Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin...are listed on the patent application entitled “Inhibitors of paxillin binding and related compositions and methods” US

1521-0111/91/1/1–13$25.00 http://dx.doi.org/10.1124/mol.116.105031MOLECULAR PHARMACOLOGY Mol Pharmacol 91:1–13, January 2017Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

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

Jordan J. Toutounchian, Jayaprakash Pagadala, Duane D. Miller, Jerome Baudry,Frank Park, Edward Chaum, and Charles R. YatesDepartment of Pharmaceutical Sciences (J.J.T., J.P., D.D.M., F.P., C.R.Y.) and Department of Ophthalmology (E.C., C.R.Y.),University of Tennessee Health Science Center, Memphis, Tennessee; Department of Biochemistry and Cellular and MolecularBiology at The University of Tennessee, Knoxville, Tennessee; and UT/ORNL Center for Molecular Biophysics, Oak RidgeNational Laboratory, Oak Ridge, Tennessee (J.B.)

Received May 9, 2016; accepted October 28, 2016

ABSTRACTTargeting vascular endothelial growth factor (VEGF) is a commontreatment strategy for neovascular eye disease, a major causeof vision loss in diabetic retinopathy and age-related maculardegeneration. However, the decline in clinical efficacy over timein many patients suggests that monotherapy of anti-VEGFprotein therapeuticsmay benefit from adjunctive treatments. Ourprevious work has shown that through decreased activation ofthe cytoskeletal protein paxillin, growth factor–induced ischemicretinopathy in the murine oxygen-induced retinopathy modelcould be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome isrequired for human retinal endothelial cell migration and pro-liferation. Specifically, the disruption of focal adhesion kinase(FAK) and paxillin interactions using the small molecule JP-153inhibited Src-dependent phosphorylation of paxillin (Y118) and

downstream activation of Akt (S473), resulting in reducedmigration and proliferation of retinal endothelial cells stimu-lated with VEGF. However, this effect did not prevent the initialactivation of either Src or FAK. Furthermore, topical application ofa JP-153-loaded microemulsion affected the hallmark features ofpathologic retinal angiogenesis, reducing neovascular tuftformation and increased avascular area, in a dose-dependentmanner. In conclusion, our results suggest that using smallmolecules to modulate the focal adhesion protein paxillin is aneffective strategy for treating pathologic retinal neovasculari-zation. To our knowledge, this is the first paradigm validatingmodulation of paxillin to inhibit angiogenesis. As such, we haveidentified and developed a novel class of small moleculesaimed at targeting focal adhesion protein interactions that areessential for pathologic neovascularization in the eye.

IntroductionDiabetic 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 ocularangiogenesis and neovascularization (Aiello et al., 1994;Ferris et al., 1984). Vascular endothelial growth factor(VEGF) plays a key role in this pathophysiology and is thetarget of current FDA-approved antiangiogenic protein ther-apeutics (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, whichis believed to result from the emergence of VEGF-independentmechanisms and expression of other growth factors andcytokines involved in maintaining the abnormal angiogenicmilieu (Bergers and Hanahan, 2008; van Beijnum et al.,2015). In addition, the further decline in visual function withlong-term anti-VEGF therapy has been linked to the loss ofthe choroidal blood supply, which is in part VEGF-dependentand which supports the integrity and health of the overlyingretinal pigment epithelium and neural retina (Marneros et al.,

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

Portions of this work were previously presented at the annual meeting of theAssociation for Research in Vision and Ophthalmology (ARVO) in Denver, CO,June 2015, and published as Toutounchian JJ, Pagadala J, Miller DD, SteinleJJ, 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.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: AV, avascular area; DAPI, 49, 6-diamidino-29-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, retinalendothelial 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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2005; Saint-Geniez et al., 2008; Tokunaga et al., 2014). Thus,targeting downstream signaling proteins linked to pathologicneovascularization represents an alternative or adjunctiveapproach to approved anti-VEGF treatments that may reducethe damaging effects of antiangiogenic therapy.VEGF activates endothelial cells, in part, by stimulating

signal transduction pathways that regulate the enzymaticturnover of adhesion complexes, or mechanotransduction“signalsomes” consisting of adaptor proteins and kinases,e.g., Src-family kinases, focal adhesion kinase (FAK), andpaxillin (Waltenberger et al., 1994; Abedi and Zachary, 1997;Provenzano and Keely, 2011). Targeting focal adhesion (FA)kinases downstream of growth factor receptor activationhas recently emerged as an effective strategy for inhibitingretinal angiogenesis (Wary et al., 2012). In ischemic modelsof retinopathy, the local silencing of Src or FAK expressioncauses a significant reduction in pathologic neovasculardisease (Kornberg et al., 2004; Werdich and Penn, 2006).However, evidence of resistance is also accumulating, as re-cently demonstratedwhen cells deficient inFAKprotein showedenhanced expression of its homolog, proline-rich tyrosine kinase2 (PYK2), which is also known to regulate gene expression andendothelial budding or sprouting via VEGF-dependent mecha-nisms (Bergers and Hanahan, 2008; Weis et al., 2008; Shenet al., 2011; Eke and Cordes, 2015). Thus, there is a critical needto identify alternative drug targets that serve as common“interface points” shared by proteins within the focal adhesioncomplex (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 char-acterizing these protein-protein interactions at the structurallevel have identified highly conserved four-helix bundledregions, or so called paxillin-binding subdomains, which spe-cifically 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 tothe cell’s leading edge where actin filaments coalesce aroundintegrins (cellular “anchors”) to provide mechanical forcesneeded to pull the cell forward. Since these complexes helpassemble and support the connections between the actincytoskeleton and the extracellular matrix, targeting theseproteins with small molecules would dismantle the FA com-plexes and obstruct proliferative and migratory signal trans-duction during angiogenesis (Fig. 9).We have identified a proliferative response phenotype of hu-

man primary retinal endothelial cells (REC) exposed to high-doseionizing radiation (Toutounchian et al., 2014). Irradiation-enhanced paxillin Y118 phosphorylation, which was reduced bymitogen-activatedproteinkinase (MAPK) inhibition.Under thesesame mechanisms, inhibiting MAPK and, thus, paxillin phos-phorylation caused a reduction in in vivo retinal angiogenesis.Ourdata suggestedadirect role for activatedpaxillin in radiation-induced retinopathy, an ischemic inflammatory disease with aneovascular component (Boozalis et al., 1987; Finger, 2008).However, the mechanisms by which paxillin coordinates

VEGF-signaling through the FAC is not well understood, asmost focus has been on targeting kinase activity of either Srcor FAK. It was shown, however, that paxillin deletion causeddysfunctional cell spreading and stunted migration, similar tothe 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-moleculemodulation of paxillin within FAs to prevent pathologic angio-genesis in neovascular disease. With this study, we haveexploited paxillin as our molecular target and have identifieda novel class of small-molecule modulators of the FA proteininteractions essential for retinal neovascularization.

Materials and MethodsReagents/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(ADPribose) 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 fromAbcam (Cambridge, MA). Mouse antibodies against human paxillin(clone 349) and FAK (clone 77) were purchased from BD Biosciences(San Jose, CA). Mouse a-tubulin primary antibody and secondaryantibodies IRDye 800CWgoat 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 stainwas purchased from ThermoFisher Scientific (Pierce; Sunnyvale, CA).6-B345TTQ and the Src kinase inhibitor SU6656 were purchasedfrom Sigma-Aldrich (St. Louis, MO). LY294002 (PI3K inhibitor) wasacquired fromCell Signaling Technologies. Primary antibody names,catalog numbers, species of origin, and dilutions are included inSupplemental Table 1.

JP-153 was synthesized in accordance with the methods devised forortho-functionalization of aniline derivatives (Houlden et al., 2010).Briefly, naphthylisocyanate 1 (5.9mmol, 1.0 g) was added to a solutionof t-butylisopropylamine (5.9 mmol, 0.9 ml) in diethyl ether (10 ml)under stirring at room temperature. The colorless solution was stirredfor 3 hours and subsequently cooled to 0°C. Tetramethylethylenedi-amine (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 wasthen 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 themixture was allowed to warm to room temperature and stirred for1 hour. The reaction mixture was then concentrated in vacuo, dilutedwith dichloromethane, and washed with saturated ammonium chlo-ride, NH4Cl (aqueous). The organic layer was evaporated onto silicaand purified by column chromatography. JP-153 purity was charac-terized with high-resolution mass and nuclear magnetic resonancespectroscopy. JP-153 and 6-B345TTQ structures and calculated LogPvalues are presented in Supplemental Fig. 1.

Primary Retinal Endothelial Cell Culture. Primary humanretinal endothelial cells (Lot 181) were purchased from Cell SystemsCorporation (Kirkland, Washington). Cells were grown on attachment-factor surfaces in M131 medium containing microvascular growthsupplements (Invitrogen, Carlsbad, CA) gentamicin (10 mg/ml) andamphotericin B (0.25mg/ml). Only primary cells up to passage six wereused. For immunoassays, RECs were plated into six-well plates,cultured for 2 days, and serum-deprived using 0.1% bovine serumalbumin (Sigma-Aldrich) overnight prior to experiments. RECs werepretreated with inhibitors, SU6656 (1 mM), LY294002 (10 mM), orJP-153 (1 mM), for 1 hour prior to VEGF (100 ng/ml) stimulation,unless mentioned otherwise. All chemical compounds were solubi-lized in dimethyl sulfoxide (DMSO) and further diluted into serum-free cell culture medium, reaching a final vehicle concentrationof ,0.01% (v/v) DMSO.

REC Proliferation Assays. To evaluate paxillin-dependentmod-ulation of retinal endothelial cell proliferation, 50,000 cells wereseeded into each well of a 96-well dish and allowed to adhere

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overnight. RECs were serum-deprived for 1 hour in 0.1% bovineserum albumin, stimulated with VEGF (100 ng/ml), treated withvehicle, kinase inhibitors, or test compounds and incubated for24 hours. Cellular proliferation was determined using the tetra-zolium salt WST-1 according to the assay manufacturer’s instruc-tions (Quick Cell Proliferation Assay Kit II; Abcam, Cambridge,MA). Optical density as a measure of cellular proliferation wasmeasured using a microplate reader at an absorbance of 450 nm.Data represent mean optical density (OD) 6 S.D., n 5 8 per group.In parallel to the 24-hour viability experiments, RECs were incu-bated with calcein-AM for 30 minutes and imaged using the EVOSFL Cell Imaging System (ThermoFisher Scientific) to observe cellnumbers.

Annexin V/Fluorescein Isothiocyanate Staining and FlowCytometry Analysis for Apoptosis. REC apoptosis was measuredby detection of phosphatidylserine translocation to the externalsurface of the cell membrane (Fadok et al., 1992). Annexin V/propidium iodide (PI) staining was performed according to manu-facturer’s instructions (BioLegend, San Diego, CA). Briefly, RECstreated with either JP-153 or vehicle for 24 hours were trypsinizedand washed twice with ice-cold phosphate-buffered saline (PBS)containing two-percent fetal bovine serum. Pelleted RECs wereresuspended in Annexin V Binding Buffer at 5.0 � 106 cells/ml andincubated with fluorescein isothiocyanate–annexin V and PI stain-ing solution (BioLegend) at room temperature for 15 minutes in thedark. Cells were then resuspended in binding buffer and analyzed byfluorescence flow cytometry using the BD LSRII Flow CytometryAnalyzer (BD Biosciences). Data were statistically assessed usingFlowJo 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 ana-lyzed by Western blotting after SDS-PAGE using human specificprimary antibodies, as previously described (Toutounchian et al.,2014).Whole REC lysates were collected in radioimmunoprecipitationassay 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 buffercontaining 2.5% 2-mercaptoethanol (Sigma-Aldrich), heated to 70°C for10 minutes, and loaded into NuPAGE 4–12% Bis-Tris Gels (Invitrogen/ThermoFisher Scientific). Immunoblotting was performed with nitrocel-lulosemembranes (Bio-Rad,Hercules, CA), blocked usingOdysseyBlockingBuffer (LI-COR), and then incubated with specific primary antibodiesovernight at 4°C. Analysis of phosphorylation is presented as a ratioof phosphorylated protein to total protein (e.g., P-Y397 FAK/total FAK);cellular lysates analyzed for both phosphorylated and nonphosphorylatedprotein were normalized to total cellular/housekeeping proteins, i.e.,GAPDHora-tubulin. Secondaryantibodies (IRDye800CWgoat anti-rabbitand IRDye 680LT goat anti-mouse; 1:12,500; LI-COR) were incubated inthe dark at room temperature for 45 minutes. Dual-channel infrared scanand quantitation of immunoblots were conducted using the Odyssey Sainfrared imaging system with Image Studio (Ver. 3.1.4; LI-COR).

In Vitro Scratch-Wound Assay. REC migration was performedin accordance with methods previously described (Ghosh et al., 2013).RECs (106 cells/well) were seeded to 12-well plates and cultured toconfluence. RECs were washed twice with 1� PBS and prewarmedserum-freeMedium 131 (Invitrogen) was introduced to wells for 1 hour toremove any residual effects of supplemented growth factors. Using asterile 200-ml pipette tip, a straight scratch down the center of the wellprovided the baseline for the analysis andquantification ofRECmigrationand proliferation over 24 hours. Wells were then washed one time withPBS to remove any detached cells. Growth factor–supplemented mediumwith or without JP-153 (0.10–10 mM) was added to each well, and plates

Fig. 1. VEGF-induced FA signaling in RECs. (A) Retinalendothelial cells were stimulated with VEGF (100 ng/ml)and cellular lysates were collected over four hours andfocal adhesion protein activation was measured usingWestern blotting as described in Materials and Methods.Initially, VEGFR-2 is activated at Y1175 upon VEGFligation which triggers FAK Y397 autophosphorylation(representativeWestern blots on the left, analysis of FAKpY397 levels on the right) (*P , 0.05, ***P , 0.001).Subsequently, Src-kinase binds to FAK and furtheractivates the kinase-domain loop FAK Y576/577 andthe focal adhesion targeting domain FAK Y925. (B) Src-dependent activation of FAK coincides with paxillin Y118phosphorylation over 4 hours (**P, 0.01, ***P, 0.001).Data represent mean 6 S.D., n = 4–8.

Targeting Src/FAK/Paxillin Signalsome in Neovascular Disease 3

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were immediately imaged using a CoolSNAP charge-coupled devicecamera (Roper Technologies, Inc., Sarasota, FL) mounted on an EclipseTE300 Inverted Microscope (Nikon, Melville, NY). Using 4� magnifica-tion anda computer-controlled stage, images at three specific coordinatesper well at the time of the initial wounding were obtained inMetamorphsoftware (Universal Imaging,West Chester, PA). Plateswere returned toincubator for 24 hours. The next day, previous coordinates were recalledand images were again collected in Metamorph and then transferred toAdobe Photoshop (CS5 Extended, Ver. 12.1; Adobe Systems, Inc., SanDiego, CA). Using the magnetic lasso tool in Photoshop, the outline ofprotruding/migrating cells from the periphery of the scratch toward thecenter was measured. The area devoid of migrating cells was recordedand quantified as a percentage change from the previous day’s areaquantification:

% Area 5�12

A24 hoursA0 hours

�(1)

Data represent mean percent wound closure 6 S.D. RECs from eachgroupwere fixed at 24 hours, stainedwithDAPI, and imaged using the

EVOS FL Cell Imaging System (ThermoFisher Scientific). Arepresentative image from each group was used to depict extent ofwound closure.

Transwell Cellular Migration Assays. Cell migration wasperformed 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 and8.0-mm pore size (Corning) were placed in a 24-well tissue cultureplate (Corning). The upper surface of the porousmembranewas coatedwith attachment factor at 37°C for 1 hour. Human RECs were serum-starved overnight in medium 131 containing 0.1% bovine serumalbumin, trypsinized, pelleted, and resuspended in medium 131 withvehicle (0.1% DMSO) or JP-153 at respective concentrations. RECswere then seeded into the upper chamber at 1� 105 cells/well.Medium131 containing either vehicle or VEGF (100 ng/ml) 1/2 JP-153 wasadded to the lower chamber. After 24 hours of incubation at 37°C,nonmigrated cells were removed from the upper side of the membranewith cotton swabs and the cells on the lower surface of the membranewere fixed in 4% paraformaldehyde for 15 minutes and washed twicewith 1� PBS. Nuclei were then stained with DAPI in PBS for five

Fig. 2. Src-dependent activation of FAKandpaxillin inRECs. (A)Src-inhibitionwithSU6656 (1mM) inhibited VEGF’s activationof FAK Y576/577, Y861, and Y925 andpaxillin Y118 (* P , 0.05,††P , 0.01) butdid not prevent autophosphorylation of FAKY397 (P . 0.05). Data (n = 3) representmean 6 S.D. (B) VEGF-mediated prolifera-tion of RECs was performed as described inMaterials and Methods. VEGF-induced pro-liferation in RECs was reduced in the pres-ence of SU6656 (1 mM), which correlatedwith FA activation in panel A (***,†††P ,0.001). Data represent mean 6 S.D., n = 8.


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minutes and images were collected using the EVOS FL Cell ImagingSystem (ThermoFisher Scientific). Images were imported into AdobePhotoshop (Adobe Systems, Inc.) and cells were counted using batchimage processing with automation. Briefly, the batches of imagesfrom all experimental groups were processed using color correction toenhance DAPI signal against background. Nuclei were outlinedusing the color-selection tool. The automation protocol was estab-lished on the basis of the first image processed in Photoshop to ensurethat the processing of each subsequent image was done without anybiasing or manipulation of quality and/or integrity. Migrating RECs

were quantified from six random fields (n5 3). Data represent meannumber of migrating cells/field 6 S.D.

Retinal Angiogenesis: Murine Oxygen-Induced RetinopathyModel. C57BL/6N (Charles River Laboratories, Wilmington, MA)mice were used in all experiments. All animal studies were performedunder the guidelines of the Association for Research in Vision andOphthalmology for the humane use of animals in vision research,and under the guidance and approval of the Institutional AnimalCare and Use Committee at the University of Tennessee HealthScience Center.

Fig. 3. Discovery of JP-153 as a potent inhibitor of VEGF-induced proliferation. (A) REC proliferation was used to investigate compound 6-B345TTQ, aknown paxillin disruptor, which was found to inhibit REC proliferation at concentrations greater than 10 mM (†P, 0.05, †††P, 0.001). Owing to potencyissues, we redesigned a derivative, JP-153, that inhibits REC proliferation substantially in concentrations as low as 0.25 mM (†††P , 0.001). Datarepresentmean6 S.D., n = 3. (B)We observed cell numbers using calcein-AM as described inMaterials andMethods. (C)We investigated apoptosis usingcleaved-PARP signaling inWestern blots and showed that JP-153 (1 mM) did not significantly enhance apoptotic signaling (panel a, P = 0.239 versus 10%fetal bovine serum controls; data are presented as the mean6 S.D.; n = 3). Flow cytometry quantified apoptotic cells within the population treated withJP-153 (1 mM, 24 hours to confirm that cell death was not induced with treatment, compared with controls; panel b, n = 50,000 cells).


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Retinal angiogenesis was induced using a mouse model ofoxygen-induced retinopathy (OIR), as previously described (Smithet al., 1994; Toutounchian et al., 2014). Five independent litters onthree separate occasions were used for OIR experiments. Mousepups exposed to the oxygen chamber were shuffled into threegroups prior to dosing (P12) to provide intralitter controls. Exper-imental groups were as follows: 1) mice reared in normal atmo-spheric conditions (negative-control; normoxia); 2) mice exposed toOIR/hyperoxic chamber and treated with vehicle microemulsion(1 ml/g; positive-control); 3) OIR-mice treated with JP-153-loadedmicroemulsion at 0.5 mg/kg; and 4) JP-153 at 5.0 mg/kg. Mousepups were exposed to 75% oxygen at postnatal day seven (P7) for5 days and then returned to normal oxygen (P12). Ocular micro-emulsion 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, aspreviously described (Toutounchian et al., 2014). JP-153 was firstloaded into the oil-phase and then incorporated into the finalmicroemulsion formulation and stored at room temperature awayfrom light until dosing. OIR mice were weighed prior to receivingeach daily dose to both eyes using either JP-153 or vehicle-loadedmicroemulsion from P12 to P17 (vehicle control, N 5 8; JP-1530.5 mg/kg,N5 14; JP-153 5.0 mg/kg,N5 14). On P17, retinas wereremoved, dissected, mounted, and stained for endothelial cells toinvestigate retinal angiogenesis. At the conclusion of the study,anesthetized animals were humanely euthanized according IACUCguidelines.

Retinal Whole-Mount Imaging and Analysis. Enucleatedwhole eyes from P17 mouse pups underwent immediate weakfixation in 4% paraformaldehyde in PBS for 1 hour and washedthree times in ice-cold PBS. Retinas were carefully isolated under aLeica S6E dissecting stereomicroscope (Leica Microsystems, Buf-falo Grove, IL) and mounted onto microscope slides. Whole retinaswere incubated overnight at 4°C with isolectin B4-594 (Alexa Fluor594; Molecular Probes, Eugene, OR), as previously described (Connoret al., 2009; Toutounchian et al., 2014). Isolectin-stained retinas werethen washed three times in 1� PBS, sealed under coverslips usingVectashield mounting medium (Vector Laboratories, Inc.), and storedat 4°C until imaging.

Images were acquired using a Zeiss LSM 710 system attachedto a Zeiss Axio Observer inverted microscope with Zen 2010 v.6.0software (Carl Zeiss Microscopy, Peabody, MA). Multidimensionalacquisition was carried out using Z-stacks with ,4-mm slicingintervals and tile-scan automation with an 8% tile overlap at aresolution of at least 512� 512 pixels per tile and digitally stitchedtogether. Quantification of avascular area (AV) and neovasculariza-tion (NV) in retinal whole mounts was performed in Adobe Photo-shop (Adobe Systems, Inc.), as previously described (Toutounchianet al., 2014). The area devoid of vascularization around the optic discwas characterized as percentage of total retinal area (%AV). Photo-shop color-range analysis tool were used to outline NV formationsafter 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 theexact quantified outlined areas and layered back into place onto theoriginal whole-retina image. Using the linear light-blending methodin Photoshop, both avascular and neovascular areas were trans-posed in white.

Statistical Analyses. All data represented herein were per-formed in replicates of three or more and presented as the mean 6S.D., unless otherwise indicated. Differences among groups wereanalyzed using one-way analysis of variance. When overall analysisrevealed significance among groups, means were compared andtested using Tukey’s posthoc analysis. Statistical significance wasset at P, 0.05. All statistical analyses were performed in SigmaPlot12.0 software (Systat Software, Inc., San Jose, CA). P valuesrepresenting significances of ,0.05, 0.01, and 0.001 are denoted

with symbols *, **, ***, whereas significances ,0.05, 0.01, 0.001among treatment arms are represented with †, ††, †††, respectively.

ResultsSrc/FAK-Paxillin Signaling Pathway in REC

Proliferation. FAK and paxillin are coordinators of FA turn-over during VEGF-induced proliferation and migration—twoseminal events of angiogenesis (Brown et al., 2005). Toconfirm the relevance of these two players in VEGF-inducedproliferation of RECs, we stimulated RECs with VEGF andanalyzed cell lysates for FAK and paxillin phosphorylationover time. Fig. 1A shows that rhVEGF (100 ng/ml) activatesVEGF receptor-2 (VEGFR-2) with maximal phosphoryla-tion occurring within 15minutes at amajor phosphorylationsite, Tyr-1175. Activation of VEGFR-2 triggers autophos-phorylation of FAK Y397 (as seen in Western blot images,with analysis to the right; *P , 0.05, ***P , 0.001), whichpromotes association of Src with FAK (Schaller et al., 1994)and subsequently leads to Src-dependent FAK phosphoryla-tion of its kinase domain loop, Y576/577 and focal adhesiontargeting domain Y925 (Fig. 1A). Src-dependent activationand binding of FAK forms the Src/FAK focal adhesioncomplex (FAC), which phosphorylates paxillin Y118 (Fig.1B, **P , 0.01, ***P , 0.001).To determine if the Src/FAK complex is necessary for

paxillin activation in RECs and thus proliferation, we exam-ined FAK and paxillin phosphorylation in VEGF-stimulatedRECs treated with Src-kinase inhibitor SU6656 (1 mM) (Blakeet al., 2000). In Fig. 2A, we show that inhibiting Src kinasereduces the phosphorylation of FAK Y576/577, Y925, andY861 (††P , 0.01) but does not affect autophosphorylation ofY397. An inactive Src/FAK complex fails to phosphorylatepaxillin Y118 (Fig. 2A, ††P , 0.01). We again treated RECswith SU6656 (1 mM) for 24 hours and showed that inhibitionof Src-mediated phosphorylation of FA proteins leads to a

Fig. 4. JP-153 inhibits VEGF-induced activation of paxillin Y118. (A)REC lysates were collected at 4 hours post-VEGF activation, andphosphorylation of paxillin Y118 was measured using Western blotting.(B) JP-153 significantly reduced phosphorylation in cells stimulated withVEGF (**,††P , 0.01) but did not affect constitutive/unstimulated levels(P = 0.749 versus vehicle control). Data represent mean 6 S.D.; n = 3.


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significant decrease in VEGF-induced proliferation (Fig. 2B,††† P , 0.001).Discovery of JP-153 as a Potent Inhibitor of VEGF-

Induced Proliferation. Src-dependent FAK and paxillinphosphorylation correlated with VEGF-induced prolifera-tion in RECs (Fig. 2B). We used this phenotypic responseto derive compounds related to a known paxillin proteindisruptor, 6-B345TTQ (Kummer et al., 2010). Our initiallead identification efforts yielded the analog JP-153, whichwas ∼50 times more potent than 6-B345TTQ in REC pro-liferation 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 morepharmaceutically favorable (Supplemental Fig. 1) (Lipinskiet al., 2001). We used calcein-AM staining (Fig. 3B) to showthat live cell number is reduced with JP-153 treatmentsin addition to reduced proliferative activity, as measuredby WST-1 in Fig. 3A. Yet, JP-153 does not promote apopto-sis in cells, as characterized by PARP cleavage (Fig. 3C,panel a, *P , 0.05 versus serum-free controls) and annexinV/PI staining at 1-mM concentration over 24 hours (Fig. 3C,panel b).

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 15minutes) show FA and effector signaling after one hour pre-treatments with JP-153 (1mM), Src-inhibitor SU6656 (1 mM) or PI3K inhibitor LY294002 (10 mM). JP-153 and SU6656 significantly reduce levels ofVEGF-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 anysignificant change to VEGF-induced pERK1/2 activation (panel c;P. 0.05), while LY294002 caused an increase in activation of ERK (†P, 0.01 vs. VEGFcontrols). 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 datapresentation consistency. B) We confirmed Akt-dependent REC-proliferation by treating cells with LY294002 which resulted in the potent inhibition ofproliferation in a more pronounced manner than JP-153 or SU6656 (***, †††P , 0.001, n = 8). Data represent mean 6 SD.


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Effector Signaling through an Activated Src/FAK-Paxillin Signaling Complex during VEGF-Induced Pro-liferation Is Akt-Dependent. We postulated that JP-153inhibits REC proliferation through disruptions in FA pro-tein interactions, as shown by Kummer et al. (2010) with6-B345TTQ. Disrupting Src/FAK binding to paxillin resultsin decreased activation of paxillin Y118 (Richardson et al.,1997). Thus, we treated RECs with JP-153 (1 mM) for 1 hourand then stimulated them with VEGF for 4 hours. In cellsJP-153 significantly reduces Y118 phosphorylation (Fig. 4,**,††P , 0.01) but did not inhibit constitutive levels ofunstimulated RECs treated with JP-153 (P 5 0.749).Next, we examined downstream FA effector signaling

during early VEGF activation at 15 minutes. We pretreatedRECs with JP-153 for 1 hour prior to VEGF-activation andmeasured phosphorylation of FAK phosphorylation sites,as well as downstream angiogenic markers AKT and ERK.Our results again confirmed that JP-153 reduces activationof paxillin Y118 compared with VEGF controls (Fig. 5A, panel a;*, †P, 0.05) but does not change autophosphorylation of FAKY397; these results mimic the activity of SU6656 (††P, 0.01).However, when we probed for FAK Y576/577, Y861, and Y925in cells, JP-153 did not affect levels of Src-dependent FAKphosphorylation sites (Fig. 5A, panels d–f; P . 0.05), whereasSU6656 inhibited these levels strongly (†P, 0.05, ††P, 0.01).To rule out kinase inhibition, we show that JP-153 was not adirect kinase inhibitor of FA signaling effectors per se, asmeasured by the Z9-LYTE SelectScreen Single Point bio-chemical assay (ThermoFisher Scientific) (SupplementalTable 2).Src-mediated activation of paxillin Y118 primes the com-

plex for recruitment to focal contacts, where interactions withPI3K and MEK activate their respective downstream sub-strates, AKT and ERK, to promote cytoskeletal rearrange-ments during proliferation and migration (Fujikawa et al.,1999; Akagi et al., 2002; Du et al., 2011). Thus, we comparedRECs treated with JP-153 and SU6656 with those treatedwith PI3K inhibitor LY294002 (10 mM). Both p-Akt (Ser473)and p-ERK 1/2 levels rose under VEGF, but only Akt waseffectively blocked by SU6656 and JP-153 (Fig. 5A, panels band c; *,†P , 0.05, ††P , 0.01), since neither show significantinhibition of p-ERK 1/2 at concentrations tested (P . 0.05).However, complete inhibition of Akt phosphorylation byLY294002 caused no reductions in FAK or paxillin activa-tion, suggesting the Src/FAK/paxillin activation cascadeprecedes PI3K-induced Akt phosphorylation. However,unlike JP-153 or SU6656, LY294002 significantly inducedERK activation (†P , 0.05; LY294002 versus VEGF). Tovalidate an Akt-dependent proliferation pathway, cellstreated 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 inhibitREC proliferation through an Akt-dependent but ERK-independent mechanism.PaxillinModulationwith JP-153 Inhibits VEGF-Induced

Migration of Retinal Endothelial Cells. We have shownthat JP-153 inhibited REC proliferation through disruptionsin Src/FAK activation of paxillin Y118 and pAkt (Fig. 5). Sinceangiogenesis requires two distinct but cooperative mecha-nisms, proliferation and migration, we examined JP-153’seffect on migration using the standard scratch wound assay.

VEGF-induced REC migration was significantly inhibited inJP-153 treatments over a range of concentrations (0.10–10mM) (Fig. 6; *,†P , 0.05, ††† P , 0.001). Next, we validatedour scratch-wound results with the Transwell migration/invasion assaywithVEGFas the chemotactic inducer (Yoshidaet al., 1996). Our results show that JP-153 inhibits REC inva-sion at submicromolar concentrations (0.10–0.50 mM) (Fig. 7,***,†††P , 0.001).Signal Disruption of Src/FAK/Paxillin Complex by

JP-153 In Vivo Inhibits Retinal Neovascularization inthe Murine Oxygen-Induced Retinopathy Model. Ourin vitro mechanism of action studies in RECs suggestedthat JP-153 inhibited proliferation and migration by dis-rupting Src/FAK/paxillin signaling pathway. Therefore, wehypothesized that JP-153 could inhibit retinal angiogenesisin vivo by reducing Src/FAK/paxillin activity. We used themurine OIR model of retinal neovascularization (RNV) totest JP-153 at low and high topical doses applied daily toeach eye during the hypoxic period (P17 retinal whole-mounts in Fig. 8A, and subsequent analysis in Fig. 8B). Ourdata shows that JP-153 inhibits neovascularization by40 and 45% in a dose-dependent manner (0.5 and 5 mg/kg,respectively), compared with vehicle-treated eyes (panelsa–c, ***P, 0.001). However, only JP-153 at the higher dose

Fig. 6. JP-153 inhibited VEGF-induced REC migration in the scratch-wound assay. The scratch-wound migration assay was performed in RECsexposed to VEGF for 24 hours, as described inMaterials and Methods. (A)Data analysis show JP-153 inhibits VEGF-induced migration in aconcentration-dependent manner. (B) Representative DAPI-stained im-ages after 24 hours. Data are presented as the mean 6 S.D. (n = 6; *,†P ,0.05, †††P , 0.001).


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enhanced the AV compared with vehicle (panels d ande, ***P, 0.001). Mouse pups kept outside the OIR chamberfor the entire study were also dosed with JP-153 (5 mg/kg)under identical age-based regimens to evaluate any impacton retinal vascular development. There were no obviousdifferences between vehicle and JP-153-treated retinas inmice not exposed to the OIR chamber (Supplemental Fig. 2).These findings suggest that JP-153 can act to regulatepathologic RNV without affecting normal retinal bloodvessel growth or vasculogenesis.

DiscussionIn previous work, paxillin Y118 activation in high-dose radi-

ation injurywas an important signaling component drivingRECproliferation in ischemic retinopathy (Toutounchian et al., 2014).We demonstrated in this study that VEGF-dependent activationof the Src/FAK/paxillin signaling complex, or signalsome, drivesREC migration and proliferation (Fig. 9). Moreover, we showedthat modulation of the Src/FAK/paxillin signaling complex withsmall molecule JP-153 reduced paxillin Y118 activation andinhibited migration and proliferation of RECs; and that thiseffect did not interferewithVEGF-dependent activation of eitherSrc or FAK. Furthermore, topical application of a JP-153-loadedocular microemulsion inhibited hallmark features of pathologicretinal angiogenesis in mice; both neovascular tuft formationand vascular regrowth in themurineOIRmodel were reduced ina dose-dependent manner.

A major finding in this study was that in human primaryRECs, Src/FAK activation of paxillin directs VEGF-inducedsignaling during REC proliferation and migration, a signalingpathway well characterized in cancer cells and other trans-formed cell lines but previously undescribed in primaryhuman RECs (Abedi and Zachary, 1997; Birukova et al.,2009; Yang et al., 2015). We hypothesized that targetingREC Src/FAK or paxillin would limit the activation ofdownstream effector proteins important for retinal angio-genesis. First, we showed VEGF induces activation of Srckinase leading to the phosphorylation of FAK and paxillin,which could be prevented by pharmacological inhibition ofSrc. We then used a small-molecule probe of paxillin bindinginteractions, 6-B345TTQ (Kummer et al., 2010), to investi-gate paxillin’s role during VEGF-induced REC proliferation.Blocking interactions that involve paxillin effectively re-duced REC proliferation in vitro, but owing to inherently lowpotency and solubility, we derived a more effective deriva-tive, JP-153.An unexpected and novel finding during in vitro mech-

anistic studies was that JP-153 reduced phosphorylationof paxillin Y118, a critical tyrosine activation site, but didnot affect FAK phosphorylation, distinguishing JP-153’sactivity from Src inhibitor SU6656. Thus, we have shownthat paxillin Y118 is an important downstream biomarkerfor VEGF-induced REC proliferation. Additionally, JP-153did not inhibit the kinase activities of Src or FAK (Supple-mental Table 2); strongly suggesting that JP-153’s antipro-liferative phenotype in RECs is through paxillin-dependent

Fig. 7. JP-153 inhibited VEGF-induced REC invasionusing the Transwell migration assay. RECs were seededonto porous membranes and chemotactic factor VEGFwas used to stimulate REC migration, as described inMaterials and Methods. (A) Results show that JP-153inhibited REC invasion in a concentration-dependentmanner (data are mean 6 S.D.; ***,†††P , 0.001; n = 6).(B) Cells traversing the membrane were fixed andstained with DAPI, and representative images of eachgroup are shown (image labels A–E: serum-free, VEGF,V + 0.10, V + 0.25, V + 0.50 mM, respectively).


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signaling, independent of kinases that may regulate its phos-phorylation. In fact, mutagenesis of FAK- or paxillin-bindingdomains are known to inhibit their interaction and preventactivation of paxillin and other downstream proteins (Subausteet 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-inhibitorLY294002 remained unchanged, although effective at pre-venting both Akt phosphorylation and REC proliferation,levels of FAK, or paxillin phosphorylation. SU6656 andJP-153 both caused reductions in Akt phosphorylation,suggesting that activation of FAK and paxillin precedesVEGF-induced activation of Akt in RECs. However, sinceJP-153 did not disrupt FAK phosphorylation levels and stillreduced p-Akt, we concluded that paxillin Y118 plays acrucial role in coordinating events that drive Akt-dependentangiogenesis in RECs. These results are in agreement with

other studies that established the important stepwise roleof 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 showthat paxillin is an important signaling intermediary thatconnects the activated Src/FAK complex and Akt inangiogenesis.The uncoupling of an active Src/FAK complex from paxillin

suggested it is a key regulator of pathologic FA signal trans-duction and potentially represents a novel in vivo targetdistinct from anti-VEGF therapies aimed at silencingreceptor-mediated kinase signaling. Studies using tar-geted deletions of FA proteins FAK and Src in the mouseretina disrupt the progression of RNV (Kornberg et al.,2004; Werdich and Penn, 2006); these findings correspondwith our in vitro results using the Src inhibitor SU6656,which affects all downstream binding and activationpartners. We show similar in vitro effects with JP-153 on

Fig. 8. JP-153 inhibited retinal angiogenesisin the murine oxygen-induced retinopathymodel. P17 retinal whole-mounts were stainedfor endothelial cells using isolectin B4-594 asdescribed in Materials and Methods. Micewere dosed daily from P12-17 using eithertopicalmicroemulsion-loaded vehicle, 0.5mg/kg,or 5.0 mg/kg JP-153. (A) Representative im-ages of retinal whole-mounts depicting: neo-vascular area (a–c) and AV (d–f). (B) Dataanalysis of retinal vasculature revealed thatJP-153 inhibited NV and increased AV in adose-dependent manner. Data representmean 6 S.D.; ***P, 0.001; N = 8–14/group.


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proliferation as with SU6656, specifically with decreasedpaxillin Y118 phosphorylation and inhibition of p-Aktdownstream, resulting in potent inhibition of movementand growth. From these studies, we can assert that theactivation of the FAC may be a crucial component in theregulation of pathologic retinal angiogenesis, in vivo. Wetested this hypothesis by administering JP-153 topically inthe OIR model, which resulted in significantly reducedretinal angiogenesis, as measured by both neovasculariza-tion and the AV. Intriguingly, we found that only the higherdoses of JP-153 were able to significantly enhance AV,suggesting perhaps that our small molecule affects patho-logic neovascularization more than vasculogenesis. How-ever, since genetic knockdown of paxillin in mice leads toearly embryonic lethality (Hagel et al., 2002), paxillin hasbeen conditionally silenced in the developing mouse retina.These studies actually showed that paxillin knockdowninduced migration and endothelial cell sprouting duringdevelopment (German et al., 2014). Thus, knocking downpaxillin may not be a strategy as clear as one would expect,since the coordination of FAs, and thus angiogenesis, mayrely on differential or contextual interactions and/or phos-phorylation patterns (Birukova et al., 2009). We are cur-rently investigating the effects of JP-153 on paxillin withrespect to its critical binding partners and how theseinteractions trigger differential phosphorylation that pro-mote 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 tothe retinal layers, despite their prevention of classic neo-vascular pathology (Tokunaga et al., 2014). These findingsraise concerns as to whether enhancing the AV, or preventingrevascularization with anti-VEGF treatment, may exacerbateischemic injury in neuroretinal tissues (Bautch and James,2009). We used the same dosing regimen of JP-153 in micereared in atmospheric conditions (room air) and found thateven high-dose treatments did not affect normal vasculo-genesis, as there were no obvious defects in “normal” vesselgrowth patterns (Supplemental Fig. 2). Our findings point toan important difference between anti-VEGF therapies andJP-153 with respect to dose effect on vasculogenesis, findingsthat suggest that JP-153 might help to avoid adverse effectsassociated with anti-VEGF monotherapy in patients long-term by sparing normal physiologic homeostasis and neuro-retinal 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 aggres-sive hyperproliferative diseases, including cancer metastasisand polycystic kidney disease, making the Src/FAK/paxillinsignalsome 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 inhumans, which suggests FA signal transduction can beeffectively and safely modulated in humans (Sulzmaieret al., 2014; Taylor et al., 2015). Paxillin, however, hasnever been successfully targeted by pharmacologic inter-vention for the treatment of any proliferative disease, eventhough its expression has been correlated with highlyinvasive cancers (Jagadeeswaran et al., 2008). Moreover,the ability of paxillin to function as a scaffold that bindsmultiple FA proteins makes it an interesting target fordevelopment of novel inhibitors of pathologic neovasculari-zation. Since adaptive resistance is a major obstacle plagu-ing the efficacy of current antiangiogenic treatments (Bergersand Hanahan, 2008), the novelty of this current study canbe characterized by two major findings: 1) paxillin is an im-portant and viable target in pathologic retinal angiogenesis;and 2) JP-153 effectively modulates paxillin-dependent sig-naling in vitro and in vivo to treat RNV. Thus, the targetand mechanism of JP-153 has extensive applicability across awide range of proliferative indications and warrants furtherpharmaceutical development and refinement as a noveltherapeutic.

Acknowledgments

The authors thank Drs. Bilal Aleiwi and Shivaputra Patil for helpwith the synthetic chemistry of JP-153 and the University ofTennessee College of Pharmacy and the University of TennesseeResearch 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.

Fig. 9. Summary diagram of JP-153’s proposed target of action. Datasuggests that JP-153 targets the interaction between an active Src/FAKsignaling complex and paxillin. Inhibiting this interaction resulted indecreased paxillin activation (Y118), preventing activation of down-stream effector protein Akt. This effect translated into potent inhibitionof REC proliferation and migration, in vitro, and inhibition of RNV,in vivo.


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Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin...are listed on the patent application entitled “Inhibitors of paxillin binding and related compositions and methods” US