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Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin Signaling Complex to Inhibit VEGF-Induced Retinal Angiogenesis

Jordan J. Toutounchian, Jayaprakash Pagadala, Duane D. Miller, Jerome Baudry, Frank Park, Edward Chaum and Charles R. Yates
Molecular Pharmacology January 2017, 91 (1) 1-13; DOI: https://doi.org/10.1124/mol.116.105031

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Abstract

Targeting vascular endothelial growth factor (VEGF) is a common treatment strategy for neovascular eye disease, a major cause of vision loss in diabetic retinopathy and age-related macular degeneration. However, the decline in clinical efficacy over time in many patients suggests that monotherapy of anti-VEGF protein therapeutics may benefit from adjunctive treatments. Our previous work has shown that through decreased activation of the cytoskeletal protein paxillin, growth factor–induced ischemic retinopathy in the murine oxygen-induced retinopathy model could be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome is required for human retinal endothelial cell migration and proliferation. Specifically, the disruption of focal adhesion kinase (FAK) and paxillin interactions using the small molecule JP-153 inhibited Src-dependent phosphorylation of paxillin (Y118) and downstream activation of Akt (S473), resulting in reduced migration and proliferation of retinal endothelial cells stimulated with VEGF. However, this effect did not prevent the initial activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded microemulsion affected the hallmark features of pathologic retinal angiogenesis, reducing neovascular tuft formation and increased avascular area, in a dose-dependent manner. In conclusion, our results suggest that using small molecules to modulate the focal adhesion protein paxillin is an effective strategy for treating pathologic retinal neovascularization. To our knowledge, this is the first paradigm validating modulation of paxillin to inhibit angiogenesis. As such, we have identified and developed a novel class of small molecules aimed at targeting focal adhesion protein interactions that are essential for pathologic neovascularization in the eye.

Introduction

Diabetic retinopathy and age-related macular degeneration are among the most common causes of blindness in adults (Pascolini and Mariotti, 2012). Vision loss occurs in the advanced stages of both diseases owing to aberrant ocular angiogenesis and neovascularization (Aiello et al., 1994; Ferris et al., 1984). Vascular endothelial growth factor (VEGF) plays a key role in this pathophysiology and is the target of current FDA-approved antiangiogenic protein therapeutics (Ozaki et al., 1999; Osborne et al., 2004; Nowak, 2006; Wilkinson-Berka et al., 2013; http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm433392.htm). However, prospective studies show a decline in long-term efficacy, which is believed to result from the emergence of VEGF-independent mechanisms and expression of other growth factors and cytokines involved in maintaining the abnormal angiogenic milieu (Bergers and Hanahan, 2008; van Beijnum et al., 2015). In addition, the further decline in visual function with long-term anti-VEGF therapy has been linked to the loss of the choroidal blood supply, which is in part VEGF-dependent and which supports the integrity and health of the overlying retinal pigment epithelium and neural retina (Marneros et al., 2005; Saint-Geniez et al., 2008; Tokunaga et al., 2014). Thus, targeting downstream signaling proteins linked to pathologic neovascularization represents an alternative or adjunctive approach to approved anti-VEGF treatments that may reduce the damaging effects of antiangiogenic therapy.

VEGF activates endothelial cells, in part, by stimulating signal transduction pathways that regulate the enzymatic turnover of adhesion complexes, or mechanotransduction “signalsomes” consisting of adaptor proteins and kinases, e.g., Src-family kinases, focal adhesion kinase (FAK), and paxillin (Waltenberger et al., 1994; Abedi and Zachary, 1997; Provenzano and Keely, 2011). Targeting focal adhesion (FA) kinases downstream of growth factor receptor activation has recently emerged as an effective strategy for inhibiting retinal angiogenesis (Wary et al., 2012). In ischemic models of retinopathy, the local silencing of Src or FAK expression causes a significant reduction in pathologic neovascular disease (Kornberg et al., 2004; Werdich and Penn, 2006). However, evidence of resistance is also accumulating, as recently demonstrated when cells deficient in FAK protein showed enhanced expression of its homolog, proline-rich tyrosine kinase 2 (PYK2), which is also known to regulate gene expression and endothelial budding or sprouting via VEGF-dependent mechanisms (Bergers and Hanahan, 2008; Weis et al., 2008; Shen et al., 2011; Eke and Cordes, 2015). Thus, there is a critical need to identify alternative drug targets that serve as common “interface points” shared by proteins within the focal adhesion complex (FAC).

Paxillin is a multidomain adaptor protein that binds to both FAK and PYK2, as well as numerous other FA proteins (e.g., GIT-1, vinculin, and actopaxin) (Turner, 2000). Studies characterizing these protein-protein interactions at the structural level have identified highly conserved four-helix bundled regions, or so called paxillin-binding subdomains, which specifically engage the paxillin N-terminal leucine-rich domains (Brown et al., 1998; Arold et al., 2002; Vanarotti et al., 2014). Paxillin, together with Src and FAK, recruit other proteins to the cell’s leading edge where actin filaments coalesce around integrins (cellular “anchors”) to provide mechanical forces needed to pull the cell forward. Since these complexes help assemble and support the connections between the actin cytoskeleton and the extracellular matrix, targeting these proteins with small molecules would dismantle the FA complexes and obstruct proliferative and migratory signal transduction during angiogenesis (Fig. 9).

We have identified a proliferative response phenotype of human primary retinal endothelial cells (REC) exposed to high-dose ionizing radiation (Toutounchian et al., 2014). Irradiation-enhanced paxillin Y118 phosphorylation, which was reduced by mitogen-activated protein kinase (MAPK) inhibition. Under these same mechanisms, inhibiting MAPK and, thus, paxillin phosphorylation caused a reduction in in vivo retinal angiogenesis. Our data suggested a direct role for activated paxillin in radiation-induced retinopathy, an ischemic inflammatory disease with a neovascular component (Boozalis et al., 1987; Finger, 2008).

However, the mechanisms by which paxillin coordinates VEGF-signaling through the FAC is not well understood, as most focus has been on targeting kinase activity of either Src or FAK. It was shown, however, that paxillin deletion caused dysfunctional cell spreading and stunted migration, similar to the phenotypes of cells without FAK (Eliceiri et al., 1999; Brown and Turner, 2004; Brown et al., 2005). To our knowledge, this report is the first paradigm validating small-molecule modulation of paxillin within FAs to prevent pathologic angiogenesis in neovascular disease. With this study, we have exploited paxillin as our molecular target and have identified a novel class of small-molecule modulators of the FA protein interactions essential for retinal neovascularization.

Materials and Methods

Reagents/Antibodies.

Recombinant human VEGF-165A protein was purchased from R&D Systems (Minneapolis, MN). Total VEGFR-2, Akt, and p44/42 MAPK [extracellular signal-regulated kinase (ERK1/2)] as well as phosphorylated VEGFR-2 (Tyr1175), FAK (Y397, Y576/577, Y925), Akt (Ser473), cleaved and total poly(ADP ribose) polymerase (PARP), GAPDH, and ERK 1/2 (Thr202/Tyr204) were acquired from Cell Signaling Technologies (Danvers, MA). Phosphorylated paxillin (Y118) and FAK (Y861) were purchased from Abcam (Cambridge, MA). Mouse antibodies against human paxillin (clone 349) and FAK (clone 77) were purchased from BD Biosciences (San Jose, CA). Mouse α-tubulin primary antibody and secondary antibodies IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse were purchased from LI-COR Biotechnology (Lincoln, NE). Calcein-AM was obtained from BD Biosciences. DAPI nuclear stain was purchased from ThermoFisher Scientific (Pierce; Sunnyvale, CA). 6-B345TTQ and the Src kinase inhibitor SU6656 were purchased from Sigma-Aldrich (St. Louis, MO). LY294002 (PI3K inhibitor) was acquired from Cell Signaling Technologies. Primary antibody names, catalog numbers, species of origin, and dilutions are included in Supplemental Table 1.

JP-153 was synthesized in accordance with the methods devised for ortho-functionalization of aniline derivatives (Houlden et al., 2010). Briefly, naphthylisocyanate 1 (5.9 mmol, 1.0 g) was added to a solution of t-butylisopropylamine (5.9 mmol, 0.9 ml) in diethyl ether (10 ml) under stirring at room temperature. The colorless solution was stirred for 3 hours and subsequently cooled to 0°C. Tetramethylethylenediamine (12.98 mmol, 2.0 ml) was added followed by n-butyllithium (11.8 mmol, 2.43 M in hexanes, 3.0 ml). The clear yellow solution was then stirred for 3 hours, during which time a white precipitate formed. The reaction mixture was cooled to –78°C and aldehyde 2 (8.85 mmol, 1.7 g) in tetrahydrofuran (5 ml) was added dropwise over 4 minutes. Following the addition, ethanol (5 ml) was added rapidly and the mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction mixture was then concentrated in vacuo, diluted with dichloromethane, and washed with saturated ammonium chloride, NH4Cl (aqueous). The organic layer was evaporated onto silica and purified by column chromatography. JP-153 purity was characterized with high-resolution mass and nuclear magnetic resonance spectroscopy. JP-153 and 6-B345TTQ structures and calculated LogP values are presented in Supplemental Fig. 1.

Primary Retinal Endothelial Cell Culture.

Primary human retinal endothelial cells (Lot 181) were purchased from Cell Systems Corporation (Kirkland, Washington). Cells were grown on attachment-factor surfaces in M131 medium containing microvascular growth supplements (Invitrogen, Carlsbad, CA) gentamicin (10 mg/ml) and amphotericin B (0.25 mg/ml). Only primary cells up to passage six were used. For immunoassays, RECs were plated into six-well plates, cultured for 2 days, and serum-deprived using 0.1% bovine serum albumin (Sigma-Aldrich) overnight prior to experiments. RECs were pretreated with inhibitors, SU6656 (1 μM), LY294002 (10 μM), or JP-153 (1 μM), for 1 hour prior to VEGF (100 ng/ml) stimulation, unless mentioned otherwise. All chemical compounds were solubilized in dimethyl sulfoxide (DMSO) and further diluted into serum-free cell culture medium, reaching a final vehicle concentration of <0.01% (v/v) DMSO.

REC Proliferation Assays.

To evaluate paxillin-dependent modulation of retinal endothelial cell proliferation, 50,000 cells were seeded into each well of a 96-well dish and allowed to adhere overnight. RECs were serum-deprived for 1 hour in 0.1% bovine serum albumin, stimulated with VEGF (100 ng/ml), treated with vehicle, kinase inhibitors, or test compounds and incubated for 24 hours. Cellular proliferation was determined using the tetrazolium salt WST-1 according to the assay manufacturer’s instructions (Quick Cell Proliferation Assay Kit II; Abcam, Cambridge, MA). Optical density as a measure of cellular proliferation was measured using a microplate reader at an absorbance of 450 nm. Data represent mean optical density (OD) ± S.D., n = 8 per group. In parallel to the 24-hour viability experiments, RECs were incubated with calcein-AM for 30 minutes and imaged using the EVOS FL Cell Imaging System (ThermoFisher Scientific) to observe cell numbers.

Annexin V/Fluorescein Isothiocyanate Staining and Flow Cytometry Analysis for Apoptosis.

REC apoptosis was measured by detection of phosphatidylserine translocation to the external surface of the cell membrane (Fadok et al., 1992). Annexin V/propidium iodide (PI) staining was performed according to manufacturer’s instructions (BioLegend, San Diego, CA). Briefly, RECs treated with either JP-153 or vehicle for 24 hours were trypsinized and washed twice with ice-cold phosphate-buffered saline (PBS) containing two-percent fetal bovine serum. Pelleted RECs were resuspended in Annexin V Binding Buffer at 5.0 × 106 cells/ml and incubated with fluorescein isothiocyanate–annexin V and PI staining solution (BioLegend) at room temperature for 15 minutes in the dark. Cells were then resuspended in binding buffer and analyzed by fluorescence flow cytometry using the BD LSRII Flow Cytometry Analyzer (BD Biosciences). Data were statistically assessed using FlowJo analysis software (V10.0.6; Tree Star Inc., Ashland, OR). Apoptotic cells were defined as annexin V-positive and PI-negative, and necrotic cells are defined as annexin V-positive and PI-positive. Viable cells were considered annexin V and PI-negative.

Immunoblot (Western) Analysis.

Cellular proteins were analyzed by Western blotting after SDS-PAGE using human specific primary antibodies, as previously described (Toutounchian et al., 2014). Whole REC lysates were collected in radioimmunoprecipitation assay lysis buffer with protease/phosphatase inhibitor (1×) cocktail (Roche, Indianapolis, IN). Total protein was measured by BCA assay (Pierce/ThermoFisher Scientific) then processed in 4× LDS loading buffer containing 2.5% 2-mercaptoethanol (Sigma-Aldrich), heated to 70°C for 10 minutes, and loaded into NuPAGE 4–12% Bis-Tris Gels (Invitrogen/ThermoFisher Scientific). Immunoblotting was performed with nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked using Odyssey Blocking Buffer (LI-COR), and then incubated with specific primary antibodies overnight at 4°C. Analysis of phosphorylation is presented as a ratio of phosphorylated protein to total protein (e.g., P-Y397 FAK/total FAK); cellular lysates analyzed for both phosphorylated and nonphosphorylated protein were normalized to total cellular/housekeeping proteins, i.e., GAPDH or α-tubulin. Secondary antibodies (IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse; 1:12,500; LI-COR) were incubated in the dark at room temperature for 45 minutes. Dual-channel infrared scan and quantitation of immunoblots were conducted using the Odyssey Sa infrared imaging system with Image Studio (Ver. 3.1.4; LI-COR).

In Vitro Scratch-Wound Assay.

REC migration was performed in accordance with methods previously described (Ghosh et al., 2013). RECs (106 cells/well) were seeded to 12-well plates and cultured to confluence. RECs were washed twice with 1× PBS and prewarmed serum-free Medium 131 (Invitrogen) was introduced to wells for 1 hour to remove any residual effects of supplemented growth factors. Using a sterile 200-μl pipette tip, a straight scratch down the center of the well provided the baseline for the analysis and quantification of REC migration and proliferation over 24 hours. Wells were then washed one time with PBS to remove any detached cells. Growth factor–supplemented medium with or without JP-153 (0.10–10 μM) was added to each well, and plates were immediately imaged using a CoolSNAP charge-coupled device camera (Roper Technologies, Inc., Sarasota, FL) mounted on an Eclipse TE300 Inverted Microscope (Nikon, Melville, NY). Using 4× magnification and a computer-controlled stage, images at three specific coordinates per well at the time of the initial wounding were obtained in Metamorph software (Universal Imaging, West Chester, PA). Plates were returned to incubator for 24 hours. The next day, previous coordinates were recalled and images were again collected in Metamorph and then transferred to Adobe Photoshop (CS5 Extended, Ver. 12.1; Adobe Systems, Inc., San Diego, CA). Using the magnetic lasso tool in Photoshop, the outline of protruding/migrating cells from the periphery of the scratch toward the center was measured. The area devoid of migrating cells was recorded and quantified as a percentage change from the previous day’s area quantification:Embedded Image(1)Data represent mean percent wound closure ± S.D. RECs from each group were fixed at 24 hours, stained with DAPI, and imaged using the EVOS FL Cell Imaging System (ThermoFisher Scientific). A representative image from each group was used to depict extent of wound closure.

Transwell Cellular Migration Assays.

Cell migration was performed using Transwell polycarbonate membranes (Corning, Corning, NY), as previously described (Cheranov et al., 2008). Briefly, cell-culture inserts containing membranes 6.5 mm in diameter and 8.0-μm pore size (Corning) were placed in a 24-well tissue culture plate (Corning). The upper surface of the porous membrane was coated with attachment factor at 37°C for 1 hour. Human RECs were serum-starved overnight in medium 131 containing 0.1% bovine serum albumin, trypsinized, pelleted, and resuspended in medium 131 with vehicle (0.1% DMSO) or JP-153 at respective concentrations. RECs were then seeded into the upper chamber at 1 × 105 cells/well. Medium 131 containing either vehicle or VEGF (100 ng/ml) +/− JP-153 was added to the lower chamber. After 24 hours of incubation at 37°C, nonmigrated cells were removed from the upper side of the membrane with cotton swabs and the cells on the lower surface of the membrane were fixed in 4% paraformaldehyde for 15 minutes and washed twice with 1× PBS. Nuclei were then stained with DAPI in PBS for five minutes and images were collected using the EVOS FL Cell Imaging System (ThermoFisher Scientific). Images were imported into Adobe Photoshop (Adobe Systems, Inc.) and cells were counted using batch image processing with automation. Briefly, the batches of images from all experimental groups were processed using color correction to enhance DAPI signal against background. Nuclei were outlined using the color-selection tool. The automation protocol was established on the basis of the first image processed in Photoshop to ensure that the processing of each subsequent image was done without any biasing or manipulation of quality and/or integrity. Migrating RECs were quantified from six random fields (n = 3). Data represent mean number of migrating cells/field ± S.D.

Retinal Angiogenesis: Murine Oxygen-Induced Retinopathy Model.

C57BL/6N (Charles River Laboratories, Wilmington, MA) mice were used in all experiments. All animal studies were performed under the guidelines of the Association for Research in Vision and Ophthalmology for the humane use of animals in vision research, and under the guidance and approval of the Institutional Animal Care and Use Committee at the University of Tennessee Health Science Center.

Retinal angiogenesis was induced using a mouse model of oxygen-induced retinopathy (OIR), as previously described (Smith et al., 1994; Toutounchian et al., 2014). Five independent litters on three separate occasions were used for OIR experiments. Mouse pups exposed to the oxygen chamber were shuffled into three groups prior to dosing (P12) to provide intralitter controls. Experimental groups were as follows: 1) mice reared in normal atmospheric conditions (negative-control; normoxia); 2) mice exposed to OIR/hyperoxic chamber and treated with vehicle microemulsion (1 μl/g; positive-control); 3) OIR-mice treated with JP-153-loaded microemulsion at 0.5 mg/kg; and 4) JP-153 at 5.0 mg/kg. Mouse pups were exposed to 75% oxygen at postnatal day seven (P7) for 5 days and then returned to normal oxygen (P12). Ocular microemulsion used for drug delivery comprised Capryol 90 (10.5% v/v), Triacetin (10.5% v/v), Tween-20 (24.5% v/v), and Transcutol P (24.5% v/v) (Gattefossé Pharmaceuticals, Saint-Priest, France) generated via homogenization and water titration methods, as previously described (Toutounchian et al., 2014). JP-153 was first loaded into the oil-phase and then incorporated into the final microemulsion formulation and stored at room temperature away from light until dosing. OIR mice were weighed prior to receiving each daily dose to both eyes using either JP-153 or vehicle-loaded microemulsion from P12 to P17 (vehicle control, N = 8; JP-153 0.5 mg/kg, N = 14; JP-153 5.0 mg/kg, N = 14). On P17, retinas were removed, dissected, mounted, and stained for endothelial cells to investigate retinal angiogenesis. At the conclusion of the study, anesthetized animals were humanely euthanized according IACUC guidelines.

Retinal Whole-Mount Imaging and Analysis.

Enucleated whole eyes from P17 mouse pups underwent immediate weak fixation in 4% paraformaldehyde in PBS for 1 hour and washed three times in ice-cold PBS. Retinas were carefully isolated under a Leica S6E dissecting stereomicroscope (Leica Microsystems, Buffalo Grove, IL) and mounted onto microscope slides. Whole retinas were incubated overnight at 4°C with isolectin B4-594 (Alexa Fluor 594; Molecular Probes, Eugene, OR), as previously described (Connor et al., 2009; Toutounchian et al., 2014). Isolectin-stained retinas were then washed three times in 1× PBS, sealed under coverslips using Vectashield mounting medium (Vector Laboratories, Inc.), and stored at 4°C until imaging.

Images were acquired using a Zeiss LSM 710 system attached to a Zeiss Axio Observer inverted microscope with Zen 2010 v.6.0 software (Carl Zeiss Microscopy, Peabody, MA). Multidimensional acquisition was carried out using Z-stacks with <4-μm slicing intervals and tile-scan automation with an 8% tile overlap at a resolution of at least 512 × 512 pixels per tile and digitally stitched together. Quantification of avascular area (AV) and neovascularization (NV) in retinal whole mounts was performed in Adobe Photoshop (Adobe Systems, Inc.), as previously described (Toutounchian et al., 2014). The area devoid of vascularization around the optic disc was characterized as percentage of total retinal area (%AV). Photoshop color-range analysis tool were used to outline NV formations after intensity thresholds were set to exclude normal vasculature. Data were recorded as a percentage of total retinal area (%NV). Representative whole-mounted retinas were displayed using the exact quantified outlined areas and layered back into place onto the original whole-retina image. Using the linear light-blending method in Photoshop, both avascular and neovascular areas were transposed in white.

Statistical Analyses.

All data represented herein were performed in replicates of three or more and presented as the mean ± S.D., unless otherwise indicated. Differences among groups were analyzed using one-way analysis of variance. When overall analysis revealed significance among groups, means were compared and tested using Tukey’s posthoc analysis. Statistical significance was set at P < 0.05. All statistical analyses were performed in SigmaPlot 12.0 software (Systat Software, Inc., San Jose, CA). P values representing significances of <0.05, 0.01, and 0.001 are denoted with symbols *, **, ***, whereas significances <0.05, 0.01, 0.001 among treatment arms are represented with †, ††, †††, respectively.

Results

Src/FAK-Paxillin Signaling Pathway in REC Proliferation.

FAK and paxillin are coordinators of FA turnover during VEGF-induced proliferation and migration—two seminal events of angiogenesis (Brown et al., 2005). To confirm the relevance of these two players in VEGF-induced proliferation of RECs, we stimulated RECs with VEGF and analyzed cell lysates for FAK and paxillin phosphorylation over time. Fig. 1A shows that rhVEGF (100 ng/ml) activates VEGF receptor-2 (VEGFR-2) with maximal phosphorylation occurring within 15 minutes at a major phosphorylation site, Tyr-1175. Activation of VEGFR-2 triggers autophosphorylation of FAK Y397 (as seen in Western blot images, with analysis to the right; *P < 0.05, ***P < 0.001), which promotes association of Src with FAK (Schaller et al., 1994) and subsequently leads to Src-dependent FAK phosphorylation of its kinase domain loop, Y576/577 and focal adhesion targeting domain Y925 (Fig. 1A). Src-dependent activation and binding of FAK forms the Src/FAK focal adhesion complex (FAC), which phosphorylates paxillin Y118 (Fig. 1B, **P < 0.01, ***P < 0.001).

Fig. 1.
Fig. 1.

VEGF-induced FA signaling in RECs. (A) Retinal endothelial cells were stimulated with VEGF (100 ng/ml) and cellular lysates were collected over four hours and focal adhesion protein activation was measured using Western blotting as described in Materials and Methods. Initially, VEGFR-2 is activated at Y1175 upon VEGF ligation which triggers FAK Y397 autophosphorylation (representative Western blots on the left, analysis of FAK pY397 levels on the right) (*P < 0.05, ***P < 0.001). Subsequently, Src-kinase binds to FAK and further activates the kinase-domain loop FAK Y576/577 and the focal adhesion targeting domain FAK Y925. (B) Src-dependent activation of FAK coincides with paxillin Y118 phosphorylation over 4 hours (**P < 0.01, ***P < 0.001). Data represent mean ± S.D., n = 4–8.

To determine if the Src/FAK complex is necessary for paxillin activation in RECs and thus proliferation, we examined FAK and paxillin phosphorylation in VEGF-stimulated RECs treated with Src-kinase inhibitor SU6656 (1 μM) (Blake et al., 2000). In Fig. 2A, we show that inhibiting Src kinase reduces the phosphorylation of FAK Y576/577, Y925, and Y861 (††P < 0.01) but does not affect autophosphorylation of Y397. An inactive Src/FAK complex fails to phosphorylate paxillin Y118 (Fig. 2A, ††P < 0.01). We again treated RECs with SU6656 (1 μM) for 24 hours and showed that inhibition of Src-mediated phosphorylation of FA proteins leads to a significant decrease in VEGF-induced proliferation (Fig. 2B, ††† P < 0.001).

Fig. 2.
Fig. 2.

Src-dependent activation of FAK and paxillin in RECs. (A) Src-inhibition with SU6656 (1 μM) inhibited VEGF’s activation of FAK Y576/577, Y861, and Y925 and paxillin Y118 (* P < 0.05,††P < 0.01) but did not prevent autophosphorylation of FAK Y397 (P > 0.05). Data (n = 3) represent mean ± S.D. (B) VEGF-mediated proliferation of RECs was performed as described in Materials and Methods. VEGF-induced proliferation in RECs was reduced in the presence of SU6656 (1 μM), which correlated with FA activation in panel A (***,†††P < 0.001). Data represent mean ± S.D., n = 8.

Discovery of JP-153 as a Potent Inhibitor of VEGF-Induced Proliferation.

Src-dependent FAK and paxillin phosphorylation correlated with VEGF-induced proliferation in RECs (Fig. 2B). We used this phenotypic response to derive compounds related to a known paxillin protein disruptor, 6-B345TTQ (Kummer et al., 2010). Our initial lead identification efforts yielded the analog JP-153, which was ∼50 times more potent than 6-B345TTQ in REC proliferation assays (Fig. 3A, panel a, †P < 0.05, †††P < 0.001; panel b, †††P < 0.001). JP-153 and 6-B345TTQ structures, IC50, and calculated Log P values depict JP-153 as more pharmaceutically favorable (Supplemental Fig. 1) (Lipinski et al., 2001). We used calcein-AM staining (Fig. 3B) to show that live cell number is reduced with JP-153 treatments in addition to reduced proliferative activity, as measured by WST-1 in Fig. 3A. Yet, JP-153 does not promote apoptosis in cells, as characterized by PARP cleavage (Fig. 3C, panel a, *P < 0.05 versus serum-free controls) and annexin V/PI staining at 1-μM concentration over 24 hours (Fig. 3C, panel b).

Fig. 3.
Fig. 3.

Discovery of JP-153 as a potent inhibitor of VEGF-induced proliferation. (A) REC proliferation was used to investigate compound 6-B345TTQ, a known paxillin disruptor, which was found to inhibit REC proliferation at concentrations greater than 10 μM (†P < 0.05, †††P < 0.001). Owing to potency issues, we redesigned a derivative, JP-153, that inhibits REC proliferation substantially in concentrations as low as 0.25 μM (†††P < 0.001). Data represent mean ± S.D., n = 3. (B) We observed cell numbers using calcein-AM as described in Materials and Methods. (C) We investigated apoptosis using cleaved-PARP signaling in Western blots and showed that JP-153 (1 μM) did not significantly enhance apoptotic signaling (panel a, P = 0.239 versus 10% fetal bovine serum controls; data are presented as the mean ± S.D.; n = 3). Flow cytometry quantified apoptotic cells within the population treated with JP-153 (1 μM, 24 hours to confirm that cell death was not induced with treatment, compared with controls; panel b, n = 50,000 cells).

Effector Signaling through an Activated Src/FAK-Paxillin Signaling Complex during VEGF-Induced Proliferation Is Akt-Dependent.

We postulated that JP-153 inhibits REC proliferation through disruptions in FA protein interactions, as shown by Kummer et al. (2010) with 6-B345TTQ. Disrupting Src/FAK binding to paxillin results in decreased activation of paxillin Y118 (Richardson et al., 1997). Thus, we treated RECs with JP-153 (1 μM) for 1 hour and then stimulated them with VEGF for 4 hours. In cells JP-153 significantly reduces Y118 phosphorylation (Fig. 4, **,††P < 0.01) but did not inhibit constitutive levels of unstimulated RECs treated with JP-153 (P = 0.749).

Fig. 4.
Fig. 4.

JP-153 inhibits VEGF-induced activation of paxillin Y118. (A) REC lysates were collected at 4 hours post-VEGF activation, and phosphorylation of paxillin Y118 was measured using Western blotting. (B) JP-153 significantly reduced phosphorylation in cells stimulated with VEGF (**,††P < 0.01) but did not affect constitutive/unstimulated levels (P = 0.749 versus vehicle control). Data represent mean ± S.D.; n = 3.

Next, we examined downstream FA effector signaling during early VEGF activation at 15 minutes. We pretreated RECs with JP-153 for 1 hour prior to VEGF-activation and measured phosphorylation of FAK phosphorylation sites, as well as downstream angiogenic markers AKT and ERK. Our results again confirmed that JP-153 reduces activation of paxillin Y118 compared with VEGF controls (Fig. 5A, panel a; *, †P < 0.05) but does not change autophosphorylation of FAK Y397; these results mimic the activity of SU6656 (††P < 0.01). However, when we probed for FAK Y576/577, Y861, and Y925 in cells, JP-153 did not affect levels of Src-dependent FAK phosphorylation sites (Fig. 5A, panels d–f; P > 0.05), whereas SU6656 inhibited these levels strongly (†P < 0.05, ††P < 0.01). To rule out kinase inhibition, we show that JP-153 was not a direct kinase inhibitor of FA signaling effectors per se, as measured by the Z′-LYTE SelectScreen Single Point biochemical assay (ThermoFisher Scientific) (Supplemental Table 2).

Fig. 5.
Fig. 5.

JP-153 acts by reducing effector signaling through Src/FAK/paxillin FA complex to inhibit VEGF-induced proliferation. A) Western blot images (left) and respective analyses (right, panels a-f) of RECs activated by VEGF (100 ng/mL for 15 minutes) show FA and effector signaling after one hour pre-treatments with JP-153 (1µM), Src-inhibitor SU6656 (1 µM) or PI3K inhibitor LY294002 (10 µM). JP-153 and SU6656 significantly reduce levels of VEGF-induced paxillin Y118 phosphorylation (panel a; **, ††P < 0.01), but only SU6656 inhibits FAK phosphorylation at Y576/577 (panel d; *, †P < 0.05), Y861 (panel e; *, †P < 0.05), and Y925 (panel f; *P < 0.05, ††P < 0.01), in agreement with earlier experiments shown in Figure 3. VEGF-induced pAKT (S473) phosphorylation was inhibited by JP-153, SU6656 and LY294002 (panel b; **, ††P < 0.01,†††P < 0.001). Neither SU6656 nor JP-153 causes any significant change to VEGF-induced pERK 1/2 activation (panel c; P > 0.05), while LY294002 caused an increase in activation of ERK (†P < 0.01 vs. VEGF controls). The dividing lines in the Western blot panel convey where samples from the same blot were shifted over to the left by one lane for data presentation consistency. B) We confirmed Akt-dependent REC-proliferation by treating cells with LY294002 which resulted in the potent inhibition of proliferation in a more pronounced manner than JP-153 or SU6656 (***, †††P < 0.001, n = 8). Data represent mean ± SD.

Src-mediated activation of paxillin Y118 primes the complex for recruitment to focal contacts, where interactions with PI3K and MEK activate their respective downstream substrates, AKT and ERK, to promote cytoskeletal rearrangements during proliferation and migration (Fujikawa et al., 1999; Akagi et al., 2002; Du et al., 2011). Thus, we compared RECs treated with JP-153 and SU6656 with those treated with PI3K inhibitor LY294002 (10 μM). Both p-Akt (Ser473) and p-ERK 1/2 levels rose under VEGF, but only Akt was effectively blocked by SU6656 and JP-153 (Fig. 5A, panels b and c; *,†P < 0.05, ††P < 0.01), since neither show significant inhibition of p-ERK 1/2 at concentrations tested (P > 0.05). However, complete inhibition of Akt phosphorylation by LY294002 caused no reductions in FAK or paxillin activation, suggesting the Src/FAK/paxillin activation cascade precedes PI3K-induced Akt phosphorylation. However, unlike JP-153 or SU6656, LY294002 significantly induced ERK activation (†P < 0.05; LY294002 versus VEGF). To validate an Akt-dependent proliferation pathway, cells treated with LY294002 potently inhibited proliferation, with levels far exceeding serum starvation, Src-inhibition, and JP-153 treatments (Fig. 5B, ***,†††P < 0.001). Together, these data suggest JP-153 acts to inhibit REC proliferation through an Akt-dependent but ERK-independent mechanism.

Paxillin Modulation with JP-153 Inhibits VEGF-Induced Migration of Retinal Endothelial Cells.

We have shown that JP-153 inhibited REC proliferation through disruptions in Src/FAK activation of paxillin Y118 and pAkt (Fig. 5). Since angiogenesis requires two distinct but cooperative mechanisms, proliferation and migration, we examined JP-153’s effect on migration using the standard scratch wound assay. VEGF-induced REC migration was significantly inhibited in JP-153 treatments over a range of concentrations (0.10–10 μM) (Fig. 6; *,†P < 0.05, ††† P < 0.001). Next, we validated our scratch-wound results with the Transwell migration/invasion assay with VEGF as the chemotactic inducer (Yoshida et al., 1996). Our results show that JP-153 inhibits REC invasion at submicromolar concentrations (0.10–0.50 μM) (Fig. 7, ***,†††P < 0.001).

Fig. 6.
Fig. 6.

JP-153 inhibited VEGF-induced REC migration in the scratch-wound assay. The scratch-wound migration assay was performed in RECs exposed to VEGF for 24 hours, as described in Materials and Methods. (A) Data analysis show JP-153 inhibits VEGF-induced migration in a concentration-dependent manner. (B) Representative DAPI-stained images after 24 hours. Data are presented as the mean ± S.D. (n = 6; *,†P < 0.05, †††P < 0.001).

Fig. 7.
Fig. 7.

JP-153 inhibited VEGF-induced REC invasion using the Transwell migration assay. RECs were seeded onto porous membranes and chemotactic factor VEGF was used to stimulate REC migration, as described in Materials and Methods. (A) Results show that JP-153 inhibited REC invasion in a concentration-dependent manner (data are mean ± S.D.; ***,†††P < 0.001; n = 6). (B) Cells traversing the membrane were fixed and stained with DAPI, and representative images of each group are shown (image labels A–E: serum-free, VEGF, V + 0.10, V + 0.25, V + 0.50 μM, respectively).

Signal Disruption of Src/FAK/Paxillin Complex by JP-153 In Vivo Inhibits Retinal Neovascularization in the Murine Oxygen-Induced Retinopathy Model.

Our in vitro mechanism of action studies in RECs suggested that JP-153 inhibited proliferation and migration by disrupting Src/FAK/paxillin signaling pathway. Therefore, we hypothesized that JP-153 could inhibit retinal angiogenesis in vivo by reducing Src/FAK/paxillin activity. We used the murine OIR model of retinal neovascularization (RNV) to test JP-153 at low and high topical doses applied daily to each eye during the hypoxic period (P17 retinal whole-mounts in Fig. 8A, and subsequent analysis in Fig. 8B). Our data shows that JP-153 inhibits neovascularization by 40 and 45% in a dose-dependent manner (0.5 and 5 mg/kg, respectively), compared with vehicle-treated eyes (panels a–c, ***P < 0.001). However, only JP-153 at the higher dose enhanced the AV compared with vehicle (panels d and e, ***P < 0.001). Mouse pups kept outside the OIR chamber for the entire study were also dosed with JP-153 (5 mg/kg) under identical age-based regimens to evaluate any impact on retinal vascular development. There were no obvious differences between vehicle and JP-153-treated retinas in mice not exposed to the OIR chamber (Supplemental Fig. 2). These findings suggest that JP-153 can act to regulate pathologic RNV without affecting normal retinal blood vessel growth or vasculogenesis.

Fig. 8.
Fig. 8.

JP-153 inhibited retinal angiogenesis in the murine oxygen-induced retinopathy model. P17 retinal whole-mounts were stained for endothelial cells using isolectin B4-594 as described in Materials and Methods. Mice were dosed daily from P12-17 using either topical microemulsion-loaded vehicle, 0.5 mg/kg, or 5.0 mg/kg JP-153. (A) Representative images of retinal whole-mounts depicting: neovascular area (a–c) and AV (d–f). (B) Data analysis of retinal vasculature revealed that JP-153 inhibited NV and increased AV in a dose-dependent manner. Data represent mean ± S.D.; ***P < 0.001; N = 8–14/group.

Discussion

In previous work, paxillin Y118 activation in high-dose radiation injury was an important signaling component driving REC proliferation in ischemic retinopathy (Toutounchian et al., 2014). We demonstrated in this study that VEGF-dependent activation of the Src/FAK/paxillin signaling complex, or signalsome, drives REC migration and proliferation (Fig. 9). Moreover, we showed that modulation of the Src/FAK/paxillin signaling complex with small molecule JP-153 reduced paxillin Y118 activation and inhibited migration and proliferation of RECs; and that this effect did not interfere with VEGF-dependent activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded ocular microemulsion inhibited hallmark features of pathologic retinal angiogenesis in mice; both neovascular tuft formation and vascular regrowth in the murine OIR model were reduced in a dose-dependent manner.

Fig. 9.
Fig. 9.

Summary diagram of JP-153’s proposed target of action. Data suggests that JP-153 targets the interaction between an active Src/FAK signaling complex and paxillin. Inhibiting this interaction resulted in decreased paxillin activation (Y118), preventing activation of downstream effector protein Akt. This effect translated into potent inhibition of REC proliferation and migration, in vitro, and inhibition of RNV, in vivo.

A major finding in this study was that in human primary RECs, Src/FAK activation of paxillin directs VEGF-induced signaling during REC proliferation and migration, a signaling pathway well characterized in cancer cells and other transformed cell lines but previously undescribed in primary human RECs (Abedi and Zachary, 1997; Birukova et al., 2009; Yang et al., 2015). We hypothesized that targeting REC Src/FAK or paxillin would limit the activation of downstream effector proteins important for retinal angiogenesis. First, we showed VEGF induces activation of Src kinase leading to the phosphorylation of FAK and paxillin, which could be prevented by pharmacological inhibition of Src. We then used a small-molecule probe of paxillin binding interactions, 6-B345TTQ (Kummer et al., 2010), to investigate paxillin’s role during VEGF-induced REC proliferation. Blocking interactions that involve paxillin effectively reduced REC proliferation in vitro, but owing to inherently low potency and solubility, we derived a more effective derivative, JP-153.

An unexpected and novel finding during in vitro mechanistic studies was that JP-153 reduced phosphorylation of paxillin Y118, a critical tyrosine activation site, but did not affect FAK phosphorylation, distinguishing JP-153’s activity from Src inhibitor SU6656. Thus, we have shown that paxillin Y118 is an important downstream biomarker for VEGF-induced REC proliferation. Additionally, JP-153 did not inhibit the kinase activities of Src or FAK (Supplemental Table 2); strongly suggesting that JP-153’s antiproliferative phenotype in RECs is through paxillin-dependent signaling, independent of kinases that may regulate its phosphorylation. In fact, mutagenesis of FAK- or paxillin-binding domains are known to inhibit their interaction and prevent activation of paxillin and other downstream proteins (Subauste et al., 2004; Kadaré et al., 2015).

Activation of Src/FAK drives proliferation and migration through intermediates ERK and Akt (Yan et al., 2008; Pylayeva et al., 2009). Our data show that PI3K-inhibitor LY294002 remained unchanged, although effective at preventing both Akt phosphorylation and REC proliferation, levels of FAK, or paxillin phosphorylation. SU6656 and JP-153 both caused reductions in Akt phosphorylation, suggesting that activation of FAK and paxillin precedes VEGF-induced activation of Akt in RECs. However, since JP-153 did not disrupt FAK phosphorylation levels and still reduced p-Akt, we concluded that paxillin Y118 plays a crucial role in coordinating events that drive Akt-dependent angiogenesis in RECs. These results are in agreement with other studies that established the important stepwise role of the Src/FAK complex as a crucial activator of the PI3K-Akt pathway (Thakker et al., 1999; Bullard et al., 2003; Thamilselvan et al., 2007). Therefore, our results show that paxillin is an important signaling intermediary that connects the activated Src/FAK complex and Akt in angiogenesis.

The uncoupling of an active Src/FAK complex from paxillin suggested it is a key regulator of pathologic FA signal transduction and potentially represents a novel in vivo target distinct from anti-VEGF therapies aimed at silencing receptor-mediated kinase signaling. Studies using targeted deletions of FA proteins FAK and Src in the mouse retina disrupt the progression of RNV (Kornberg et al., 2004; Werdich and Penn, 2006); these findings correspond with our in vitro results using the Src inhibitor SU6656, which affects all downstream binding and activation partners. We show similar in vitro effects with JP-153 on proliferation as with SU6656, specifically with decreased paxillin Y118 phosphorylation and inhibition of p-Akt downstream, resulting in potent inhibition of movement and growth. From these studies, we can assert that the activation of the FAC may be a crucial component in the regulation of pathologic retinal angiogenesis, in vivo. We tested this hypothesis by administering JP-153 topically in the OIR model, which resulted in significantly reduced retinal angiogenesis, as measured by both neovascularization and the AV. Intriguingly, we found that only the higher doses of JP-153 were able to significantly enhance AV, suggesting perhaps that our small molecule affects pathologic neovascularization more than vasculogenesis. However, since genetic knockdown of paxillin in mice leads to early embryonic lethality (Hagel et al., 2002), paxillin has been conditionally silenced in the developing mouse retina. These studies actually showed that paxillin knockdown induced migration and endothelial cell sprouting during development (German et al., 2014). Thus, knocking down paxillin may not be a strategy as clear as one would expect, since the coordination of FAs, and thus angiogenesis, may rely on differential or contextual interactions and/or phosphorylation patterns (Birukova et al., 2009). We are currently investigating the effects of JP-153 on paxillin with respect to its critical binding partners and how these interactions trigger differential phosphorylation that promote FA signaling during angiogenesis.

VEGF participates in both pathologic and physiologic growth. Thus, it is not surprising that anti-VEGF therapeutics can potently inhibit vascular growth and retinal function. These deficits were a result of significant structural changes to the retinal layers, despite their prevention of classic neovascular pathology (Tokunaga et al., 2014). These findings raise concerns as to whether enhancing the AV, or preventing revascularization with anti-VEGF treatment, may exacerbate ischemic injury in neuroretinal tissues (Bautch and James, 2009). We used the same dosing regimen of JP-153 in mice reared in atmospheric conditions (room air) and found that even high-dose treatments did not affect normal vasculogenesis, as there were no obvious defects in “normal” vessel growth patterns (Supplemental Fig. 2). Our findings point to an important difference between anti-VEGF therapies and JP-153 with respect to dose effect on vasculogenesis, findings that suggest that JP-153 might help to avoid adverse effects associated with anti-VEGF monotherapy in patients long-term by sparing normal physiologic homeostasis and neuroretinal function.

In conclusion, our results detail an effective strategy to treat pathologic RNV using the small molecule JP-153. Aberrations in FA protein signaling underlie many aggressive hyperproliferative diseases, including cancer metastasis and polycystic kidney disease, making the Src/FAK/paxillin signalsome an attractive therapeutic target (Ischenko et al., 2007; Sweeney et al., 2008; Lee et al., 2015). Recently, small-molecule kinase inhibitors of paxillin binding partners, Src and FAK have advanced to late-stage clinical trials in humans, which suggests FA signal transduction can be effectively and safely modulated in humans (Sulzmaier et al., 2014; Taylor et al., 2015). Paxillin, however, has never been successfully targeted by pharmacologic intervention for the treatment of any proliferative disease, even though its expression has been correlated with highly invasive cancers (Jagadeeswaran et al., 2008). Moreover, the ability of paxillin to function as a scaffold that binds multiple FA proteins makes it an interesting target for development of novel inhibitors of pathologic neovascularization. Since adaptive resistance is a major obstacle plaguing the efficacy of current antiangiogenic treatments (Bergers and Hanahan, 2008), the novelty of this current study can be characterized by two major findings: 1) paxillin is an important and viable target in pathologic retinal angiogenesis; and 2) JP-153 effectively modulates paxillin-dependent signaling in vitro and in vivo to treat RNV. Thus, the target and mechanism of JP-153 has extensive applicability across a wide range of proliferative indications and warrants further pharmaceutical development and refinement as a novel therapeutic.

Acknowledgments

The authors thank Drs. Bilal Aleiwi and Shivaputra Patil for help with the synthetic chemistry of JP-153 and the University of Tennessee College of Pharmacy and the University of Tennessee Research Foundation for financial support.

Authorship Contributions

Participated in research design: Toutounchian, Miller, Yates.

Conducted experiments: Toutounchian, Pagadala.

Contributed new reagents or analytic tools: Yates, Miller.

Performed data analysis: Toutounchian, Park, Chaum, Yates.

Wrote or contributed to the writing of the manuscript: Toutounchian, Park, Baudry, Chaum, Yates.

Footnotes

    • Received May 9, 2016.
    • Accepted October 28, 2016.

  • This work was funded by the University of Tennessee College of Pharmacy (Pharmaceutical Sciences) Research Enhancement Seed Grant (2014) and the University of Tennessee Research Foundation’s Technology Maturation Fund Program (2015). Conflict of interest statement: Jordan J. Toutounchian, Jayaprakash Pagadala, Duane D. Miller, Frank Park and Charles R. Yates are listed on the patent application entitled “Inhibitors of paxillin binding and related compositions and methods” US Patent Application number 61/935,616. JP-153 is a patent-pending technology owned by the University of Tennessee Research Foundation. No competing financial interests exist for authors Jerome Baudry or Edward Chaum.

  • Portions of this work were previously presented at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO) in Denver, CO, June 2015, and published as Toutounchian JJ, Pagadala J, Miller DD, Steinle JJ, and Yates R (2015) The role of a Src/FAK-paxillin signalsome in VEGF-induced retinal neovascularization. Invest Ophthalmol Vis Sci 56:208–208.

  • dx.doi.org/10.1124/mol.116.105031.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

AV
avascular area
DAPI
4′, 6-diamidino-2′-phenylindole
DMSO
dimethyl sulfoxide
ERK
extracellular signal-regulated kinase
FA
focal adhesion
FAC
focal adhesion complex
FAK
focal adhesion kinase
GIT-1
ADP ribosylation factor GTPase-activating protein
LY294002
2-morpholin-4-yl-8-phenylchromen-4-one
MAPK
mitogen-activated protein kinase
NV
neovascularization
OIR
oxygen-induced retinopathy
PARP
poly(ADP ribose) polymerase
PBS
phosphate-buffered saline
PI
propidium iodide
PI3K
phosphatidylinositol-4,5-bisphosphate 3-kinase
REC
retinal endothelial cell
RNV
retinal neovascularization
6-B345TTQ
6-Bromo-3,4-dihydro-4-(3,4,5-trimethoxyphenyl)-benzo[h]quinolin-2(1H)-one
SU6656
(3Z)-N,N-dimethyl-2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylidene)-2,3-dihydro-1H-indole-5-sulfonamide
VEGF
vascular endothelial growth factor

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Targeting Src/FAK/Paxillin Signalsome in Neovascular Disease

Jordan J. Toutounchian, Jayaprakash Pagadala, Duane D. Miller, Jerome Baudry, Frank Park, Edward Chaum and Charles R. Yates
Molecular Pharmacology January 1, 2017, 91 (1) 1-13; DOI: https://doi.org/10.1124/mol.116.105031
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