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Mol Cancer Ther. 2005;4:1670-1680
© 2005 American Association for Cancer Research

Biological and molecular properties of a new {alpha}vß3/{alpha}vß5 integrin antagonist

Laura Belvisi1, Teresa Riccioni3, Marcella Marcellini3, Loredana Vesci3, Ilaria Chiarucci3, Daniela Efrati3, Donatella Potenza1, Carlo Scolastico1, Leonardo Manzoni2, Katia Lombardo3, M. Antonietta Stasi3, Augusto Orlandi4, Alessandro Ciucci4, Beatrice Nico5, Domenico Ribatti5, Giuseppe Giannini3, Marco Presta6, Paolo Carminati3 and Claudio Pisano3

1 Organic and Industrial Chemistry Department, Centre for Biomolecular Interdisciplinary Studies and Industrial Applications, University of Milan; 2 Institute of Molecular Science and Technologies, Consiglio Nazionale delle Ricerche, Milan, Italy; 3 R&D Sigma-Tau S.p.A., Pomezia, Italy; 4 Institute of Pathology, Tor Vergata University, Rome, Italy; 5 Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy; and 6 Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Brescia, Italy

Requests for reprints: Claudio Pisano, Area of Oncology, R&D Sigma-Tau S.p.A., Via Pontina km 30.400, 00040 Pomezia, Italy. Phone: 39-06-91-39-37-60; Fax: 39-06-91393988. E-mail: claudio.pisano{at}sigma-tau.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of the present study was to identify specific {alpha}vß3/{alpha}vß5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for {alpha}vß3 and {alpha}vß5 integrins with negligible interacting with {alpha}5ß1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-D-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of {alpha}vß3/{alpha}vß5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of {alpha}vß3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-{alpha}vß3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-{alpha}vß3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual {alpha}vß3/{alpha}vß5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
New blood vessel formation or angiogenesis plays a pivotal role in tumor growth and metastatic spread. Tumor angiogenesis is a multistep process characterized by the chemotactic and mitogenic response of endothelial cells to angiogenic growth factors, proteolytic degradation of extracellular matrix, and modulation of endothelial cell interaction with extracellular matrix mediated by integrin receptors (1–3). Each of these steps may represent a potential target for the development of antiangiogenic and antimetastatic therapies (4).

Integrins are a family of membrane-spanning adhesion receptors composed of noncovalently linked {alpha} and ß subunits, which combine to originate a variety of heterodimers with different ligand recognition properties. Several integrins interact with polypeptide domains containing the Arg-Gly-Asp (RGD) amino acid sequence present in various extracellular matrix–associated adhesive glycoproteins (5). Besides cell adhesion to extracellular matrix, integrins also mediate intracellular events (6, 7) that control cell migration, proliferation, and survival (8–10).

{alpha}vß3 Integrin plays a key role in angiogenesis (11). It interacts with several extracellular matrix proteins (e.g., vitronectin, fibrinogen, fibronectin, thrombin, thrombospondin, and von Willebrand factor) and cooperates with molecules endowed with different biological functions, including metalloproteases, growth factors, and their receptors. Due to its numerous functions and to its relatively limited cellular distribution, {alpha}vß3 integrin represents an attractive target for therapeutic intervention. Indeed, {alpha}vß3 is not generally expressed on epithelial cells and normal quiescent endothelial cells but is significantly up-regulated on activated endothelial cells and in metastatic tumor cells. Growth factors, such as fibroblast growth factor-2 (FGF2) and tumor necrosis factor-{alpha}, stimulate {alpha}vß3 expression in the developing chicken chorioallantoic membrane (CAM) and in the angiogenesis model of the rabbit cornea (11, 12). Up-regulation of {alpha}vß3 expression is also induced in the vasculature of human tumors cultured on the CAM, implanted into severe combined immunodeficient mice, or grown on the rabbit cornea (13–15). Recent studies have provided evidence that integrins and growth factors may act synergistically following physical interaction. Phosphorylated platelet-derived growth factor receptor ß, vascular endothelial growth factor (VEGF) receptor 2, and insulin receptor have been shown to interact selectively with {alpha}vß3 and this interaction results in a synergistic signaling effect (16).

The importance of {alpha}vß3 in tumor angiogenesis is also shown by the fact that {alpha}vß3 antagonists, including cyclic RGD peptides and monoclonal antibodies, were successfully used to inhibit blood vessel development and tumor growth in different models (15, 17, 18). Systemic administration of {alpha}vß3 antagonists to animals with ongoing angiogenesis induces apoptosis in newly formed blood vessels (19). In this regard, administration of {alpha}vß3 antagonists in the chick CAM causes p53 activation and increased expression of the cell cycle inhibitor p21WAF1 (20). Recently, direct activation of caspases-3 has been proposed as an alternative explanation for the proapoptotic activity of RGD peptides (21).

Besides {alpha}vß3, {alpha}vß5 integrin has been implicated in the angiogenic process possibly via a signaling pathway distinct from that of {alpha}vß3. Indeed, neutralizing anti-{alpha}vß5 antibody inhibits VEGF-stimulated angiogenesis in the CAM assay, whereas anti-{alpha}vß3 antibody inhibits FGF2-induced angiogenesis (14). The existence of distinct angiogenic pathways can be explained with the prevalence of specific growth factors and/or cell-adhesive proteins in different conditions. Thus, experimental evidence suggests that dual {alpha}vß3/{alpha}vß5 antagonists may represent a multitarget approach for the inhibition of tumor angiogenesis and tumor growth (18, 22, 23).

Recently, a library of RGD-containing pseudopeptides has been synthesized (Fig. 1 ; ref. 24). These compounds are characterized by the replacement of the D-Phe-Val or the D-Phe-[NMe]Val dipeptide present in the lead structures c(RGDfV) or cyclo(Arg-Gly-Asp-D-Phe-[NMe]Val) (EMD121974), also known as cilengitide (25), respectively, with a 6,5- and 7,5-fused bicyclic lactam. In comparison with D-Phe-Val or D-Phe-[NMe]Val dipeptide, bicyclic lactams show different reverse-turn mimetic properties that could constrain the RGD sequence into different conformations and possibly provide the required activity and selectivity for integrin antagonism.



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  		Figure 1. Structures of the RGD-containing pseudopeptide library.

 
The use of conformationally constrained cyclic peptides or rigid peptidomimetic scaffolds that match biologically active conformation might enhance ligand binding for entropic reasons. Recently, the crystal structure of the extracellular segment of integrin {alpha}vß3 in complex with the cyclic pentapeptide ligand EMD121974 has been reported (26). The crystal structure of the peptide/integrin complex provides the exact conformation of EMD121974 bound to {alpha}vß3 integrin and can serve as a basis for understanding the general mode of interaction of integrins with other RGD-containing ligands.

The purpose of the present study was to determine whether the cyclic RGD pseudopentapeptides containing stereoisomeric 6,5- and 7,5-fused bicyclic lactams could provide the required activity and selectivity for {alpha}vß3/{alpha}vß5 integrin antagonism and to assess their antiangiogenic and antitumor activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
Primary cultures of bovine microvascular endothelial cells were obtained from bovine adrenal glands as described (27) and maintained in DMEM supplemented with 20% FCS, 50 units/mL heparin (Sigma, St. Louis, MO), 50 µg/mL bovine brain extract, and 100 units/mL gentamicin. Human umbilical vein vascular endothelial cells (HUVEC) and human microvascular dermal endothelial cells were obtained from BioWhittaker (Walkersville, MD) and grown in EGM-2 (BioWhittaker). Human mammary carcinoma MDA-MB435 cells (a generous gift of Dr. R. Giavazzi, Mario Negri Institute, Bergamo, Italy) were cultured in DMEM plus 10% FCS, 10 mmol/L L-glutamine, and 100 units/mL gentamicin. A2780 ovarian carcinoma cells (a generous gift of Dr. F. Zunino, Istituto Nazionale dei Tumori, Milan, Italy) were maintained in RPMI 1640 containing 10% fetal bovine serum and 50 µg/mL gentamicin sulfate. LNCaP prostate carcinoma cells (European Collection of Animal Cell Cultures, Salisbury, United Kingdom) and DAOY medulloblastoma and U87MG glioblastoma cells (both from American Type Culture Collection, Manassas, VA) were grown according to the manufacturer's instructions.

Solid-Phase Receptor-Binding Assay
The receptor-binding assays were done as described previously (24, 28, 29). Briefly, purified {alpha}vß3 and {alpha}vß5 (Chemicon International, Inc., Temecula, CA) and {alpha}5ß1 (Sigma-Aldrich, Milan, Italy) receptors were diluted to 0.5 or 1 µg/mL in coating buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 1 mmol/L MnCl2 in the presence ({alpha}vß3 and {alpha}vß5) or absence ({alpha}5ß1) of 2 mmol/L CaCl2 and 1 mmol/L MgCl2. An aliquot of diluted receptors (100 µL/well) was added to 96-well microtiter plates and incubated overnight at 4°C. Then, the plates were incubated with blocking solution (coating buffer plus 1–2% bovine serum albumin) for additional 2 hours at room temperature or at 37°C ({alpha}5ß1) to block nonspecific binding followed by 3-hour incubation in the presence of increasing concentrations of test compounds at room temperature with 0.05 and 0.1 nmol/L [125I]echistatin (Amersham Pharmacia, Little Chalfort, Buckinghamshire, United Kingdom) or at 37°C with 3 µg/mL fibronectin (Sigma-Aldrich, Milan, Italy) biotinylated using EZ-Link Sulfo-NHS-Biotynilation kit (Pierce, Rockford, IL) for {alpha}vß3/{alpha}vß5 and {alpha}5ß1 receptor-binding assays, respectively. After washing, the plates coated with {alpha}vß3/{alpha}vß5 were sealed and counted in the gamma counter (Packard Instrument, Meriden, CT). The plates coated with {alpha}5ß1 were instead incubated for 1 hour at room temperature with streptavidin-biotinylated peroxidase complex (Amersham Biosciences, Uppsala, Sweden) followed by 30-minute incubation with 100 µL Substrate Reagent Solution (R&D Systems, Minneapolis, MN) before stopping the reaction by addition of 50 µL of 1 N H2SO4. Absorbance at 415 nm was read in a microplate reader (1420 Multilabel Counter, Wallac, Turku, Finland).

Each data point is the result of the average of triplicate wells and was analyzed by nonlinear regression analysis with Allfit program.

Platelet Aggregation Assay
Guinea pig platelet aggregation response to the 11-mer thrombin receptor–activating peptide (25–100 µmol/L) was measured in platelet-rich plasma. The platelet concentration in platelet-rich plasma was adjusted to 3 x 108/mL. Platelet aggregation was determined within 1 hour by a turbidimetric method (30) in a dual-channel aggregometer. Vehicle or peptide solutions at different concentrations were added to platelet-rich plasma 1 minute before the beginning of aggregation. The extent of platelet aggregation was quantified as the maximum change in light transmittance within 4 minutes after the addition of the agonist. The results were plotted and expressed as the antagonist concentration that inhibited 50% platelet aggregation.

Cell Proliferation Assay
For the proliferation assay, cells were seeded in 96-well plates in complete medium at different concentrations according to the cell type. After 24 hours, the medium was removed and replaced with fresh medium containing scalar concentrations of the compounds. Then, the plates were incubated for additional 72 hours. On the last day of the experiments, cells were stained with toluidine blue and plates were read with a microplate reader at 595 nm. Results were plotted and expressed as mean ± SE compound concentration that inhibited 50% of cell proliferation.

Cell Adhesion Assay
Plates (96 wells) were coated with either fibronectin (Sigma, St. Louis, MO) or vitronectin (Calbiochem, San Diego, CA), both at 5 µg/mL in PBS, overnight at 4°C. Cells (~4 x 104–5 x 104/100 µL) were seeded in each well and allowed to adhere for 1 to 3 hours at 37°C in the presence of various concentration of RGD peptides. Nonadherent cells were removed with PBS and the remaining cells were fixed with 4% paraformaldehyde for 10 minutes. Adherent cells were stained with 1% toluidine blue for 10 minutes and rinsed with water. Stained cells were solubilized with 1% SDS and quantified on a microtiter plate reader at 600 nm. Experiments were done in quadruplicate and repeated at least thrice. Results are expressed as mean ± SE compound concentration that inhibited 50% of cell adhesion.

CAM Assay
Fertilized chicken eggs were maintained under constant humidity at 37°C. On day 4 of incubation, a square window was opened into the shell after removing 2 to 3 mL albumen. The opening was closed with a glass and sealed with paraffin. On day 9 of incubation, a sterile gelatin sponge (1 mm3) was placed onto the CAM immediately followed by topical administration (2–3 µL) of FGF2 or VEGF (both at 500 ng/egg) in the presence of vehicle or 30 µg/egg of either ST1646 or EMD121974. Treatments were repeated daily until day 12. Quantification of CAM vascular network was assessed at days 9 and 12 by counting the number of vessels converging toward the implant under a stereomicroscope. The angiogenic response was calculated and the results were expressed as the number of eggs out of total with an inhibition in the angiogenic response greater than 90% compared with controls.

Histology on Bouin's fluid-fixed CAMs was also done. In brief, serially sectioned paraffin-embedded small pieces of CAMs were stained with a 1% aqueous solution of toluidine blue (Merck, Darmstadt, Germany) and angiogenesis was analyzed (31) by a 144-point mesh inserted in the eyepiece of a Leitz-Dialux 20 photomicroscope at x250 magnification.

Mouse Dorsal Air Sac Assay
The dorsal air sac method was done on CD1 mice as described previously (32). Briefly, 1 x 106 MDA-MB435 cells resuspended in HBSS were placed in a Millipore chamber and then implanted into a dorsal air sac produced by s.c. injection of 10 mL air. The animals were treated i.p. twice daily with 15 mg/kg ST1646 or once daily with 60 mg/kg suramin for 7 days. At this time, the mice were sacrificed and the skin areas in contact with the dorsal side of the chamber were trimmed off. Quantification of the total vascularized area was done by image analysis with the Image Pro Plus software (Media Cybernetics, Silver Springs, MD).

Tumor Growth Assay and Histology
Human ovarian carcinoma A2780 cells were inoculated s.c. (2 x 106 per mouse) in the right flank of CD1 nude mice. After 2 days, mice were divided in two groups of eight mice each, anesthetized by Zoletil (50 mg/10 mL/kg i.p.), and implanted s.c. in the left flank with an osmotic pump (Alzet, Mini-Osmotic Pump model 2002, 0.5 µL/h, release of 14 days) filled with a sterile saline solution with or without ST1646 (16 mg/mL).

Tumor diameters were measured biweekly with a caliper and the tumor volume (in mm3) was calculated using the formula [length (mm) x width (mm)2] / 2, where the width and the length were the shortest and longest diameters, respectively. Animals were sacrificed 14 days after pump implantation. The Mann-Whitney's test was used for statistical analysis.

For histologic analysis, tumors were placed in zinc fixative for 24 hours, dehydrated, and paraffin embedded. Sections (4 µm thick) were then stained for CD31 and factor VIII. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide and methanol, and nonspecific antibody binding was blocked by incubation with 10% normal rabbit or goat serum in TBS. Sections were then incubated at room temperature for 1 hour with a 1:40 dilution of a rat monoclonal CD31 antibody (BD PharMingen, San Diego, CA) or for 30 minutes with a 1:100 dilution of a rabbit polyclonal factor VIII (DAKO, Glostrup, Denmark) antibody. This step was followed by the application of biotinylated rabbit anti-rat or goat anti-rabbit IgG followed by a streptavidin-horseradish peroxidase conjugate. Bound antibody was revealed using the substrate 3,3'-diaminobenzidine as chromogen. Sections were counterstained with hematoxylin. The number of tumor vessels per mm2 was evaluated under the microscope at 200-fold magnification by two observers.

The percentage of necrotic area was evaluated on H&E-stained tumor sections by computer-assisted image analysis. Scion Image software was used and images were captured at x20 magnification by a Hamamatsu (Hamamatsu City, Tokyo, Japan) camera connected to a Nikon (Nikon Corporation, Tokyo, Japan) microscope.

Animal Husbandry
Animals were housed in a limited access animal facility. Animal room temperature and relative humidity were set at 22 ± 2°C and 55 ± 10%, respectively. Artificial lighting provided a 24-hour cycle of 12 hours light/12 hours dark (7 a.m.–7 p.m.). The care and husbandry of animals were in accordance with European Directives no. 86/609 and with Italian D.L. 116 (January 27, 1992). All experiments were approved by the Sigma-Tau veterinarian.

Molecular Modeling Studies
Conformational preferences of ST1646 have been investigated by molecular mechanics calculations performing Monte Carlo/Energy Minimization conformational searches (33) and Monte Carlo/Stochastic Dynamics free simulations (34). Molecular mechanics calculations were done within the framework of MacroModel (35) version 5.5 using the MacroModel implementation of the Amber all-atom force field (36) and the implicit water GB/SA solvation model of Still et al. (37). Simulations of the RGD cyclic peptide were done at 300 K. A time step of 1 fs was used for the Stochastic Dynamics part of the algorithm. The total simulation time was 10 ns and samples were taken at 1-ps intervals, yielding 10,000 conformations for analysis. Docking studies of ST1646 in the ligand-binding site of the crystal structure of the extracellular segment of integrin {alpha}vß3 were done within the framework of the Analyze mode of the MacroModel interactive molecular modeling package.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ST1646 Is a Potent and Selective {alpha}vß3/{alpha}vß5 Integrin Inhibitor
Seven compounds (Fig. 1) belonging to a library of novel RGD-containing pseudopeptides were screened in a solid-phase assay for their capacity to compete with radiolabeled echistatin for the binding to purified human {alpha}vß3 and {alpha}vß5 and to compete with biotinylated fibronectin for the binding to {alpha}5ß1 integrin. The well-characterized integrin antagonists cyclic pentapeptide c(RGDfV) and EMD121974 (25, 38) as well as the natural integrin ligands vitronectin, fibronectin, and fibrinogen were used as positive controls (Table 1 ). The most active compound ST1646 inhibited radiolabeled echistatin binding to {alpha}vß3 and {alpha}vß5 with IC50 of 3.8 ± 0.9 and 1.4 ± 0.2 nmol/L, respectively. Selectivity of this compound was shown by its low capacity to compete with biotinylated fibronectin for {alpha}5ß1 interaction (IC50, 1,420 ± 201 nmol/L).


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  		Table 1. In vitro binding of RGD-containing pseudopeptides to purified integrin receptors

 
{alpha}vß3 and {alpha}IIbß3 Integrins share an identical ß subunit and highly homologous {alpha} subunits (39, 40). Because {alpha}IIbß3 integrin plays a critical role in hemostasis by mediating platelet aggregation, inhibition of this receptor may cause unwanted bleeding. On this basis, we evaluated the ability of ST1646 to prevent guinea pig platelet aggregation in response to thrombin receptor–activating peptide in vitro. To this purpose, increasing concentrations of ST1646, c(RGDfV), EMD121974, or echistatin were assessed by a turbidimetric platelet aggregation assay, and the antagonist concentration ± SD that inhibited 50% platelet aggregation (IC50) was calculated. Similar to c(RGDfV) (IC50, 11 ± 2 µmol/L) and EMD121974 (IC50, 10 ± 1 µmol/L), ST1646 (IC50, 12 ± 2 µmol/L) was 600 times less active than echistatin (IC50, 0.02 ± 0.001 µmol/L) in inhibiting thrombin receptor–activating peptide–induced platelet aggregation.

To assess the capacity of ST1646 to behave as an integrin antagonist for endothelial cells, bovine microvascular endothelial cells were allowed to adhere to immobilized vitronectin or fibronectin in the presence of increasing concentrations of the synthetic RGD-containing pseudopeptides (ranging between 0.1 and 100 µmol/L). As shown in Table 2 , ST1646 significantly inhibited cell adhesion to either vitronectin or fibronectin in a dose-dependent manner, with IC50 of 0.9 and 37.5 µmol/L, respectively. For both cell adhesion proteins, the activity of ST1646 was higher, on a molar basis, than that of c(RGDfV) and EMD121974. In contrast, all the other RGD-containing pseudopeptides did not exert a significant antagonist activity. The most active compound ST1646 in comparison with EMD121974 was also tested on HUVEC, human microvascular dermal endothelial cells, and a panel of human tumor cell lines (Table 3 ). In all these cells, ST1646 inhibited cell adhesion to either vitronectin or fibronectin with a lower IC50 than EMD121974. As expected, prostate LNCaP carcinoma cell line (41) did not adhere to vitronectin and showed, as the other cell lines tested, a modest sensitivity to ST1646 and EMD121974 in the adhesion assay to fibronectin.


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  		Table 2. Effect of RGD-containing pseudopeptides on bovine endothelial cell adhesion to vitronectin and fibronectin

 

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  		Table 3. Effect of ST1646 and EMD121974 on human endothelial and tumor cell adhesion to vitronectin and fibronectin

 
In vitro Antiangiogenic Activity of ST1646
To evaluate the effect of ST1646 on endothelial cell proliferation, subconfluent HUVEC cultures were grown for 72 hours in complete medium containing increasing concentrations of the compound. As shown in Fig. 2 , ST1646 inhibited HUVEC proliferation with a potency similar to EMD121974 (IC50, 2.9 and 4.4 µmol/L for the two compounds, respectively). The inhibitory effect was reversible. Indeed, when adherent cells were treated with ST1646 for 24 hours and detached cells were then washed with PBS and plated in the absence of the molecule, ST1646 removal completely abolished the antiproliferative effect of the molecule, leaving the cells free to adhere to the plate and proliferate (data not shown).



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  		Figure 2. Effect of ST1646 and EMD12974 on endothelial cell proliferation. Adherent HUVECs were seeded in 96-well plates in complete medium. After 24 h, the medium was removed and replaced with fresh medium containing various concentrations of ST1646 or EMD121974 and incubated for further 72 h. Then, cells were stained with crystal violet and plates were read with a microplate reader at 595 nm. Representative of three separate experiments.

 
In vivo Antiangiogenic Activity of ST1646
In a first set of experiments, the antiangiogenic activity of ST1646 was evaluated in a gelatin sponge/CAM assay using FGF2 or VEGF as an angiogenic stimulus (31). As shown in Table 4 , daily administration of ST1646 (30 µg/embryo) caused a significant inhibition of the angiogenic response triggered by both growth factors (P < 0.001). A similar inhibition of VEGF-induced angiogenesis was observed in EMD121974-treated embryos. Accordingly, histologic analysis of CAM specimens followed by blood vessel counting indicated that FGF2-loaded sponges were characterized by numerous blood vessels (20 ± 4 vessels per field) and a dense collagenous matrix among the sponge trabeculae (Fig. 3A ). No blood vessels were instead detectable inside FGF2 plus ST1646–loaded sponges (Fig. 3B).


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  		Table 4. Effect of ST1646 on FGF2- and VEGF-induced angiogenesis in the CAM assay

 


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  		Figure 3. Effect of ST1646 on angiogenesis in vivo. A and B, CAM assay. FGF2 (500 ng/embryo) was applied on the CAM at day 9 via a gelatin sponge implant. CAMs were then treated daily with vehicle or ST1646 (30 µg/egg). At day 12, histologic analysis was done. Note the absence of blood vessels among the sponge trabeculae in ST1646-treated implants (B) compared with the significant angiogenic response observed in vehicle-treated samples (A). C and D, murine dorsal air sac assay. Millipore diffusion chambers loaded with mammary carcinoma MDA-MB435 cells were implanted s.c. into a dorsal air sac. Then, mice were treated i.p. with vehicle (C) or ST1646 (D) at 15 mg/kg twice daily. After 7 d, skin areas in contact with the dorsal side of the chamber were photographed.

 
Next, tumor cell–induced vascularization in the mouse air sac model (32) was used to assess the effect of systemic administration of ST1646 on tumor-triggered neovascularization. Mammary carcinoma MDA-MB435 cells produce high levels of VEGF (data not shown). They induce a robust angiogenic response in the otherwise poorly vascularized s.c. area when inserted in a Millipore diffusion chamber and then implanted in a dorsal air sac (Fig. 3C). To assess its antiangiogenic capacity, ST1646 was given i.p. at 15 mg/kg twice daily for 7 days starting 1 hour after implantation of the Millipore chamber. Control animals received no treatment or were treated with 60 mg/kg suramin. At the end of treatment, animals were sacrificed, the chambers were removed, and viability of MDA-MB435 cells was assessed by trypan blue exclusion test. Vascularization in the skin in contact with the chamber was estimated by computer-assisted image analysis. As shown in Table 5 and Fig. 3D, systemic administration of ST1646 caused a 44% decrease of the vascularized area (P < 0.01), without affecting the in vivo viability of MDA-MB435 cells (>90%). Suramin administration resulted in a similar inhibition of vascularization (Table 5). However, part of antiangiogenic activity of suramin could reflect the direct cytotoxic effect on tumoral cells. In fact, the evaluation of in vivo viability of MDA-MB 435 at the end of experiments was reduced to ~65%.


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  		Table 5. Effect of ST1646 on mammary carcinoma MDA-MB435 cell–induced neovascularization in the mouse air sac model

 
Antitumor Activity of ST1646
The in vivo antitumor activity of ST1646 was investigated on human ovarian carcinoma A2780 cells xenografted in nude mice. As observed for various tumor cell types of different origin (42–46), A2780 cells express significant levels of both {alpha}vß3 and {alpha}vß5 integrins (data not shown), thus representing a suitable target for integrin antagonists. Two days after tumor cell implantation, ST1646 was delivered by continuous infusion via an osmotic pump releasing 8 µg/h of the compound under test. After 14 days, ST1646 treatment resulted in a significant inhibition of tumor growth (Fig. 4A ).



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  		Figure 4. Antitumor and antiangiogenic activity of ST1646. A, human ovarian carcinoma A2780 cells were inoculated s.c. (2 x 106 per mouse) in the right flank of CD1 nude mice. After 2 d, mice were implanted s.c. in the left flank with an osmotic pump releasing 8 µg/h ST1646 or vehicle. Tumor size was measured after 14 d. **, P < 0.01 versus vehicle (Mann-Whitney). B, immunohistochemical evaluation of CD31+ blood vessels in control (a) and ST1646-treated tumors (b). Original magnification, x200.

 
To assess whether the inhibitory effect of ST1646 on the tumor growth was paralleled by an inhibition in tumor vascularization, sections of control and ST1646-treated xenografts were immunostained with anti-CD31 antibodies to visualize tumor vascularity. To overcome possible differences in vascularization due to differences in tumor size, all lesions were examined when they reached a tumor mass equal to ~500 mg (corresponding to days 8 and 12 in control and ST1646-treated animals, respectively). The results show a significant decrease in CD31+ blood vessel density (46.1 ± 3.8 versus 60.2 ± 5.1 vessels per mm2; P < 0.05) and total CD31+ blood vessel area (2.6 ± 0.2% versus 4.4 ± 0.4% of tissue section area) in ST1646-treated tumors when compared with control lesions (Fig. 4B). Evaluation of factor VIII immunostaining gave similar results (data not shown). The decrease in tumor vascularity was paralleled by an increase in the size of the necrotic area observed in the central region of ST1646-treated tumors (31.9 ± 5.6%) when compared with control lesions (24.3 ± 4.3%), although the difference did not reach the statistical significance due to the intragroup variability.

Conformational Analysis and Docking Studies of ST1646
Examination of the three-dimensional structure of the cyclic pentapeptide antagonist EMD121974 bound to {alpha}vß3 integrin (Protein Data Bank entry 1L5G; ref. 25) reveals a conformation characterized by an inverse {gamma}-turn with Asp at position (i + 1) and by a distorted ßII'-turn with Gly and Asp at the (i + 1) and (i + 2) positions (Fig. 5A ). A 8.9-Å distance between Asp and Arg Cß atoms is observed in this pentapeptide-bound conformation. The backbone conformations of the ligand in the integrin complex and in solution (as determined by nuclear magnetic resonance spectroscopy in water) are very similar (47), thus showing the validity of the ligand-based design strategy.



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  		Figure 5. X-ray conformation of EMD121974 and ST1646. A, X-ray conformation of {alpha}vß3-bound EMD121974 [from 1L5G (26)]. B, conformation of ST1646 sampled during the 10-ns Monte Carlo/Stochastic Dynamics simulation after energy minimization.

 
Remarkably, ST1646 showed high structural preorganization for binding, assuming a preferred cyclopeptide conformation very similar to the X-ray-binding conformation of EMD121974. The average of the Arg/Asp Cß/Cß distance is 8.5 Å over the 10-ns Monte Carlo/Stochastic Dynamics simulation. An energy-minimized conformation of ST1646 obtained from a frame featuring the binding requirements and corresponding to the most populated geometry of the same trajectory (48) is shown in Fig. 5B. The root-mean-square deviation in the rigid superimposition between this conformation and the X-ray structure of bound EMD121974 is only 0.16 Å for the backbone atoms of the RGD sequence. Docking studies of ST1646 in the ligand-binding site of {alpha}vß3 integrin were attempted starting from the crystal structure of the {alpha}vß3 integrin-EMD121974 complex. The minimum energy conformation of ST1646 shown in Fig. 5B was docked in the ligand-bound integrin by superimposing the C{alpha} atoms of the cyclopentapeptide backbone and the guanidine and carboxy moieties of ST1646 to the corresponding elements of EMD121974. The resulting complex seemed to maintain almost the same ligand-receptor distances and interactions observed in the crystalline complex of EMD121974 with {alpha}vß3 (25, 39). The main interactions are between the positively charged arginine of ST1646 and the negatively charged side chains of Asp150 and Asp218 in the {alpha} subunit and between the anionic aspartic acid and the metal cation in the metal ion–dependent adhesion site region of the ß subunit (Fig. 6 ). Further stabilization occurs through hydrogen bonds between the NH group of the Asp residue and the carbonyl oxygen atom of Arg216 in the ß subunit as well as between the Asp side chain and the NH group of Asn215 in the ß subunit (Fig. 6). Moreover, the central Gly residue is in close contact with the integrin surface (data not shown).



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  		Figure 6. ST1646 docked in the crystal structure of the extracellular domain of {alpha}vß3 integrin. A, the {alpha} subunit is red and the ß subunit is blue (ribbon representation). Carbon atoms of integrin residues involved in the interactions are gray, those of the ligand peptide are green, nitrogens are blue, and oxygens are red. The two visible Mn2+ ions at metal ion–dependent adhesion site and adjacent to metal ion–dependent adhesion site are cyan and magenta, respectively. B, the ribbon representations of {alpha} and ß subunits have been omitted for a better view of the significant ST1646-integrin interactions. ST1646 integrin residues involved in the interactions and Mn2+ ions are shown.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Experimental evidence indicates that dual {alpha}vß3/{alpha}vß5 antagonists may represent a multitarget approach for the inhibition of tumor angiogenesis and growth. In this regard, integrin selectivity represents a major concern for the development of antiangiogenic RGD-related molecules. Indeed, when present in linear peptides, the RGD sequence can flexibly associate with different types of integrin receptors by changing its conformation, thus resulting in nonselective binding. Conformationally constrained cyclic RGD peptides can be synthesized with relatively higher specificity for different integrin receptors (47).

With the aim to identify highly selective dual {alpha}vß3/{alpha}vß5 integrin antagonists, we tested a series of compounds belonging to a RGD-containing cyclic pseudopeptide library (24) in which the RGD sequence was conformationally constrained by the presence of a bicyclic lactam. Among the compounds tested, we identified ST1646 as a potent and selective antagonist endowed with antiangiogenic activity in vitro and in vivo. Remarkably, the 7,5-fused bicyclic scaffold of ST1646 forced the cyclopeptide to assume a preferred conformation very similar to the X-ray conformation of {alpha}vß3-bound EMD121974. Binding of ST1646 might actually be enhanced by the high structural preorganization. The structure model built for the ST1646-{alpha}vß3 complex through docking studies confirmed that, similarly to the crystal structure of the EMD121974-{alpha}vß3 complex, the ligand seems to interact mainly through electrostatic forces in a rather shallow cleft and that essentially no hydrophobic interactions can be observed.

ST1646 prevents the binding of radiolabeled echistatin to immobilized {alpha}vß3 and {alpha}vß5 integrins with high affinity. In addition, it inhibits with high potency the adhesion of human endothelial cells to vitronectin and to a lesser extent to fibronectin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and EMD121974 and the natural ligand vitronectin. ST1646, like EMD121974, was instead unable to interact with {alpha}5ß1 integrin when compared with the natural ligand fibronectin. As stated above, {alpha}IIbß3 integrin plays a critical role in hemostasis by mediating platelet aggregation. Thus, inhibition of this receptor may cause unwanted bleeding. However, ST1646 is 600 times less active than echistatin in inhibiting thrombin receptor–activating peptide–induced platelet aggregation. Accordingly, no bleeding-related side effects were observed in animals treated with ST1646 delivered by i.p. injection or by s.c. minipumps during the in vivo angiogenesis and tumor assays (data not shown).

ST1646 inhibited the proliferation of endothelial cells in vitro. In addition, it prevented their capacity to invade a three-dimensional collagen gel and to form tube-like structures on Matrigel (data not shown). These effects occurred in the absence of a detectable cytotoxic activity of the compound. Integrin signaling events include the activation of nonreceptor tyrosine kinases, such as focal adhesion kinase, and the polymerization of the actin cytoskeleton, which in turn regulate cellular shape and motility (49). It must be pointed out that endothelial cells treated with ST1646 were viable and could be subcultured when the compound was removed from the cell culture medium, showing that ST1646 does not induce endothelial cell death in vitro.

In agreement with the in vitro observations, we found that ST1646 exerts a significant antiangiogenic activity in vivo. In the CAM assay, topical administration of ST1646 was able to fully prevent the angiogenic responses elicited by recombinant FGF2 or VEGF. This occurred in the absence of any effect on embryo viability, suggesting a low toxicity of the molecule in vivo. Previous observations have shown that neutralizing anti-{alpha}vß5 antibody inhibits VEGF-stimulated angiogenesis in the CAM assay, whereas anti-{alpha}vß3 antibody inhibits FGF2-induced angiogenesis (12). The capacity of ST1646 to inhibit the angiogenic activity of both growth factors seems to be in keeping with its dual {alpha}vß3/{alpha}vß5 antagonist activity. Interestingly, the systemic administration of ST1646 in mice was able to exert a significant antiangiogenic activity when neovascularization was triggered by VEGF-producing mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a Millipore chamber.

Integrin antagonists can inhibit tumor growth in vivo (18, 22, 42). This may result from their capacity to block angiogenesis and/or from a direct effect on tumor cells. Indeed, integrins are implicated in several steps that characterize the acquisition of the invasive and metastatic potential by tumor cells, such as intravasation, adhesion to the vessel wall, extravasation, infiltration, and proliferation into target tissue (50). Tumor cells express {alpha}vß3 in several malignancies and this expression correlates with tumor progression in melanoma, glioblastoma, and ovarian and breast cancer (42–46). In addition, tumor and stromal cells produce vitronectin, which is most abundant at sites of tumor invasion and neovascularization, including malignant brain tumors. Vitronectin production, in addition to supporting endothelial cell survival, may also be critical for enhancing the adhesion and invasion of tumor cells expressing {alpha}vß3 and {alpha}vß5 (51, 52). Thus, inhibition of {alpha}vß3 and {alpha}vß5 receptors, besides blocking tumor-induced angiogenesis, may affect several mechanisms involved in tumor progression. In this regard, it is noteworthy that {alpha}vß3 can modulate the activity of different proteolytic machineries in tumor cells and endothelial cells, including metalloprotease and plasminogen activator systems (53–55).

Here, we have shown that ST1646 inhibits the growth of tumor xenografts induced by s.c. injection of human ovarian carcinoma A2780 cells in nude mice. This was paralleled by a significant decrease in tumor vascularity. A2780 cells express both {alpha}vß3 and {alpha}vß5 integrins (data not shown). Thus, the action of ST1646 may result from a cooperative inhibition of both tumor cell proliferation and angiogenesis. Indeed, our data have shown that ST1646 exerts a potent inhibitory effect on cell adhesion and proliferation (data not shown) in a variety of {alpha}vß3/{alpha}vß5-expressing tumor cell lines with a potency similar or stronger that that exerted by c(RGDfV) or EMD121974. Interestingly, ST1646 does not affect the proliferation of nonadherent {alpha}vß3/{alpha}vß5-expressing NB4 and HL60 human leukemia cells, thus indicating its selectivity for anchorage-dependent cells (data not shown). These observations, together with its incapacity to interfere with platelet aggregation, provide further evidence on the possible therapeutic implications of ST1646 in the treatment of different types of cancers.

Antagonists of {alpha}v integrin, such as cilengitide (56–58), and the anti-{alpha}vß3 antibody Vitaxin (59) are in phase II clinical trials for cancer patients and several other integrin antagonists are currently under preclinical evaluation (60–62). ST1646 represents a selective dual {alpha}vß3/{alpha}vß5 antagonist with interesting biochemical and biological features to be tested in cancer therapy and, possibly, in other angiogenesis-dependent pathologies, including diabetic retinopathy, arthritis, and psoriasis.


   Acknowledgments
 
We thank Angelo Marconi, Patrizia Tobia, Silvio Zavatto, and Alessandra Rinaldi for excellent technical assistance and Consorzio Interuniversitario Lombardo per L'Elaborazione Automatica for computing facilities.


   Footnotes
 
Grant support: R&D Sigma-Tau S.p.A.; Consiglio Nazionale delle Ricerche; COFIN research programs; and Associazione Italiana per la Ricerca sul Cancro, Ministero dell'Istruzione dell'Università e della Ricerca Centro di Eccellenza "IDET," Firb 2001 and Cofin 2004, and Istituto Superiore di Sanitá Oncotechnological Program (M. Presta).

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

Note: L. Belvisi and T. Riccioni contributed equally to this work.

Received 4/21/05; revised 7/28/05; accepted 8/30/05.


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