UPARANT: A Urokinase Receptor Derived Peptide Inhibitor of ... · UPARANT: A Urokinase Receptor–Derived Peptide Inhibitor of VEGF-Driven Angiogenesis with Enhanced Stability and

Small Molecule Therapeutics

UPARANT: A Urokinase Receptor–Derived Peptide Inhibitorof VEGF-Driven Angiogenesis with Enhanced Stability andIn Vitro and In Vivo Potency

Maria Vincenza Carriero1, Katia Bifulco1, Michele Minopoli1, Liliana Lista2, Ornella Maglio2,3,Luigi Mele1, Gioconda Di Carluccio1, Mario De Rosa4, and Vincenzo Pavone2

AbstractThis work is based on previous evidence showing that chemotactic sequence of the urokinase receptor

(uPAR88-92) drives angiogenesis in vitro and in vivo in a protease-independentmanner, and that the peptideAc-

Arg-Glu-Arg-Phe-NH2 (RERF) prevents both uPAR88–92- and VEGF-induced angiogenesis. NewN-acetylated

and C-amidated peptide analogues containing a-methyl a-amino acids were designed and synthesized tooptimize the biochemical properties for therapeutic applications.Among these,Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2, namedUPARANT, adopts in solution a turned conformation similar to that found for RERF, is stable

to sterilization in 3 mg/mL sealed vials in autoclave for 20 minutes at 120�C, is stable in blood, and displays along-time resistance to enzymatic proteolysis. UPARANT competes with N-formyl-Met-Leu-Phe (fMLF) for

binding to the formyl-peptide receptor, inhibits VEGF-directed endothelial cell migration, and prevents

cytoskeletal organization and avb3 activation in endothelial cells exposed to VEGF. In vitro, UPARANTinhibits VEGF-dependent tube formation of endothelial cells at a 100� lower concentration than RERF. In vivo,UPARANT reduces to the basal level VEGF-dependent capillary sprouts originating from the host vessels that

invaded Matrigel sponges implanted in mice, and completely prevents neovascularization induced by

subcorneal implantation of pellets containing VEGF in rabbits. Both excellent stability and potency position

UPARANT as a promising new therapeutic agent for the control of diseases fueled by excessive angiogenesis,

such as cancer and inflammation. Mol Cancer Ther; 13(5); 1092–104. �2014 AACR.

IntroductionAngiogenesis is a complex multistep process leading to

the formation of new blood vessels from a preexistingvascular network. During new vessel formation, endo-thelial cells degrade their basement membrane, migrateinto the interstitial matrix, and proliferate. An imbalancein this process contributes to numerous malignant,inflammatory, ischemic, infectious, and immune disor-ders (1).

The receptor for urokinase-type plasminogen activa-tor (uPAR) plays an important role in controlling cellmigration (2, 3). uPAR is a glycosylated glycosyl-phos-phatidyl-inositol–anchored protein (4) formed by 3domains (DI, DII, and DIII) connected by short linkerregions (5). Besides being responsible for focalizinguPA-mediated plasminogen activation on cell surface(6), uPAR also promotes intracellular signaling, thusregulating physiologic processes such as wound heal-ing, immune responses, and stem cell mobilization, aswell as pathologic conditions such as inflammation andtumor progression (7–10). The role of uPAR in angio-genesis is well documented. uPAR is able to focusurokinase proteolytic activity on cell surface and tomodulate cell migration (11, 12). We found that solubleforms of uPAR, containing the 88Ser-Arg-Ser-Arg-Tyr92

sequence (uPAR88-92), as well as the synthetic peptideSer-Arg-Ser-Arg-Tyr (SRSRY), stimulates in vitro and invivo angiogenesis in a protease-independent manner(13). The uPAR88-92 sequence interacts with the formylpeptide receptors (FPR) type 1 and 2, henceforth induc-ing cell migration in an integrin-dependent manner(9, 14, 15). Upon binding to FPR, the synthetic peptideSRSRY causes FPR internalization and triggers vitro-nectin receptor activation with an inside–outside type ofmechanism (16).

Authors' Affiliations: 1Department of Experimental Oncology, IstitutoNazionale Tumori "FondazioneG.Pascale"-IRCCS; 2Department ofChem-ical Sciences, "Federico II" University of Naples; 3IBB-National ResearchCouncil Naples; and 4Department of Experimental Medicine, SecondUniversity of Naples, Naples, Italy

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Vincenzo Pavone, Department of ChemicalSciences, "Federico II" University of Naples, Naples, Italy. Phone: 39-081674399; Fax: 39-081674090; E-mail: [email protected]; andMaria Vincenza Carriero, Department of Experimental Oncology, IstitutoNazionale Tumori "Fondazione G. Pascale"-IRCCS, via M. Semmola,80131, Naples, Italy. Phone: 39-0815903569; Fax: 39-0815903814. E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0949

�2014 American Association for Cancer Research.

MolecularCancer

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Mol Cancer Ther; 13(5) May 20141092

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Published OnlineFirst April 4, 2014; DOI: 10.1158/1535-7163.MCT-13-0949

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Recently, we found that the residue Ser90 of uPAR iscritical for uPAR signaling, and that S90P and S90Esingle amino acid substitutions exert opposite effectson uPAR activities. Cells expressing membrane-associ-ated uPAR carrying Ser90 substituted with Glu exhibit areduced binding to and a decreased adhesion ontovitronectin, an impaired agonist-induced FPR internal-ization, and a dramatic reduction of in vitro and in vivocell migration and invasion (17). To inhibit uPAR func-tions, we developed, a family of penta-peptides carry-ing the S90E substitution in the uPAR88-92 sequence.These peptides block uPAR-dependent cell signaling byinterfering with the complex cross-talk involving uPAR,FPR, and integrins. The peptide containing the N-ter-minal pyro-glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH2) shares the same binding site withSRSRY and competes with fMLF for binding to FPR,thus preventing uPAR/FPR interaction. pERERY-NH2inhibits migration of various tumor cell lines in culture(18). Subsequently, new tetra-peptides having the gen-eral formula Ac-Arg-Glu-Arg-X-NH2 (X ¼ Phe, Tyr,Trp) were synthesized, and the peptide Ac-Arg-Glu-Arg-Phe-NH2 (denoted RERF) was selected for its abil-ity to potently prevent in vitro and in vivo cell migrationand invasion (19). Recently, we found that RERF alsobehaves as an antiangiogenic agent. It inhibits in vitroand in vivo responses promoted either by uPAR88-92 orby VEGF. RERF also prevents cytoskeletal organizationand the recruitment of avb3 integrin at the focal adhe-sions in endothelial cells exposed to VEGF, by forcingavb3 in an inactive state either directly or indirectly,through FPR (20).Starting from these pieces of biologic information, we

intended to generate peptide analogues with optimizedproperties for therapeutic applications. We synthesizednew peptides containing a-methyl-a-amino acids, andthey were characterized from a biologic point of view.All these new analogues are N-acetylated and C-ami-dated, as being a common way of the N- and C-terminalmodifications to enhance the stability to exopeptidase-mediated proteolysis (21). Among the new series of pep-tides, Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2, showedthe best desired activity in preliminary experiments ofVEGF-directed endothelial cell migration.It adopts in solution a turned conformation, it is quite

stable to trypsin and chymotrypsin digestion, inmice andrats blood. It is also stable to sterilization in 3 mg/mLsealed vials in autoclave for 20 minutes at 120�C, andremained stable at 25�C for 2 years.Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2, named UPAR-

ANT, competeswith fMLF for binding to FPR, and inhibitsFPR internalization in endothelial cells. It prevents F-actinpolymerization and adhesion onto vitronectin in endothe-lial cells exposed to VEGF. In vitro, UPARANT inhibitsVEGF-dependent tube formation of endothelial cells at a�100 lower concentration than RERF. In vivo, it preventsVEGF-dependent capillary sprouts originating from thehost vessels that invaded Matrigel sponges implanted in

mice and neovascularization induced by subcornealimplantation of pellets containing VEGF in rabbits.

Materials and MethodsPeptide synthesis and purification

Peptides were synthesized by the solid-phaseapproach using standard Fmoc methodology in a man-ual reaction vessel, and purified by RP-HPLC-C18 col-umn to a 99% purity. Fluoresceinated UPARANT (FITC-UPARANT) was synthesized by the on-resin procedure,using e-aminocaproic acid as spacer (19). It retains the93% of the inhibitory activity on cell migration (datanot shown). Molecular weights were confirmed by massspectrometry.

Stability during sterilization in buffer and shelf lifeA total of 90 sealed vials containing 0.5 mL of UPAR-

ANT in PBS at a concentration of 3 mg/mL were auto-claved for 20 minutes at 120�C. One sample was imme-diately analyzed by liquid chromatography/mass spec-trometry (LC/MS) to ascertain the lack of any modifiedcompound. The remaining samples were kept in a ther-mostat at 25�C. Three vials were opened everymonth andanalyzed by LC/MS.

Blood stabilityBlood stability studieswere performed at IRBMScience

Park. Technical details are reported in the SupplementaryMaterials and Methods.

Enzymatic digestion of peptidesThe peptides were subjected to digestion by trypsin at a

peptide:trypsin ratio of 1,000:1 (w:w) for RERF and Icm25,whereas a 10:1 (w:w) peptide:trypsin ratio was used forIcm25B and UPARANT. The trypsin concentration is 0.6mg/L. The peptides were also subjected to digestion bychymotrypsin at a peptide:chymotrypsin ratio of 10:1(w:w). Icm25-4 was not studied for enzyme digestion.Digestions were followed for 1 hour at 25�C in 0.1 M Tris-HCl buffer pH 8.5. The peptide consumption was fol-lowed by RP-HPLCmeasuring the area peaks at differenttimes between 0 and 60 minutes. Quantification of theintact peptide peak over time indicated that its disappear-ance seemed to follow first-order kinetics (data notshown), and the t1/2 was estimated.

Nuclear magnetic resonance spectroscopy andstructure calculations

Nuclear magnetic resonance (NMR) experiments wereperformed on Bruker Avance 600 MHz spectrometer,equipped with triple resonance cryo-probe. NMR char-acterization was performed in H2O/D2O 90:10 (v/v) andin H2O/CF3CD2OD 70:30 (v/v) at 298 K. Details arereported in the Supplementary Materials and Methods.Restrained molecular dynamic (RMD) simulations werecarried out using a hand-built starting model with struc-tural data from NMR measurements [e.g., interprotondistances from Nuclear Overhauser Effect (NOE) value].

Antiangiogenic Activity of UPARANT

www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1093




Details on structure calculations are reported in the Sup-plementary Materials and Methods.

Cell culturesHuman umbilical vein endothelial cells (HUVEC) were

purchased by Lonza (C2519A, Lot No. 0000115425),which provided a certificate of analysis for each cell lot.This guarantees the expression of CD31/105 and vonWillebrand Factor through 15 population doublings.HUVEC, used between the third to the seventh passageaccording to Arnaoutova and colleagues, (22), weregrown in Eagle basalmedium (EBM) supplementedwith4% FBS, 0.1% gentamicin, 1 mg/mL hydrocortisone, 10mg/mL EGF, and 12 mg/mL bovine brain extract(Cambrex).

Migration assayCell migration of HUVEC was performed in Boyden

chambers, using 8-mm-pore size polyvinylpyrrolidone-free-polycarbonate filters at 37�C, 5% CO2 as previouslydescribed (13). Briefly, 7 � 104 viable cells suspended inserum-free EBM were allowed to migrate for 4 hourstoward EBM or 40 ng/mL VEGF165 (Pepro Tech), mixedwithdiluents or the indicatedpeptides at 37�C ina5%CO2.At the end of assays, cells on the lower filter surface werefixed, stained with hematoxylin, and 10 random fields/filter were counted at �200 magnification.

Binding assaysHUVECs (5 � 105 cells/sample) were preincubated

with diluents or the indicated effectors for 60 minutes at4�C, extensively rinsed with PBS and then exposed to 10nmol/L N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein(FITC-fMLF) purchased from Molecular Probes for 60minutes at 4�C. Quantification of cell-associated fluores-cence was assessed by reading endothelial cell lysateswith a fluorescence plate reader Victor 3 (Perkin Elmer)using 485 nm excitation and 535 nm emission filters. Analiquot of each sample was separated on 10% SDS-PAGEand subjected to Western blot analysis for the a-tubulincontent.

Fluorescence microscopyHUVEC grown on glass slides to semiconfluence were

incubated with the indicated effectors for 30 minutes at37�C and then exposed to 10 nmol/L FITC-fMLF or 10nmol/L FITC-UPARANT diluted in serum-free EBM for30 minutes at 37�C as described (19). To analyze cytoskel-etal organization and avb3 localization, cells grown onglass coverslips to semiconfluence, were starved for 60minutes in EBM. Then, cells were exposed to EBM alone,40 ng/mL VEGF165, or 40 ng/mL VEGF165 mixed to 10nmol/L indicated peptides for 30 minutes at 37�C inhumidified air with 5% CO2. Slides were washed withPBS, fixed and permeabilized with 2.5% formaldehyde–0.1% Triton X-100 in PBS for 10 minutes at 4�C. Afterseveral washes in PBS, slides were incubated with2 mg/mL anti-vinculin mAb (clone VIIF9 purchased from

Chemicon) and then with 1:800 goat Alexa Fluor 488antimouse immunoglobulin G (IgG; Molecular Probes) at23�C for 60 and 45minutes, respectively. Thereafter, 2mg/mL LM609 anti-avb3 monoclonal antibodies (mAb; Che-micon) and then 1:800 diluted rabbit Alexa Fluor 594antimouse IgG (Molecular Probes) were applied to slidesat 23�C for 60 and 45 minutes, respectively. To analyzecytoskeletal organization, coverslips were incubatedwith0.1mg/mL rhodamine-conjugated phalloidin (Invitrogen)at 23�C for 45 minutes. In all cases, slides were mountedusing 20% (w/v) mowiol, cells visualized with a 510META-LSM confocalmicroscopy (Carl Zeiss) and z-serieswith 0.38-mm intervals were collected.

Western blottingHUVEC grown to semiconfluence were starved for 18

hours in 0.2% FBS EBM. Then, cells were exposed toEBM alone, 40 ng/mL VEGF165 with/without 10 nmol/L UPARANT at 37�C in humidified air with 5% CO2. Atthe indicated times, cells were lysed in radioimmuno-precipitation assay buffer (10 mmol/L Tris pH 7.5, 140mmol/L NaCl, 0.1% SDS, 1% Triton X-100, 0.5% NP40)containing 5 mmol/L Na3VO4 and a protease inhibitormixture (Sigma-Aldrich). Cell lysate protein contentwas measured by a colorimetric assay (Bio-Rad). West-ern blot analysis was performed as previously described(17, 20). Briefly, 50 mg proteins were separated on 10%SDS-PAGE and transferred onto a polyvinylidene fluo-ride membrane. The membranes were blocked with 5%nonfat dry milk and probed with anti-phospho-Akt(Ser473; p-AKT) Ab or anti-p-ERK1/2 (Thr202/204) Ab(Cell Signaling) and then with anti-AKT mAb (R&DSystem) or anti-ERK1/2 mAb (Millipore). In all cases,washed filters were incubated with horseradish perox-idase–conjugated antimouse or anti-rabbit antibody anddetected by Enhanced Chemiluminescence Kit (GE-Healthcare).

Cell adhesion assayCell adhesion assays were performed using 16-well

plates coated with 5 mg/mL vitronectin, diluted in PBSand the xCELLigence technology (Roche Diagnostics) asdescribed (23). HUVEC (5� 103 cells/well) were plated ineach coatedwell, in serum-free EBM in the presence or theabsence of 40 ng/mL VEGF165 with/without 10 nmol/LC-terminal amidated Ac-Glu-Arg-Phe-Arg-NH2 controlpeptide (ERFR), 10 nmol/L RERF or 10 nmol/L UPAR-ANT and allowed to adhere for 4 hours at 37�C, 5% CO2.Cell adhesion was automatically monitored by the RealTime Cell Analysis xCELLigence system (Acea Biosci-ence), which measures the impedance value of each welland expressed as a cell index value.

Cell viabilityFor the MTS assay, the CellTiter 96 AQueous Cell Pro-

liferation Assay Kit (Promega) was used following themanufacturer’s instruction. Briefly,HUVEC (2� 103/well)were seeded in 96-well tissue culture plates and left to

Carriero et al.

Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics1094




adhere in complete media for 3 hours, then rinsed twicewithPBS followedby the incubationwithEBMmixed to 40ng/mL VEGF165 with or without peptides at the indicatedconcentration. Medium was replaced every 24 hours. Atthe indicated times, suspended cells were removed and 10mL of the MTS reagent was added into each well and cellswere incubated at 37�C for 3 hours. The absorbance wasdetected at 490 nm with a Microplate Reader (Bio-Rad).With a similar experimental design, endothelial cell pro-liferation was assessed using 16-well plates and the RealTime Cell Analysis xCELLigence technology. The imped-ance value of each well was automatically monitored andexpressed as a cell index.

Endothelial cell tube formation assayThe formation of vascular-like structures was assessed

on Matrigel as described (13). HUVEC were suspended in300 mL prewarmed EBM. Diluents or effectors were addedto the cell suspension before seeding cells (4 � 104 cells/well) on plates coated with 300 mL/well growth factorreducedMatrigel (BectonDickinson).When indicated, cellswere preincubated for 30 minutes at 37�C with 1 mg/mLanti-humanVEGFAb, 2mg/mLLM609anti-avb3mAbs,ornonimmune serum. Complete capillary tube networkswere assessed within a low-magnification field observedunder light microscopy after 6-hour incubation at 37�C inhumidified air with 5% CO2. To quantify tube formation,5 random areas/well at �100 magnification were imagedand the number of tubes formed by cord-like structuresexceeding 100 mm in length (24) were visualized usingAxiovision 4.8 software (Carl Zeiss) and counted.

Matrigel sponge angiogenesis assayTwelve 6- to 8-week-old C57BL/6J male mice

(Charles River Laboratories) of 23 to 25 g 6 to 8 weeksold, were maintained in accordance with institutionalguidelines complying with national and internationallaws and policies. Briefly, VEGF165 (100 ng/mL) andheparin (50 U/mL) diluted in PBS, with or without 75 or150 mg/kg UPARANT, were added to unpolymerizedliquid Matrigel at 4�C, to a final volume of 500 mL. TheMatrigel suspension was slowly injected subcutaneous-ly into the flanks of mice, using a cold syringe, where itquickly polymerizes to form a solid gel. Matrigel withbuffer alone was used as negative control. Five daysafter injection, the animals were killed and the gels wereremoved, minced, and then diluted in water for hemo-globin content measurement with a Drabkin ReagentKit (Sigma). The final hemoglobin concentration wasnormalized to 100 mg of recovered gel and calculatedfrom a standard calibration curve after spectrophoto-metric analysis at 540 nm.

Corneal pocket assayCorneal pocket assays were performed at PRIMM.

Twelve female New Zealand White rabbits (CharlesRiver) weighing 2.5 to 3.0 kg were anesthetized byintramuscular injection of acepromazina (1 mg/kg),

ketamine hydrochloride (35 mg/kg), and xylazinehydrochloride (5 mg/kg) and 3 to 4 drops of 0,4%ossibuprocain chlorohydrate solution were topicallyapplied to the eye before micropocket surgery. A wirespeculum was positioned in the eye, and a sucralfate-hydron pellets containing PBS, 5 mg UPARANT, or 180ng VEGFwith/without 5 mg UPARANTwere implantedinto the cornea aftermaking amicropocket in the stroma,using standard surgical tools (25, 26). Tobradex (0.3%tobramycin–0.1% dexamethasone) was applied to thesurface of the cornea after pellet implantation to preventinfection. Observation and quantification of the angio-genic responses were performed by a slit-lamp stereo-microscope. The angiogenic activity was evaluated onthe basis of the number and growth rate of newly formedcapillaries. A density value of 1 corresponded to 0 to 25vessels per cornea, 2 from 25 to 50, 3 from 50 to 75, 4 from75 to 100, and 5 for >100 vessels.

Statistical analysisThe results are expressed as the mean � SD of the

number of the indicated determinations. Data were ana-lyzed by one-way analysis of variance and post hoc Bon-ferroni modified t test for multiple comparisons. P < 0.001was accepted as significant.

Ethics statementThe research work with mouse model has been

approved by Institutional Ethical Committee of IstitutoNazionale Tumori "Fondazione G. Pascale"-IRCCS,Naples, Italy (protocol no. 09, December 20, 2010).

ResultsDevelopment and synthesis of RERF analogues

Previously, we reported the NMR structure of RERFin solution (19). RERF, although displaying some con-formational flexibility, preferentially adopts an a-turn(type I-aRS) conformation (27) in water/trifluoroethanol(TFE) mixed solvent. Ca-methyl-a-amino acids withwell-defined stereo chemical properties impose localrestrictions on backbone conformation, thus conferringstructural stability (28–29). Aib residue (a-amino iso-butyric acid) is the prototype of Ca-methyl-a-aminoacids, and is well known to be characterized by arestricted conformational freedom (30) in the 310-a-helical region of the Ramachandran plot. OtherCa-methyl-a-amino acids, including Ca-methyl-phe-nylalanine (31) are b-turn and helix inducers, muchstronger than the unmethylated, phenyl-containing,protein amino acid Phe. Ca-methy-a-amino acid, whenincorporated into peptide sequences containing codedresidues, strongly facilitates and stabilizes type IIIb-turns or helicogenic a-turns as previously described(32, 33). In addition, Ca-methyl-a-amino acid substitu-tions could provide enhanced binding to a moleculartarget, resistance to proteolytic degradation and longerpersistence in circulation. On the basis of this informa-tion, we have synthesized 4 peptide analogues of RERF






containing commercially available Ca-methyl-a-aminoacids, namely:

Icm25-4 and Icm25-4c are both tetrapeptides analoguesof RERF in which the Glu residue has been replaced byAib. Icm25-4c also contains a second substitution of theterminal Phe residue with D-Ca(Me)Phe (S-configura-tion). Icm25 and Icm25B are both pentapeptides corre-sponding to N-terminal elongation of Icm25-4 and Icm25-4c with a second Aib residue.

Enzymatic digestionThe peptides were subjected to digestion by trypsin

and chymotrypsin at different peptide:enzyme ratios,depending on the digestion rates. The enzyme concen-tration is 0.6 mg/L, and is similar to trypsin concentra-tion determined in plasma 117 to 637 mg/L (34). Icm25-4was not studied for enzyme digestion. The peptideconsumption was determined by measuring the HPLCarea peaks during digestion. It seemed to follow first-order kinetics (data not shown), and the t1/2 wereestimated. We found that RERF and Icm25 at a pep-

tide:trypsin ratio of 1,000:1 are rapidly digested, the t1/2being 3.05 and 6.19 minutes, respectively. No digestionof Icm25B and Icm25-4c was detected at a peptide:trypsin ratio of 1,000:1, whereas at a peptide:trypsinratio of 10:1 the t1/2 were 37.5 and 11.4 minutes forIcm25B and Icm25-4c, respectively. Both RERF andIcm25, containing the natural Phe residue at the C-terminus, were digested by chymotrypsin with t1/2being 23.6 and 20.0 minutes, respectively. Conversely,no digestion of Icm25B and Icm25-4c, both containingthe unnatural C-terminal Ca(Me)Phe, was detectedwithin 60 minutes.

Screening of the peptides as inhibitors of VEGF-dependent endothelial cell migration

RERF has been shown to inhibit endothelial cell migra-tion driven by VEGF (20). We compared the effects ofRERF and Aib containing peptides in inhibiting endothe-lial cell migration directed toward 40 ng/mLVEGF165. Byconventional Boyden chamber assays, we found that 40ng/mL VEGF165 elicited endothelial cell migration up to235% of the basal cell migration. When used as chemoat-tractant at 10 nmol/L concentration, Aib containing pep-tides, as well as RERF, slightly reduced basal cell migra-tion, whereas the control peptides ARARY or ERFR wereineffective (Table 1). As expected, a 57% and 54%

Table 1. Peptide inhibitory activity on VEGF-dependent endothelial cell migration

Effector Chemoattractants Cell migration (%) Inhibition (%)

EBM CTL 10040 ng/mL VEGF 235 � 1410 nmol/L ERFR 101 � 2a10 nmol/L ARARY 110 � 4b10 nmol/L RERF 99 � 5b10 nmol/L Icm25 95 � 9b10 nmol/L Icm25B 94 � 9b10 nmol/L Icm25-4 105 � 12b10 nmol/L ICM25-4c 89 � 6b

1 mg/mL VEGF Ab EBM 90 � 340 ng/mL VEGF 102 � 2b 57

10 nmol/L ERFR 40 ng/mL VEGF 231 � 9 210 nmol/L Icm25-4 40 ng/mL VEGF 121 � 8a 4910 nmol/L RERF 40 ng/mL VEGF 110 � 5b 5410 nmol/L Icm25 40 ng/mL VEGF 96 � 5b 5910 nmol/L Icm25B 40 ng/mL VEGF 90 � 8b 6210 nmol/L Icm25-4c 40 ng/mL VEGF 75 � 3b 68NOTE: HUVEC (4 � 104 cells/well) were allowed to migrate toward chemoattractants, in the presence of the indicated effectors for 4hours at 37�C, 5%CO2.Quantitative analysis of cellmigrationwas calculatedas apercentageof cellmigration assessed in the absenceof anyeffector or chemoattractant (CTL). The inhibitory effect of eachpeptidewas reported relative to the extent of cellmigration towardVEGF (taken as 100%).Data are themean�SDof 2 independent experiments, performed in duplicate. Theywere analyzed byone-wayANOVA and post hocBonferonnimodified t test for multiple comparisons. Statistical significancewithP valueswas calculated against40 ng/mL VEGF.aStatistical significance with P < 0.001.bStatistical significance with P < 0.0001.

Icm25-4 Ac-Arg-Aib-Arg-Phe-NH2Icm25 Ac-Aib-Arg-Aib-Arg-Phe-NH2Icm25B Ac-Aib-Arg-Aib-Arg-D-Ca(Me)Phe-NH2Icm25-4c Ac-Arg-Aib-Arg-D-Ca(Me)Phe-NH2

Carriero et al.





inhibition of VEGF-directed endothelial cell migrationwas elicited by anti-VEGF Ab and by RERF, respectively.The combination of VEGF165 with 10 nmol/L concentra-tion of Icm25, Icm25B, Icm25-4, or Icm25-4c reducedendothelial cell migration by 59%, 62%, 49%, and 68%,respectively (Table 1). Because the peptide Icm25-4cshowed a long t1/2 in enzyme digestions, and exerted thestrongest inhibition of endothelial cell migration, it wasnamed UPARANT, and further characterized for its bio-chemical properties, conformational preferences and bio-logic activity.

Stability of UPARANTWe examined the stability of UPARANT during stan-

dard autoclave sterilization. Ninety sealed vials contain-ing 0.5 mL of UPARANT in PBS at a concentration of 3mg/mL were autoclaved for 20 minutes at 120�C. Onesamplewas immediately analyzed by LC/MS to ascertainthe lack of any modified compound. The remaining sam-ples were kept in a thermostat at 25�C. Three vials wereopened everymonth and analyzed byLC/MS.As a result,no degradation after autoclaving and standing at 25�C insealed vials for 2 years was observed (data not shown).The stability of UPARANT in physiologic solution, in

mice and rats blood was also studied at 0.3 and 1 mg/mLconcentrations. Blood stability was followed for 2 hours.UPARANT resulted undegraded as shown in Supple-mentary Tables S1 and S2.

NMR analysis of UPARANTWe investigated the conformational preferences of

UPARANT inwater and in water/TFE byNMR spectros-copy (Supplementary Tables S3 and S4 and Fig. S1).ROESY spectra in water and in water/TFE essentiallydisplay the samepattern of sequential andmedium-range

NOE effects (see SupplementaryMaterials andMethods),indicating that the peptide has a quite similar conforma-tional behavior in both solvent systems. However,detailed analysis was performed in water/TFE solutiononly, because of more dispersed amide signals and fewerresonance overlaps. The pattern of NOEs and their rela-tive intensities are summarized in Fig. 1A. RMD calcula-tions, using the NMR experimental data as conformation-al restraints, gave an ensemble of conformers quite similarin terms of backbone conformation, underlining the pres-ence of one predominant and stable structure forUPARANT. Figure 1B reports the backbone atoms super-position of the ensemble of conformers along the MDtrajectory. Figure 1C reports the average backbone torsionangles of UPARANT as obtained fromRMDsimulation invacuo at 300 K.

Thepeptide conformationcorresponds toan incipient 310-helix characterized by 2 consecutive type III b-turn, fol-lowed by a type I b-turn. This structure is stabilized by thetypical COi–HNiþ3 hydrogen bonds (see Fig. 1C): a weakinteraction Ac-CO��NH-Arg3, a H-bond Arg1-CO��NH-Ca(Me)Phe4, and Aib2-CO��NH-amide. Along the trajec-tory of the RMD simulation, the Arg1 and Arg3 guanidinegroups experience intraresidue H-bonds, and the Arg1

guanidine group is also H-bonded to Ca(Me)Phe4-COgroup.

UPARANT competes with fMLF for binding to theformyl-peptide-receptor

We have previously documented that RERF competeswith fMLF for binding to FPR, being FPR the mainbinding site of RERF (19). To test whether similar toRERF (20), UPARANT competes with fMLF for bindingto FPR, HUVEC were preincubated at 4�C (to avoidreceptor internalization) with diluents, 100 nmol/L

Figure 1. NMR analysis ofUPARANT. A, summary ofsequential, short- and medium-range connectivities. Thethickness of the bar indicates therelative intensities of theNOEs, thatis, weak, medium, and strong. TheX in position 2 and the Z in position4 correspond to Aib and Ca(Me)Phe, respectively.bR andbS refer tothe protons on the Cb atoms at theproR and proS positions,respectively. Dd/DT refers to thetemperature coefficients of theamide protons. B, ensemble ofconformers along the RMDtrajectory. C, average molecularconformation of the backbonetorsion angles f, c. H-bonds areshown as broken lines with thecorresponding distances.






concentration of fMLF, RERF, or UPARANT for 60 min-utes at 4�C, and then exposed to 10 nmol/LN-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for addi-tional 60 minutes at 4�C. Quantification of cell-associatedfluorescence was assessed by reading endothelial celllysates with a fluorescence plate reader. An aliquot ofeach sample was subjected to Western blot analysis withanti-a-tubulin to control that equal amount of proteins/sample has been read (inset to Fig. 2A). Measurement ofcell-associated fluorescence revealed that the control pep-tide ERFRdid notmodify binding of FITC-fMLF,whereasan excess of unlabeled fMLF,RERF (used for comparison),or UPARANT dramatically reduced binding of fluores-cent FPR-agonist to endothelial cells at a similar extent(Fig. 2A). To evaluate the effect of UPARANT on FPR

internalization in response to agonist-stimulation, bind-ing experiments were also performed at 37�C. Uponexposure to FITC-fMLF, FPR seemed mainly internalizedwith the appearance of fluorescent spots localized in thecytoplasmas shownbyz-stack analysis of confocal images(Fig. 2B and C). Similar to fMLF or RERF, 100 nmol/LUPARANT strongly reduced internalization of fluores-cent agonist in all cell population (Fig. 2B). To assesswhether UPARANT itself causes FPR internalization,HUVECs were preincubated with diluents, or an excessof fMLF, RERF, or UPARANT for 30 minutes at 37�C, andthen exposed to 100 nmol/L FITC-UPARANT for addi-tional 30 minutes at 37�C. As shown by z-stack analysis ofconfocal images, endothelial cells exhibited only a slightlyinternalization of FITC-UPARANT,whichwas prevented

Figure 2. UPARANTprevents agonist-dependent FPR internalization in endothelial cells. A, HUVECwere preincubatedwith diluents (None), 100 nmol/L ERFR,100 nmol/L fMLF, 100 nmol/L RERF, or 100 nmol/L UPARANT for 60 minutes at 4�C, and then exposed to 10 nmol/L N-formyl-Nle-or Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for 60 minutes at 4�C. Fluorimetric measurement of cell-associated fluorescence was assessed using 485 nm excitation and535 nmemission filters. Data, expressed as a percentage of the fluorescence associated to cells exposed to FITC-fMLF (None¼ 100%), representmean�SDfrom an experiment performed in triplicate, representative of 2 replicates. Inset, Western blot analysis for the a-tubulin content on an aliquot of eachcell lysate. B–E, representative imagesofHUVEC incubatedwith diluents (None), 100 nmol/L fMLF, 100 nmol/LRERF, or 100 nmol/LUPARANT for 30minutesat 37�C, exposed to 10 nmol/L FITC-fMLF (B and C) or 10 nmol/L FITC-UPARANT (D and E) for 30 minutes at 37�C and then visualized using a Zeiss510 META LSM microscope. Z-series images represent focal planes corresponding to 0.38 mm vertical interval of HUVEC incubated with FITC-fMLF (C) orFITC-UPARANT (E) alone. Scale bar, 10 mm. Original magnifications, �630.

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by unlabeled fMLF, RERF, as well as by UPARANT (Fig.2D and E). All together, these findings suggest that uPAR-ANT competes with fMLF for binding to FPR on cellsurface and inhibits FPR activation by preventing itsinternalization.

UPARANT inhibits cytoskeletal organization, avb3recruitment at focal adhesions, and adhesion ontovitronectin of endothelial cells stimulatedwithVEGFVEGF-triggered endothelial cell motility is the result of

marked cytoskeletal reorganization and accumulation ofstress fibers associated with new actin polymerization,and rapid formation of focal adhesions, which do notoccur in the presence of RERF (20). To analyze the effectsof UPARANT on cytoskeletal organization induced byVEGF, HUVEC were exposed to 40 ng/mL VEGF165, inthe presence/absence of 10 nmol/L UPARANT, and thendouble stained for vinculin and F-actin. As expected,VEGF inducedamarked reorganization of actin into stressfibers spanning the length of the cells, most of which

colocalized with vinculin-positive focal adhesions, thatwas prevented by 10 nmol/L RERF (Fig. 3A). A total of 10nmol/L UPARANT fully abrogated VEGF-inducedeffects on cytoskeleton. In the latter case, endothelial cellshad a condensed, rounded morphology; similar tountreated cells, the F-actin was condensed into fewerfibers and was completely absent from the leading edgesof the cells (Fig. 3A). It is known that avb3 integrin has aprominent role in the activity of VEGF. In response toVEGF, b3 integrin regulates integrin-dependent actinreorganization, thus leading avb3-VEGFR2 complexes tolocalize at new formed focal adhesions (35). UPARANTcaused disappearance of avb3 at focal adhesions and theappearance of thin, avb3-positive linings at the cell edge,similar to untreated cells, whereas the addition of 10nmol/L ERFR did not modify VEGF-dependent integrinredistribution. (Fig. 3B). RERF has been proven to inhibitVEGF-dependent signaling by affecting avb3 integrin-dependent cell adhesion onto vitronectin (20). We inves-tigated whether avb3 integrin is indeed involved in the

Figure 3. UPARANT prevents cytoskeletal organization, disappearance of avb3 from focal adhesions and cell adhesion onto vitronectin in endothelial cellsstimulated with VEGF. A, representative confocal images of HUVEC grown on glass slides to semiconfluence exposed to EBM (None), or 40 ng/mL VEGF165with/without 10 nmol/L RERF or 10 nmol/L UPARANT for 30 minutes at 37�C and double stained for vinculin and F-actin. Scale bar, 20 mm. Originalmagnification, �630. B, representative confocal images of HUVEC grown on glass slides to semiconfluence, exposed to EBM (None), 40 ng/mL VEGF165mixed to 10 nmol/L ERFR or 10 nmol/L UPARANT for 30 minutes at 37�C and double stained for vinculin and avb3. Scale bar, 20 mm. Original magnification,�630. C, HUVEC (5 � 103/well) suspended in EBM mixed to diluents or 40 ng/mL VEGF165 with/without the indicated peptides were allowed toadhere for 4 hours at 37�Con 16-well plates coatedwith vitronectin. Cell adhesionwas automatically monitored by the xCELLigence system,whichmeasuresthe impedance value of each well, expressed as cell index. In the graph, the black line shows the time of peptide addition at which cell index valueswere normalized. Data, mean � SD from a quadruplicate experiment representative of 2 replicates.






inhibitory activity of UPARANT. HUVEC suspended inEBM mixed to diluents or 40 ng/mL VEGF165 with/without 10 nmol/L ERFR, 10 nmol/L RERF, or 10nmol/L UPARANT, were allowed to adhere for 4 hoursat 37�C on 16-well plates coated with vitronectin. Wefound that RERF andUPARANT reduced endothelial celladhesion onto vitronectin at a similar extent, either in thepresence or in the absence of VEGF, whereas the controlpeptide ERFRwas almost ineffective (Fig. 3C). All togeth-er, these findings show that UPARANT prevents VEGF-driven avb3 relocalization and ligand-dependent avb3activation.

UPARANT inhibits VEGF-dependent migration andintracellular protein phosphorylation of endothelialcells without affecting cell proliferation

To better characterize the inhibitory effect of UPAR-ANT on VEGF-dependent cell migration, HUVEC wereallowed tomigrate in Boyden chambers toward 40ng/mLVEGF165with/without increasing concentrations of RERF

used as positive control, ERFRused as negative control, orUPARANT.We found that unlike ERFR, bothUPARANTand RERF inhibit VEGF-directedmigration of endothelialcells in a dose-dependent manner. In both cases, theinhibition starts in the low fmol/L concentration rangeand plateaus in the nmol/L range (Fig. 4A). RERF hasbeen proven to prevent VEGF-induced phosphorylationof many proteins, such as AKT and ERK1/2 (20). Toinvestigate the effects of UPARANT on the phosphory-lation of these VEGF intracellular targets, HUVEC wereexposed to EBM or 40 ng/mLVEGF165 with or without 10nmol/L UPARANT from 0 to 60 minutes, and phosphor-ylated AKT and ERK1/2 were detected by Western blot.As shown in Fig. 4B, UPARANT consistently decreasedthe VEGF165-induced and time-dependent increase of Aktand ERK1/2 phosphorylation. Moreover, ANOVA statis-tical analysis revealed that growth of endothelial cellsexposed to VEGF was not modified by 10 nmol/L or 10mmol/L UPARANT up to 72 hours as shown by an MTSassay (Fig. 4C) as well as by proliferation curves

Figure4. UPARANT inhibitsmigration andprotein phosphorylation of endothelial cells stimulatedwith VEGFwithout affecting cell proliferation. A,HUVECwereallowed to migrate in Boyden chambers for 4 hours at 37�C in 5% CO2 toward 40 ng/mL VEGF165 plus diluents (None) or increasing concentrations of theindicated peptides. The extent of cell migration was expressed as a percentage of the net VEGF165-dependent cell migration, considered as 100%.Data, mean� SD of 3 independent experiments, performed in duplicate. Statistical significance with P values was calculated against 40 ng/mL VEGF (None).�, statistical significance with P < 0.0001; ��, statistical significance with P < 0.001. B, HUVEC grown on glass slides to semiconfluence were exposedto 40 ng/mL VEGF165 with/without 10 nmol/L UPARANT at 37�C in humidified air with 5% CO2 for the indicated times. Whole cell lysates (50 mg/sample)immunoblotted with anti-phospho-Akt (pAKT) or anti-phospho-ERK1/2 (pERK1/2) Abs and then with total anti-Akt (AKT) Ab or total anti-ERK1/2 (ERK) mAb.An experiment representative of 2 replicates is shown. C and D, HUVEC (2 � 103/well) were seeded in 96-well tissue culture (C) or 16-well plates (D),left to adhere in complete media for 3 hours at 37�C 5% CO2, rinsed twice with PBS, and then allowed to grow in EBM mixed to 40 ng/mL VEGF165 with orwithout the indicated peptides for 72 hours at 37�C 5% CO2. Medium was replaced every 24 hours. C, absorbance of adherent cells was assessedby MTT assay. Plot represents the mean � SD of 2 independent experiments, each performed in quadruplicate. D, the impedance value of each well wasautomatically monitored by the xCELLigence system and expressed as cell index. Data, mean � SD from a quadruplicate experiment representativeof 2 replicates.

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automatically monitored with the xCELLigence system(Fig. 4D). This is in agreement with our previous datashowing that 10 mmol/L RERF does not modify thegrowth of endothelial cells. Although we have not iden-tified yet the exact molecular mechanism by which RERFand UPARANT inhibit VEGF-triggered signaling, ourdata suggest that RERF and UPARANT impinge on com-mon downstream signaling mediators.

UPARANT inhibits tube formation of endothelialcells exposed to VEGF in vitroTo investigate the ability ofUPARANT to inhibit VEGF-

triggered angiogenesis in vitro, endothelial cells wereplated on Matrigel in the presence of 40 ng/mLVEGF165 mixed to diluents or increasing concentrationsof RERF, ERFR, or UPARANT. As expected, endothelialcells exposed to VEGF, formed a 3-dimensional networkof tubes resembling capillary-like structures, whichreached at 6 hours the 257 � 10% of braches counted in

the absence of any angionenic stimulus (Fig. 5A and B).Unlike ERFR control peptide or nonimmune serum, endo-thelial cell incubation with anti-VEGF Ab or anti-avb3mAb reduced VEGF-dependent tube formation to thebasal level (Fig. 5B). UPARANT reduced the extent ofVEGF-dependent tube formation in a dose-dependentmanner. Similar to RERF, the inhibitory effect of UPAR-ANT starts in the femtomolar concentration range andlevels off in the nanomolar range. Remarkably, UPAR-ANT caused an overall 75% inhibition of VEGF-depen-dent tube formation above 10 nmol/L, when comparedwith RERF, which caused a 54% inhibition. Also, the IC50of UPARANT seems to be �100-fold lower as comparedwith RERF (Fig. 5C), indicating again that UPARANTinhibits VEGF signaling more efficiently than RERF.

UPARANT inhibits angiogenesis in vivoTo assess the effect of UPARANT on angiogenesis in

vivo, first a quantitative Matrigel sponge assay was

Figure 5. UPARANT inhibits VEGF-induced angiogenesis in vitro and in vivo. A–C, HUVEC were suspended in prewarmed EBM, with or without 40 ng/mLVEGF165 in the presence or the absence of 5 mg/mL nonimmune serum (NIS), 1 mg/mL anti-human VEGF Ab, 5 mg/ml anti-avb3 mAb, clone LM609,or the indicated peptides at 10nmol/L concentration and seededonMatrigel-coatedplates for 6 hours at 37�C, 5%CO2. A, representative pictureswere takenwith an inverted microscope at �200 magnifications. B, quantitative analysis of tube formation was indicated as a percentage of tubes formed bycord-like structures exceeding 100 mm in length, counted in the absence of any angiogenic stimulus considered as 100% (CTL). Data, mean � SD of 3independent experiments performed in duplicate. �, P < 0.0001 in respect to VEGF (None). C, quantitative analysis of tube formation was indicated as apercentage of tubular branches counted in the presence of VEGF165 alone, considered as 100%. Data, mean � SD of 2 independent experimentsperformed in duplicate. D, Matrigel sponge assay in mice. Matrigel-containing sponges loaded with PBS (CTL), 100 ng/mL VEGFmixed to PBS (None), 75 or150 mg UPARANT were implanted subcutaneously into the dorsal flank of 12 C57BL/6J male mice. Five days after injection, the animals were killedand the gels were removed,minced, and diluted inwater. The hemoglobin concentrationwas determinedwith a Drabkin Reagent Kit, normalized to 100mg ofrecovered gel and calculated from a standard calibration curve after spectrophotometric analysis at 540 nm. �, P < 0.0001 in respect to VEGF alone(None). E and F, corneal pocket assay in 12 female New Zealand White rabbits. Sucralfate-hydron pellets impregnated with diluents (PBS), 5 mg UPARANT,180 ng VEGF, or a mixture of 180 ng VEGF and 5 mg UPARANT were implanted in rabbit corneas. E, representative stereomicroscopy imagesrecorded15dayafter pellet implantation. F, quantification of the angiogenic score (1 corresponds to0–25vessels per cornea, 2 from25 to50, 3 from50 to75, 4from 75 to 100, and 5 for >100 vessels). Plots represent data from a total of 6 eyes/sample. The median values are indicated.






performed. A cocktail of VEGF165 (100 ng/mL) and hep-arin (50 U/mL) promoted a hemorrhagic vascularizationof the Matrigel sponge, which was clearly detectable at 5days postimplantation (Fig. 5D). The presence in thesponge of 75 or 150 mg UPARANT produced a markedreduction of capillary sprouts originating from the hostvessels that invaded Matrigel sponges as detected byvisual inspection of the gels and quantification of hemo-globin recovered from sponges (Fig. 5D). A 64% and 72%reduction of hemoglobin was found in sponges loadedwith 75 and 150 mg, respectively.

Furthermore, antiangiogenic activity of UPARANTwas investigated by a corneal pocket assay in rabbits.Neovascular growth was evaluated in 12 rabbits uponsubcorneal implantation of slow release pellets containingPBS, 5 mg UPARANT, or 180 ng VEGF with/without 5 mgUPARANT. Implantation of pellets containing 180 ngVEGF stimulated ingrowth of blood vessels in the rabbitcorneas starting from day 2, and continuing to growprogressively up to 15 days before regressing. At day15, rabbit corneas probed with pellets impregnated withVEGF exhibited higher angiogenic score then the averagescore of those impregnated with vehicle only (PBS; 3.3 �0.6 vs. 0.1 � 0.1 with P < 0.0001; Fig. 5E and F). WhenUPARANTwas tested at 5 mg/pellet alone, it did not elicitinflammatory response. Conversely,UPARANTelicited a75% inhibition of VEGF-induced vascularization (0.84 �0.4 vs. 3.3 � 0.6 with P < 0.0001; Fig. 5E and F). Takentogether, our findings indicate thatUPARANTbehaves asan antiangiogenic factor, which prevents VEGF-inducedangiogenesis in vivo more effectively than RERF.

DiscussionThis work is based on previous evidence showing that

the chemotactic sequence of the human urokinase recep-tor (uPAR88-92) drives in vitro and in vivo angiogenesis in aprotease-independent manner (13). A few years ago wedeveloped the peptide Ac-Arg-Glu-Arg-Phe-NH2, name-ly RERF, that prevents, in vitro and in vivo, migration andinvasion of tumor cells by inhibiting the uPAR88-92-depen-dent signals (19).More recently,wehavedocumented thatRERF prevents both uPAR88–92- and VEGF-inducedangiogenesis in vitro and in vivo (13). However, RERF isexpected to have some pharmacokinetic drawbacks as

drug candidate, which are common to peptides: they areusually very susceptible to proteolytic degradation in vivoand are rapidly cleared from the circulation in minutes(36). To overcome these drawbacks, we have generated aseries of peptide analogues of RERF containing Ca-meth-yl-a-amino acids, a structural modification used in thepast to overcome these limitations (37). Among the enor-mous scenario of possible peptide modifications devel-oped in the last 50 years, we have chosen Ca-methyl-a-amino acid substitutions on the basis of the previouslyreportedNMR solution structure of RERF. In fact, when itis necessary to modify the peptide composition toimprove the pharmacologic profile, it is also mandatoryto keep, and possibly reinforce, the bioactive conforma-tion, and henceforth the pharmacophore orientation as inthe original bioactive peptide. The NMR solution struc-ture of RERF revealed that this peptide, although display-ing some conformational flexibility, preferentially adoptsa turned structure, therefore Ca-methyl a-amino acidsubstitutions were selected for their well-known propen-sity to strongly induce turned structures. Among theseries of newly synthesized analogues (Icm25, Icm25B,Icm25-4, and UPARANT), UPARANT displayed in apreliminary evaluation of the biologic activity the stron-gest inhibition of endothelial cell migration and showed along t1/2 in enzyme digestions. UPARANT was firstcharacterized from a structural point of view by NMRspectroscopy.

The solution structures of UPARANT and RERFreveals common features as can be seen by comparisonof Fig. 6A–C. The backbone atoms superimposition for allresidues of RERF and UPARANT gave root-mean-squaredeviation (RMSD) values of 0.76 Å, which underlinestheir structural similarity. Both RERF and UPARANTadopt in solution a helicogenic-turned structures; ana-turn (type I-aRS) is found for RERF and an incipient310-helix for UPARANT (Fig. 1C). However, UPARANTshows a more stable and compact turned structure withrespect to RERF. As anticipated from the design, thepresence, in the tetrapeptide UPARANT, of Ca-meth-yl-a-amino acids, such as Aib and Ca(Me)Phe, induces asignificant stabilization of the turned structure. UPAR-ANT and RERF share a common structural motif: thearomatic ring of the Phe residue is flanked by 2 positively

Figure 6. Comparison of thesolution structures of UPARANTand RERF. A, simplifiedrepresentation with backboneatoms in ribbon drawing ofUPARANT average structure. B,averagemolecular conformation ofRERF as derived from NMRanalysis in water/TFE solution,backbone atoms are in ribbondrawing. C, backbone atomssuperposition of RERF (purple) andUPARANT (cyan).

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charged residues. A major difference regards the orien-tation of Arg1 side chain. In the structure of RERF, theguanidinium group is involved in ion pairing with Glu2,whereas in UPARANT the same group is involved in H-bonding with the amide terminal CO group. In addition,it is worth mentioning that RERF and UPARANT wereboth studied by NMR spectroscopy in 2 solvent systems,water and water/TFE, for the well-known secondarystructure inducing effect of TFE. RERF is poorly struc-tured in water and its turned structure clearly appears inthe presence of TFE only. Conversely, UPARANT is wellstructured in both solvent systems. We have foreseenthat stabilization of the turned conformation for UPAR-ANT, similar to that found for RERF, would have ben-eficial effects on biologic activity. We found that UPAR-ANT inhibits VEGF-driven angiogenesis in vitro and invivo more efficiently as compared with RERF. In vitro,UPARANT inhibits VEGF-dependent tube formation ofendothelial cells at a 100� lower concentration thanRERF. Remarkably, a 75% reduction of tubes formed byendothelial cells exposed to VEGF is achieved by UPAR-ANT at 10 nmol/L concentration whereas only a 54%inhibition has been observed in the presence of 10 nmol/L RERF. In vivo, UPARANT reduces to the basal levelVEGF-dependent capillary sprouts originating from thehost vessels that invaded Matrigel sponges implanted inmice and prevents neovascularization induced by sub-corneal implantation of pellets containing VEGF inrabbits.Our previous work has documented that RERF pre-

vents uPAR/FPR and fMLF/FPR interactions, beingFPR the main binding site of RERF (19). Although wehave not investigated UPARANT/FPR association anddisassociation process, we found that an excess ofUPARANT prevents fMLF binding to FPR, suggestingthat fMLF and UPARANT share the same binding site.The pivotal role of avb3 in mediating VEGF-dependentproangiogenic stimulus is well established (35). Theinvolvement of avb3 integrin in the UPARANT inhib-itory effect is supported by the findings that cell adhe-sion to vitronectin is greatly reduced in the presence ofUPARANT. Taken together, our results confirm theinvolvement of FPR in binding and mediating inhibitionby UPARANT and suggest a mechanism by which avb3is forced into an inactive state by UPARANT and,consequently, VEGF downstream signaling mediatorsare not activated.Our findings unravel a complex picture of the mech-

anistic effects of UPARANT on endothelial cells and,more generally, highlight the role of uPAR, integrins,and VEGF-induced signaling events in the regulation ofendothelial cell functions. At molecular level, inhibitoryeffect of UPARANT on VEGF signaling is shown by thedecreased amount of phospho-AKT and phospho-ERK1/2 in VEGF-treated endothelial cells.Pharmacologic control of angiogenesis is considered

one of the most promising approaches for the treatmentof diseases sustained by excessive angiogenesis, such as

cancer and inflammation. However, several side effectshave been ascribed to antiangiogenic drugs such asbevacizumab that significantly increases the risk ofcardiac ischemic events in cancer patients (38). Withrespect to this, UPARANT may be considered a gooddrug by virtue of its ability to selectively inhibit FPRunlike bevacizumab, indicating that UPARANTbehaves differently from other VEGF inhibitors. Mech-anistically, UPARANT blocks VEGF-triggered signalingby preventing FPR and avb3 integrin activities withoutaffecting cell survival. In this respect, although we havenot ascertain whether a direct interaction betweenUPARANT and VEGF occurs, it is possible to assumethat UPARANT bocks integrin avb3 activity, thus inhi-biting only a part of the VEGF/VEGFR signaling. Thissuggests a potentially wider, but still target-specificactivity for UPARANT.

Furthermore, unlike RERF, UPARANT is stable to ster-ilization, is stable in blood, and displays a long-timeresistance to enzymatic digestion. Peptide-based thera-peutics is one of the fastest growing class of new drugs(39). Peptides have unique advantages as therapeuticagents. They have high activity per unit mass, and lowmanufacturing costs. They also offer great potency, selec-tivity, and specificity, have low off-target toxicity and lowdrug–drug interactionpotential. In this frame,UPARANTcould be considered a promising therapeutic agent for thecontrol of diseases fueled by excessive angiogenesis, suchas cancer and inflammation.

Disclosure of Potential Conflicts of InterestM.V. Carriero has ownership interest (including patents) in Pharma-

phelix s.r.l. V. Pavone is the President of Pharmaphelix s.r.l. and also hasownership interest (including patents) in the same. No potential conflictsof interest were disclosed by the other authors.

Authors' ContributionsConception and design: M.V. Carriero, V. PavoneDevelopment of methodology: K. Bifulco, L. Lista, L. Mele, G.Di CarluccioAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): O. MaglioAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M.V. Carriero, K. Bifulco, M. Minopoli,O. Maglio, V. PavoneWriting, review, and/or revision of the manuscript: M.V. Carriero,O. Maglio, V. PavoneAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Bifulco, L. ListaStudy supervision: M.V. Carriero, M. De Rosa, V. Pavone

AcknowledgmentsThe authors thank M. Paro at PRIMM for corneal pocket assays in

rabbits and EdithMonteagudo, Head of Preclinical Research, and the staffof IRBM Science Park for blood stability studies.

Grant SupportThis work was supported by Associazione Italiana per la Ricerca sul

Cancro 2013, project 14225 (M.V. Carriero) and by Italian Ministry ofHealth RF-2010-2316780 (M.V. Carriero).

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked adver-tisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

Received November 5, 2013; revised February 13, 2014; accepted March4, 2014; published OnlineFirst April 4, 2014.






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Carriero et al.




2014;13:1092-1104. Published OnlineFirst April 4, 2014.Mol Cancer Ther Maria Vincenza Carriero, Katia Bifulco, Michele Minopoli, et al.

PotencyIn Vivoand In VitroVEGF-Driven Angiogenesis with Enhanced Stability and

Derived Peptide Inhibitor of−UPARANT: A Urokinase Receptor

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