that the order of amino acid residues from the N-terminus to the C-terminus of the original peptide becomes the order of amino acid residues from the C-terminus to the N-terminus in the modified peptidomimetic. Such sequences can be referred to as “inverso” sequences. Peptides including reversed sequences can comprise, e.g., RSPKPP (SEQ ID NO:8), PRSPKPP (SEQ ID NOV). RPPKKP (SEQ ID NO: 10) and so on.

[0050] Peptidomimetics can be both the retro and inverso versions, i.e., the “retro- inverso” version of a peptide disclosed herein. The new peptidomimetics can be composed of D-amino acids arranged so that the order of amino acid residues from the N-terminus to the C-terminus in the peptidomimetic corresponds to the order of amino acid residues from the C-terminus to the N-terminus in the original peptide.

[0051] Other methods for making a peptidomimetic include replacing one or more amino acid residues in a peptide with a chemically distinct but recognized functional analog of the amino acid, i.e., an artificial amino acid analog. Artificial amino acid analogs include (3-amino acids, P-substituted (3-amino acids (“P3-amino acids”), phosphorous analogs of amino acids, such as V-amino phosphonic acids and V-amino phosphinic acids, and amino acids having non-peptide linkages. Artificial amino acids can be used to create peptidomimetics, such as peptoid oligomers (e.g., peptoid amide or ester analogues), P-peptides, cyclic peptides, oligourea or oligocarbamate peptides; or heterocyclic ring molecules. Exemplary retro-inverso targeting peptidomimetics include RSPKPP (SEQ ID NO: 8), PRSPKPP (SEQ ID NOV), RPPKKP (SEQ ID NO: 10), wherein the sequences include all D-amino acids.

[0052] Methods of Treatment - Pulmonary Arterial Hypertension (PAH)

[0053] The methods described herein include methods for the treatment of disorders associated with pulmonary arterial hypertension (PAH). Generally, the methods include administering a therapeutically effective amount of an NEDD9 peptide or peptidomimetic as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. Methods for identifying such subjects are known in the art. e.g., using ventilation / perfusion (V / Q) scintigraphy; pulmonary angiography; Dual-Energy Computed Tomography angiography (DECT); and / or Computed Tomography angiography (CTA) (see, e.g., Maron et al., JAMA Cardiol. 2016 Dec 1; 1(9): 1056-1065; Gopalan et al., European Respiratory' Review 2017 26: 160108; Kharat et al., Thromb Res. 2018 Mar; 163:260-265; Corrigan et al., Clin Exp Emerg Med. 2016 Sep; 3(3): 1 17-125; van Beek et al.. Continuing Education in Anaesthesia Critical Care & Pain, August 2009, 9(4): 119-124; Sakuma et al., Circ J. 2005 Sep;69(9): 1009-15.

[0054] As used in this context, to "‘treat” means to ameliorate at least one symptom of the disorder associated with PAH. For example, a treatment can result in treatment of, or a reduction in risk or severity of, pulmonary hypertension.

[0055] Medical therapies for pulmonary' arterial hypertension can also be administered, e.g., vasodilators and anti-proliferative agents, e.g., Epoprostenol (Flolan), Epoprostenol (Veletri). Treprostinil (Remodulin), Iloprost (Ventavis). Treprostinil (Tyvaso), Bosentan (Tracleer), Ambrisentan (Letairis), Sildenafil (Revatio), or Tadalafil (Adcirca); Calcium Channel Blockers; Blood Thinners; Diuretics; Digoxin (Lanoxin); or Oxygen. See, e.g., Pulido et al., Heart Failure Reviews May 2016, Volume 21. Issue 3, pp 273-283.

[0056] Methods of Treatment - Cancer

[0057] The methods described herein include methods for the treatment of subjects with cancer, e.g.. solid tumors. Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. In some embodiments, the methods include administering a therapeutically effective amount of a treatment comprising a peptide or peptidomimetic as described herein.

[0058] As used in this context, to ‘'treat’ ’ means to ameliorate at least one clinical parameter associated with cancer, e.g., a solid tumor. For example, a treatment can result in a reduction in tumor size or growth rate. Administration of a therapeutically effective amount of a compound described herein for the treatment of a solid tumor will result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in risk of metastasis, increased survival, and / or decreased morbidity and mortality, inter alia.

[0059] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas that include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the cancer is a carcinoma, adenocarcinoma, or sarcoma.

[0060] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory7system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary7. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An '‘adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0061] Pharmaceutical Compositions and Methods of Administration

[0062] The methods described herein include the use of pharmaceutical compositions comprising a NEDD9 peptide or peptidomimetic as described herein as an active ingredient.

[0063] Pharmaceutical compositions typically include a pharmaceutically7acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can be administered separately or can be incorporated into the compositions. For example, for treating PAH the compositions can also include epoprostenol (FLOLAN or VELETRI), treprostinil (REMODULIN). iloprost (VENTAVIS), treprostinil (TYVASO). bosentan (TRACLEER). ambrisentan (LETAIRIS). sildenafil (REVATIO), or tadalafil (ADCIRCA); calcium channel blockers; blood thinners; diuretics; digoxin (LANOXIN); or oxygen. The present methods can also include administering such additional compounds separately.

[0064] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0065] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0066] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0067] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0068] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0069] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0070] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0071] The pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery'.

[0072] In some embodiments, the therapeutic compounds are prepared with carriers, including carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.

[0073] Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.

[0074] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES

[0075] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0076] Example 1. NEDD9 expression is increased in remodeled pulmonary arterioles from PAH patients.

[0077] The relevance of NEDD9 to PAH was studied using human pulmonary artery endothelial cells from PAH patients (PAH-HPAECs) (from Pulmonary Hypertension Breakthrough Initiative) and paraffin-embedded distal pulmonary arterioles (50- 80pm) harvested from PAH patients at the time of lung explant (all work was IRB approved). Anti-NEDD9 immunohistochemistry' was performed using the 3,3'- diaminobenzidine substrate method.1For immuno-fluorescence experiments, tissue sections were stained using goat anti-human NEDD9 and rabbit anti-human collagen III antibodies (Abeam). Images were viewed using an LSM 700 Flexible Confocal Microscope (Carl Zeiss). Samples from cancer biopsy, idiopathic pulmonary fibrosis explanted lungs, or donor lung transplant tissue were used as controls. Compared to donor control HPAECs, NEDD9-positivity was increased in PAH-HPAECs, which correlated with collagen III expression measured by quantitative volumetric analysis (ImageJ; NIH) (r= +0.77, P=0.02) (Fig. 3A). Directionally similar findings were observed for collagen quantity in remodeled PAH pulmonary arterioles vs. with controls (Fig. 3B). Exosomes from HPAECs regulate NEDD9 overexpression in other vascular cell types in PAH (data not shown).1

[0078] Example 2. NEDD9 inhibition decreases pulmonary arterial fibrosis and improves PAH in vivo.

[0079] NEDD9-deficient transgenic mice were studied using the SU-5416 / Hypoxia model of PAH, which among murine models is characterized by severe vascular fibrosis. Briefly, male and female WT and NEDD9’ ' mice were administered a subcutaneous injection of the VEGFR-2 kinase inhibitor Sugen-5416 (20 mg / kg; Sigma) and placed immediately in hypoxia (O2 10%) for 21 days. Repeat SU-5416 injections were administered on protocol days 7 and 14.1Compared to WT-SU- 5416 / Hypoxia, we found that NEDD9' "-SU-5416 / Hypoxia mice had decrease endothelial collagen III and overall pulmonary arterial collagen burden (Fig. 4A) and improved pulmonary artery pressure (data not shown).1In the monocrotaline (inflammatory) model of PAH, treatment with siRNA-NEDD9 prevented fibrotic and hypertrophic remodeling of pulmonary arterioles (Fig. 4B), and normalized pulmonary vascular resistance vs. untreated PAH rats (1.8 ± 0.3 vs. 7.3 ± 2.3 Wood units, P<0.01).

[0080] Example 3. Increased NEDD9 upregulates collagen III net formation, matrix remodeling, and HPAEC stiffness.

[0081] To explore potential functional effects of increased endothelial NEDD9 on fibrosis in HPAECs and matrix remodeling, cells were treated with the pro-fibrotic hormone aldosterone (ALDO), which is increased in PAH37and used in these experiments to increased reactive oxygen species accumulation.38-39We observed that compared to cells transfected with scrambled si-RNA (negative control), si-NEDD9 decreased ALDO (10‘7mol / L)-induced collagen III net formation, cell stiffness assessed using atomic force microscopy, and 3-dimensional collagen matrigel contraction significantly (Figs. 5A-D). Compared to untransfected cells, inhibition of NEDD9 also caused a significant difference in the quantity of mRNA transcripts that code for key ECM proteins linked to focal adhesion signaling as assessed by RNA- Seq, including fibronectin 1 (-1.51 log fold-change [logFC], P<10e-29, false discovery rate [FDR]<10e-27), COLA1 (-1.55 logFC, P<0.01, FDRO.01), and others.

[0082] Example 4. In HPAECs, NEDD9 interacts with focal adhesion proteins FAKandITG5A.

[0083] We used anti-FAK (rabbit, Abeam) immunofluorescence to profile focal adhesion expression in PAH-HPAECs, (Fig. 6A). Next. anti-NEDD9 immunoprecipitation-liquid chromatography-mass spectrometry was performed on HPAECs using the in-gel trypsin digestion method of Schevchenko and colleagues40with some modifications, as reported by our laboratory previously.1-2As peptides eluted, they were subjected to electrospray ionization and then entered into a linear trap quadrapole (LTQ) Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher). Using this approach, we identified the mass spectra corresponding to FAK (Fig. 6B) as well as ITG5A (Fig. 6C), which is a key focal adhesion integrin that interacts with ECM heparan sulfate proteoglycans and regulates vascular endothelial growth factor (VEGF) signaling (Fig. 6D). Example 5. NEDD9 regulates sulfatase-1, which modifies heparan sulfate in a key step for VEGF signaling.

[0084] The removal of the 6-0-sulfate group of heparan sulfate proteoglycans by the endosulfatase, SULF1, is a critical post-translational modification that leads to release of VEGF ligands from heparan sulfate proteoglycans,41 42and subsequent VEGF- dependent angiogenesis.43We observed that inhibition of NEDD9 with siRNA decreased SULF1 expression significantly compared to si-Scr or untransfected cells (Fig. 7A). Further, pulmonary endothelial SULF1 expression was increased significantly in the SU-5416 / hypoxia (angioproliferative) and monocrotaline (inflammatory) experimental rat models of PAH in vivo, which was associated with a decrease in levels of unmodified heparan sulfate (i.e., 6-OH heparan sulfate) (Fig. 7B). We further observed a significant increase in SULF1 expression in remodeled pulmonary arterioles isolated from PAH patients compared to donor controls (Fig. 7C). In monolayers of HPAECs co-cultured with human pulmonary artery7smooth muscle cells (HPASMCs), we observed protein-protein complex formation involving heparan sulfate and neuropilin-1, which is a membrane-bound co-receptor for VEGF41'43and proposed PAH biomarker.44These complexes populated cell junctions (Fig. 7D). Taken together with our other findings suggesting SULF1 regulation by NEDD9 in HPAECs. these data implicate focal adhesion-heparan sulfate-VEGF signal transduction in the pathogenesis of PAH.45

[0085] Example 6. Measurement of affinity between NEDDtide and NEDD9.

[0086] Published chemical probes for NEDD9 are lacking; however, Teyra and colleagues reported previously a consensus motif that binds to NEDD9 with the sequence: PxKP.46We searched the NEDD9 interactome for this motif and found it in both human FAK paralogs, FAK (aka FAK1, PTK2, FRNK) and FAK2 (aka PTK2B, PYK2).46Two copies of the motif occur in the long-disordered linker between the kinase and fat domains of FAK. We synthesized 14 residue-long peptides containing these sequences (see the top panel of FIG. 8A) and measured their affinity to the NEDD9 linker between the kinase and fat domains by7isothermal titration calorimetry (ITC). The resulting isotherms were fitted with a single site model to yield thermodynamic parameters of AH. AS, stoichiometry, and Kd of about 10 pM affinity and high enthalpy values consistent with protein-protein interactions (Fig. 8A). We also syntehsised shorter NEDDtides of 10 and 6 amino acids (pep 10 and 11 in FIGs. 8A-D). Both NEDDtides 1 and 2 bound with <10 pM affinity and a high enthalpy value consistent with protein-protein interactions.

[0087] Example 7. Determination of the crystal structure ofNEDD9.

[0088] Crystals of NEDD9 grew in PEG20K and NaCitrate-pH5 and were cryoprotected for data-collection at the synchrotron. Molecular replacement and refinements produced a final model with seven molecules in the asymmetric unit and with good statistics. Nearly all atoms were resolved and alignment of the seven molecules revealed an inflexible core and flexible SRC loop. The NEDD9 SH3 crystal structure showed a canonical SH3 fold with the Cysl8 side chain in the RT- loop facing towards the putative polyproline binding pocket (FIG. 8E). Electrostatic surface representation showed an acidic polyproline binding pocket, consistent with the Class VII motif (0PxK+2P+3(-)).46The sulfur atom of Cysl8 was within 3.0, 3.3, and 4.3 A of the amide backbone atoms of the RT-loop as well as the side chain carboxyl group of Glu21, which presumably withdraws electrons from the sulfur and increases its reactivity (Fig. 8E).

[0089] Example 8. Docking model of the FA / NEDD9 complex.

[0090] At present, high-resolution crystal structures of the FAK-NEDD9 complex are lacking; therefore, to guide chemistry we used the Schrodinger Software Suite™. Computationally-generated docked models of ligand (FAK peptide) and receptor (our apo NEDD9 SH3 structure) were generated using receptor grids centered on the canonical polypropylene binding site, which was derived from a consensus of 12 preexisting SH3-polyproline complex structures. Schrodinger Glide was executed for docking in high precision mode.47,48The best poses with the lowest calculated free energy represent our docked models and initiated a structure-guided, rational development of novel chemical designs (Fig. 8E).

[0091] Example 9. NEDDtide transfection into HPAECs; NEDD9-Cysl8 undergoes covalent modification.

[0092] Biotin-tagged NEDDtide 1 (10 kDa) (0.25-0.4 pg) w as mixed into 500 ng of Chariot Transfection ReagentTM, which served as vehicle control, and loaded directly onto an SDS-PAGE gel. Separately, the NEDDtide-Chariot mixture was transfected into cultured HPAECs for 1 hr. Anti-biotin immunoblotting confirmed successful intracellular NEDDtide transfection (Fig. 9A). Example 10. TMP ligands purified NEDD9 at Cysl8.

[0093] A published fragment-based ligand discovery screen using covalent, electrophilic small-molecule probes in HEK-293T cells suggested that NEDD9-Cys 18 is liganded in cellulo by the compound N-[3,5-Bis(trifluoromethyl)phenyl] acry lamide (TMP).49To demonstrate this definitely, NEDD9 (1 pg) was incubated with 1 M dithiothreitol (DTT) for 1 hr followed by TMP (100 pM) for 3 hr. Recombinant chemically modified NEDD9 protein samples were proteolyzed using the iST in-solution proteolysis kit (PreOmics). NEDD9 tryptic products were analyzed by LC-MS. The m / z value for the TMP -modified form (960.34210) was detected based on a nucleophilic substitution reaction involving the acrylamide arm of TMP and Cysl8 of NEDD9 (FIGs. 9B-C). These data show that Cysl8 in the NEDD9 SH3 domain is reactive and, thus, a potential molecular handle to target with a covalent probe for optimizing specificity of NEDDtide targeting.

[0094] Example 11. Development of covalent NEDD9 inhibitors.

[0095] This example describes the development of (NEDD9)-binding peptides modified with electrophilic residues.

[0096] The following materials and methods were used in this example.

[0097] Peptide synthesis: Peptides corresponding to amino acids 678-686 of human FAK1 containing bromo acetamide side chain variants were generated using solidphase Fmoc chemistry on a Symphony X peptide synthesizer (Gyros Protein Technologies), with amino acids sequentially added to rink amide AM resin. Following N-terminal acetylation, deprotection, and cleavage from the resin, peptides were purified by reverse-phase high performance liquid chromatography and mass spectrometry (LC-MS) and quantified by amino acid analysis.

[0098] Mass spectroscopy: Recombinant NEDD9 SH3 domain at a concentration of lOuM was incubated at 20°C for one hour with and without lOOuM compounds in a reaction buffer containing 20mM Hepes-pH7.5 and 150mM NaCl and analyzed by LC-ESI-MS as previously described (Zhang et al., Chem Biol. 2012 Jan 27;19(1): 140- 54).

[0099] Fluorescence polarization (FP): Binding assays were performed in 33ul volume at a constant labeled peptide concentration of 100 nM, and the rigid MBP fusion of NEDD9 protein at 20uM concentrations and titrating small molecules and peptides ligands ranging between lOOnM and lOOuM in buffer containing 20 mM HEPES pH 7.6, 20 mM NaCl, 100 uM TCEP, 0.01% Pluronic acid. FP assays were performed in 384 well plates using a PherastarFS plate reader.

[0100] Computationally-generated docked models of the FAK1 peptide in the NEDD9 SH3 domain were generated using the Schrodinger Software Suite™ highlighting the receptor (our apo NEDD9 SH3 structure). This was accomplished using receptor grids centered on the canonical polyproline binding site, derived from a consensus of 12 pre-existing SH3 -polyproline complex structures. Schrodinger Glide was executed for docking in high precision mode. The best poses with the lowest calculated free energy represented our docked models and showed that the P1.K3 residues straddled Arg59 and P4.R6 straddled Trp43. Importantly, K3 and R6 pointed in the same direction towards the RT-loop, forming hydrogen and ionic bonds with Glut 7, Glu21, and Cysl8 (Fig. 8E, 10A). The model emphasized the proximity of the position +5 Arginine guanidinium group to Cysl8. Sequence alignment includes human isoforms of FAK1 centered highlighting cross-paralog and cross-site consensus motifs (P.KP.R). These permutations were used to align sequence compatibility for FAK1 peptide modification with a synthetic covalent R-group, the bromo-acetamide warhead (Fig. 10B). This analysis directed focus to three peptides that were synthesized by replacing the +5 arginine with an alternative residue containing the electrophilic bromo-acetamide. Liquid chromatography -mass spectrometry demonstrated that a 10-fold excess of peptides reacted completely with the SH3 domain of NEDD9 after 1 hr incubation at room temperature (Fig. IOC). The shortest peptide (FAKlp31) was counterscreened using mass spec against six randomly-chosen available proteins (TIRR, NSP10, ORFN, BRD4, KRAS, SENP1), showing that the peptide had exquisite specificity for NEDD9 (Fig. 10D). The functional effect of FAK1 modified with a bromo-acetamide warhead on the NEDD9- FAK protein-protein interaction was explored using fluorescence polarization (Fig. 10E). These data demonstrated marked inhibition of NEDD9-FAK binding in the presence of FAK1 modified by the bromo-acetamide warhead compared to a reversible fluorescence FAK1 peptidyl probe. Taken together, these data demonstrate that FAK1 -bromo-acetamide interacts with NEDD9 through a mechanism involving peptide-protein interaction and covalent binding to Cysl8 to inhibit FAK-NEDD9 protien-protein interaction, which is a functional effect of relevance to various human diseases, particularly pulmonary hypertension and solid tumor expansion. Based on the modeling, a group of electrophile-containing fragments intended to form a covalent bond with Cysl8 of the NEDD SH3 domain were designed (Fig. 10F). The overall synthetic scheme is to prepare various electrophilic arginine analogs (e.g., as shown in Fig. 10F-H) and couple them to a standard side chain protected, N- capped, free-carboxyl pentapeptide (PPKPS). The electrophilic arginine analogs will be synthesized to optimize trajectory of the electrophile. Peptides containing electrophilic warheads were generated using standard C-to-N fluorenylmethyloxy carbonyl (Fmoc) solid-phase chemistry on a Symphony X peptide synthesizer (Gyros Protein Technologies), with amino acids sequentially added to rink amide AM resin. Following N-terminal acetylation, deprotection, and cleavage from the resin, peptides were purified by reverse-phase high performance liquid chromatography and mass spectrometry (LC-MS) and quantified by amino acid analysis. A synthetic scheme for an exemplary' compound is listed in Fig. 10G; similar methods can be used to synthesize the other compounds. Exemplary' electrophilic arginine analogs are shown in Fig. 10H.

[0101] Example 12. Effects of NEDD9 inhibition on focal adhesion assembly in cancer and pulmonary endothelial fibrosis and PAH in vitro and in vivo

[0102] NEDD9-SH3 is a modifiable target by which to inhibit ECM remodeling, heparan sulfate- VEGF angioproliferative signaling, and, thus, vascular remodeling in PAH, as NEDD9 has been shown to regulate focal adhesion assembly in cultured cancer cell lines, and NEDD9 inhibition improves pulmonary' endothelial fibrosis and PAH in vivo.

[0103] To evaluate the effects of treatment with NEDDtides as described herein, control HPAECs (Lonza) and HPAECs isolated from PAH patients (PAH-HPAECs),1and lung fibroblasts (Lonza) are grown to confluence in monolayers in EGM-2 or DMEM supplemented with 5% FBS, respectively, at 37 °C, 5% CO2. To increase oxidant stress and induce FAK activation,16cells are transfected with the chemogenetic D-amino acid oxidase HyPer system. This system allows for on-off switching of endogenous H2O2 generation, enabling analysis of FAK-NEDD9 interaction onset and offset across a nuanced time course, and permits delivery' of covalent NEDDtide prior to onset of oxidizing conditions.74’75In experiments involving HyPer. we also treat cells with mPEG-catalase (100 lU / ml) to reduce H2O2 to water, as a negative control. As an additional method to activate FAK with relevance to PAH, cells are stimulated with FGF2 (1-100 pM) (Sigma) for 12-24 hr.

[0104] SU-5416 / Hypoxia PAH mice, an angioproliferative / ECM expansion model of PAH, are also used. Male and female WT and NEDD9" ' mice (12-14 weeks) are administered the VEGFR-2 kinase inhibitor SU-5416 (Sigma) (20mg / kg) by subcutaneous injection and placed in hypoxia (02 10%) until cardiac catheterization and tissue harvest experiments are performed 21 days later. To maximize pulmonary hypertension and vascular remodeling, repeat SU-5416 subcutaneous injection is administered on day 7 and day 14.1

[0105] Monocrotaline PAH rats, an inflammatory vascular injury / ECM expansion model of PAH. is also used, in which male and female rats are administered a single dose of vehicle control or monocrotaline (MCT) (50 mg / kg) (Sigma) by intraperitoneal injection. Catheterization and tissue harvest experiments are performed 21-23 days thereafter.2-77

[0106] Pharmacological inhibition of FAK-NEDD9 interaction is achieved by administration of NEDDtide or an analog thereof, e.g., as shown in FIGs. 10A-H, be solubilized in KH2PO4 buffer (pH 7.4), formulated in human microsomes (Life Technologies),80and administered by intratracheal injection. This approach allows for microsomal payload diffusion across the alveolar-capillary interface, an established delivery method for PAH drugs.81A fluorescently-labeled covalent NEDDtide is synthesized and used to monitor the distribution of NEDDtide (1-50 mg / kg) analyzed by immunofluorescence in lung sections co-stained for vWF (Abeam), a-smooth muscle actin (Abeam), and vimentin (Abeam) antibodies to identify PAECs, PASMCs, and lung fibroblasts, respectively.1,2As a positive control, the FAK inhibitor PF-562271 (25 mg / kg in 0.1 mL) will be given twice daily by oral gavage.19

[0107] Changes in ECM are assessed using three specific assays: cell proliferation, three-dimensional collagen matrix contraction, and atomic force microscopy (AFM). The BrdU incorporation assay will be performed using the cell proliferation ELISA colorimetric kit (Roche) per the manufacturer’s instructions and as reported.83Briefly, 48 hr after treatments, cells are seeded at the concentration of 2x103 per well into 96- well plates overnight. After serum starvation for 4 hr with EBM-2 medium, cells are labeled with BrdU (10 pM) for 2 hr and fixed. Absorbance is measured in a microplate reader. For three-dimensional collagen matrix contraction, we will follow methods reported previously. For AFM, cell stiffness will be analyzed using MFP-3- dimensional AFM (Asylum Research; HMS Core). Elastic moduli are calculated using software e.g., Igor Pro 6.36. For each sample, >5 areas will be analyzed.1

[0108] Additionally, we will profile changes in ECM proteins by proximity ligase and immunogold electron microscopy using antibodies against ITGA5, fibronectin, collagen I / III, laminin, and matrix metalloproteinase (MMP)-2 / 9 (see Aim 2. 1 for methods). High resolution imaging will provide data on ECM localization of these proteins, as well the formation of protein-protein complexes involving ITGA5- fibronectin, fibronectin-collagen and others reported in pathogenic matrix remodeling.

[0109] Changes in VEGF biofunctionality is assessed using three specific assays: cell migration, scratch assay, tube formation. For cell migration studies, cells (1.5x104 per well) are seeded into the ibidi Culture-Insert 2 Well. After cell attachment, inserts are incubated for 7 h in the presence or absence of the VEGFR-A (Fit- 1) receptor antagonist AAL-993 (lOmM) (Santa Cruz) (100 ng / ml) as negative control in EBM-2 with 1% FBS. Cell migration is captured by live cell imaging microscopy and expressed as the percentage of area migrated (ImageJ, NIH).83 For tube formation, 96-well cell culture cluster half area plates are coated with 30 pl of growth factor reduced Matrigel (BD Bioscience) and incubated at 37 °C for 30 min. Next, cells are seeded (4 x!03 per well) in the presence or absence of AAL-993 (10 mM) for 12 h.85 Then, cells are stained with 2 pM of Calcein AM (Invitrogen) and tube-like structures are imaged with an inverted fluorescence microscope. Total tube length is quantified by ImageJ. The scratch assay will be performed per published methods.86

[0110] The effects of covalent NEDDtide(s) vs. PF-573,228 on pulmonary arterial remodeling and PAH in vivo. Methods for small particle delivery to the lung vasculature have been published previously.87Pilot experiments will be performed for each model to confirm adequate delivery of covalent NEDDtide. This will be accomplished by quantifying immunofluorescence colocalization of fluorescent- labeled covalent NEDDtide with signals for vWF. SMA, and vimentin to profile endothelial, vascular smooth muscle cell, and fibroblast uptake of covalent NEDDtide, respectively, in distal pulmonary arterioles of paraffin-embedded lung sections isolated from mouse / rat lung. At the completion of the treatment protocols for mice / rats entered into the main experiments, differences in right ventricular systolic pressure, pulmonary vascular resistance, cardiac index, mean arterial pressure, right ventricular-pulmonary arterial coupling and other measures will be assessed between groups using a Millar Mikro-TipTM pressure catheter-pressure transducer unit as reported extensively by our laboratory.1’2’39,77’88

[0111] At the completion of these studies, lungs are perfused with 10-20 mL PBS prior to tissue harvesting. Differences in global NEDD9, FAK, P-FAK(Y397), P-FAK (Y576), paxillin, c-Src, and ITGA5 in distal pulmonary arterioles will be quantified by immunofluorescence, and complexes between proteins by proximity ligase. Celltype specific analyses will also be performed to monitor changes in focal adhesion complex formation in PAECs, pulmonary artery’ smooth muscle cells and fibroblasts. The expression profile of focal adhesion proteins and protein-protein complexes will be compared to the expression profile of ECM remodeling proteins fibronectin, collagen I / III, laminin, and matrix metalloproteinases-2 / 9, fibrosis extent and distribution using Gomori trichrome and Picrosirius red stains to quantity’ the ratio of cross-linked to total collagen levels in the pulmonary’ arteriole wall vs. ECM space, and vascular stiffness assessed by AFM.1

[0112] Example 13. NEDD9 peptides bind specifically to NEDD9.

[0113] The structure of the SH3 domain of human BCAR1 (92% homology' with NEDD9-SH3) was determined in complex with a peptide containing the first KPXR motif of human FAKE Crystals formed in 2M NH4SO4 and 0.1M BisTris pH 6.5 generating datasets with excellent statistics. A high quality electron density7surface representation of the first 7 residues of the peptide ligand in the FAK binding groove of BCAR-SH3 in FIG. 11 A shows the complex structure of the FAK peptide bound to the BCAR1 SH3 domain revealing the proximity of Arg to Ser64; this electrostatic surface representation shows the complementarity of the charges between the positively charged basic peptide ligand and the negatively charge acidic BCAR1 binding pocket, with an atomic closeup of the interaction between the ionic side chains and the SH3 domain showing salt bonds.

[0114] HPAEC lysates were transfected with aNEDDtide (PPKPSRP, SEQ ID NO:2) conjugated with phthalimide at -COOH or H2N terminus, or non-conjugated control for 1 hr and changes to NEDD9 expression were measured by anti-NEDD9 immunoblot. The results, shown in FIG. 1 IB, show dose-dependent degradation of NEDD9 in the phthalimide-peptide transfected conditions compared to control, provided important supporting evidence for NEDD9 targeting by the NEDDtide. To demonstrate specificity, the effect of peptide Pl on other proteins was evaluated. As shown in FIG. 11C, the Pl peptide (Thalidomyl-(Ahx)-PPKPSRP-NH2, SEQ ID NO:2 modified by thalidomide on the N-terminal end and by C-terminal amidation) degraded NEDD9 over time, but failed to degrade another SH3 domain protein, GBR2, which (based on predictive models) lacks a binding pocket that would recognize the proline repeat binding motif of our peptide. FIG. 1 ID shows the degrading effect of Pl at the maximum concentration (600 ng) for 10 min.

[0115] In addition, HPAEC monolayers were subjected to anti-NEDDtide and anti- NEDD9 immunofluorescence per standard methods.

[0116] Cultured human pulmonary artery endothelial cells were transfected with a NEDDtide (DEAPPKPSRPGYPS. SEQ ID NO:7) with an N-terminal modification of Biotin-Ahx (1 ng / ul) for 2 hr and then anti-NEDD9 and anti-Biotin immunofluorescence was performed. The images of HPAECs in FIG. 1 IE show subcellular colocalization of NEDDtide and NEDD9 in areas that are structurally consistent with focal adhesions, which is absent in the bottom row of images in cells lacking the peptide.

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[0220] OTHER EMBODIMENTS

[0221] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:1 . A peptide comprising or consisting of a sequence PX1KPX2R, where Xi is P, Q or K, and X2is S or P, optionally PPKPSR (SEQ ID NO:1); PPKPSRP (SEQ ID NO:2); PPKPSRPG ((SEQ ID NO:3); PPKPSRPGY (SEQ ID NO:4); PPKPSRPGYP (SEQ ID NO:5); PKKPPR (SEQ ID NO:6), or DEAPPKPSRPGYPS (SEQ ID NO:7), or a peptidomimetic thereof.

2. The peptide of claim 1, comprising or consisting of 6-14 or 6-20 amino acids, optionally 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

16. 17, 18, 19, or 20 amino acids.

3. The peptide of claim 1, wherein the peptide comprises an electrophilic arginine analog, optionally comprising the structure:

4. The peptide of claims 1-3, which is modified.

5. The peptide of claim 4, wherein the peptide is modified by conjugation with phthalimide or 4-nitrophthalimide optionally on the amino (N) or carboxy (C) terminus, by biotinylation of the amino terminus and / or amidation of the carboxy terminus, or by conjugation to a linear or branched-chain monomethoxy polyethylene glycol.

6. The peptide of claim 1 , comprising SEQ ID NO:2 modified by thalidomide on the N-terminal end and by C-terminal amidation (Thalidomyl-(Ahx)-PPKPSRP- NH2).

7. A composition comprising the peptide of claims 1 -6.

8. A pharmaceutical composition comprising the peptide of claims 1-6, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the carrier comprises liposomes.

10. A method of treating a subject who has a disorder associated with pulmonary arterial hypertension (PAH), the method comprising administering to the subject a therapeutically effective amount of the peptide of claims 1 -6, composition of claims 7-8, or the pharmaceutical composition of claim 9.

11. A method of treating a subject who has a solid tumor, the method comprising administering to the subject a therapeutically effective amount of the peptide of claims 1-6, composition of claims 7-8. or the pharmaceutical composition of claim 9.

12. The method of claim 11, wherein the solid tumor is breast cancer, gastric cancer, or pancreatic cancer.

13. The method of claims 11 or 12, wherein the therapeutically effective amount results in a reduction in risk of metastasis.

14. The peptide of claims 1-6, composition of claims 7-8. or the pharmaceutical composition of claim 9, for use in a method of treating a subject who has a disorder associated with pulmonary arterial hypertension (PAH), the method comprising administering to the subject a therapeutically effective amount of the peptide of claims 1-6, composition of claims 7-8, or the pharmaceutical composition of claim 9.

15. The peptide of claims 1-6, composition of claims 7-8, or the pharmaceutical composition of claim 9, for use in a method of treating a subject who has a solid tumor, the method comprising administering to the subject a therapeutically effective amount of the composition.

16. The peptide, pharmaceutical composition, or composition for the use of claim 15, wherein the solid tumor is breast cancer, gastric cancer, or pancreatic cancer.

17. The peptide, pharmaceutical composition, or composition for the use of claims 15 or 16. wherein administering the therapeutically effective amount results in a reduction in risk of metastasis.

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