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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.
1
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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.
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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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Address correspondence to: Dr. Charles R. Yates, The University
of Tennessee,Memphis, Department of Pharmaceutical Sciences, 881
Madison Avenue, Phar-macy Building Room 446, Memphis, TN 38163.
E-mail: [email protected]
Targeting Src/FAK/Paxillin Signalsome in Neovascular Disease
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