Bioconjugate Chem. 2009, 20, 1611–1617
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Functionalized Cyclic RGD Peptidomimetics: Conjugable ligands for rvβ3 Receptor Imaging Daniela Arosio,*,† Leonardo Manzoni,*,† Elena M. V. Araldi,# Andrea Caprini,§ Eugenia Monferini,§ and Carlo Scolastico‡ CNR-Istituto di Scienze e Tecnologie Molecolari (ISTM), Via Fantoli 16/15, I-20138, Milan, Italy, Universita` di Milano, Dipartimento di Chimica Organica e Industriale, Via Venezian 21 and Centro Interdipartimentale Studi Biomolecolari e Applicazioni Industriali (CISI), Via Fantoli 16/15, I-20138, Milano, Italy, and CISI scrl, Via Fantoli 16/15, I-20138, Milano, Italy. Received April 8, 2009; Revised Manuscript Received May 29, 2009
The Rvβ3 integrin is an adhesion molecule involved in physiological and pathological angiogenesis as well as in tumor invasion and metastasis, and therefore, there is a strong interest in developing novel agents interacting with this molecule. We report the synthesis and characterization of fluorescent Rvβ3 integrin probes and their use to visualize integrin Rvβ3 expression on human normal and cancer cells. The fluorescent probes we describe here may be of use for noninvasive imaging of RVβ3 integrin expression also in ViVo.
INTRODUCTION Integrins are heterodimeric transmembrane proteins expressed at the cell surface and involved in cellular adhesion to the extracellular matrix (1). A deregulated expression and/ or function of integrins contributes to the progression of lethal pathologies including cancer (2, 3). Among the integrin superfamily, the Rvβ3 integrin has been the focus of considerable attention because of its expression in a restricted number of normal and malignant cell types such as platelets (4), osteoclasts (5), melanoma (6), glioblastoma (7), and osteosarcoma cells (8), and its elevated expression on activated endothelial cells during physiological and pathological angiogenesis (9). The Rvβ3 integrin interacts with specific proteins of the extracellular matrix such as fibronectin, vitronectin, von Willebrand factor, and fibrinogen through the tripeptide Arg-Gly-Asp (RGD) sequence (10, 11). Therefore, RGD-containing peptides targeting the Rvβ3 integrin are attractive for studying and managing diseases related to its overexpression. Many research groups have in fact focused on developing RGD peptides and analogous compounds for biomedical studies (12-16). Linear peptide sequences containing the RGD motif are known to bind integrins, and these have been used as celltargeting agents (17). Due to the low affinity and promiscuity of such linear peptides, however, their utility for selective cell targeting is limited. Peptide derivatives, such as cyclo(RGDfV) or Cilengitide have been used as tumor-homing agents due to their selectivity for Rvβ3 integrin (18, 19). Our group previously reported a library of cyclic RGD pentapeptide mimics based on 1-aza-2-oxobicyclo[X.3.0]alkane amino acids as dipeptide analogues (20, 21). The replacement of the D-Phe-Val dipeptide in the lead structure cyclo(RGDfV) with such azabicycloalkane scaffolds showing different reverseturn mimetic properties constrains the RGD sequence into * Corresponding authors. Tel.: +39 02 50320906, Fax: +39 02 50320945, E-mail:
[email protected], Tel.: +39 02 50320907, Fax: +39 02 50320945, E-mail:
[email protected]. † CNR-ISTM. ‡ CISI scrl. § University of Milano and CISI. # University of Milano and CISI, Doctorate School of Molecular Medicine.
Figure 1. Integrin inhibitors and scaffolds for their preparation.
different conformations and provides the required activity and selectivity for integrin antagonism. This library was found to contain specific high-affinity ligands for Rvβ3 integrin, which are currently under evaluation as very promising antiangiogenic drugs. One of the peptides tested, ST1646, displayed the highest affinity to Rvβ3, inhibiting echistatin binding to Rvβ3 with an IC50 of 3.7 ( 0.6 nM (Figure 1). The functionalization of such azabicycloalkane scaffolds with heteroalkyl substituents enabled us to conjugate different diagnostic or therapeutic entities. Our group has recently reported an efficient and simple synthesis of functionalized azabicycloalkane amino acids mimicking a homoSer-Pro dipeptide (22) and their use in the preparation of cyclic RGD compounds (5, Figure 1) (23). These mimics present a heteroalkyl side chain ending with a hydroxy group that can easily be converted into other suitable functional groups (i.e., azide, amine, etc.) or directly used for conjugations (23, 24). Such molecules are then attractive tools for the homing of drugs and biological tracers in cells highly expressing Rvβ3 integrin. To this end, we explored the possibility to use these compounds as “markers” of the Rvβ3 integrin expression in human cells. We selected the green fluorescent dye Fluorescein as a probe, because it is approved for human use in diagnostic techniques such as retinal angiography (25). Fluorescein is therefore advantageous because of its known safety profile in humans. Furthermore, the use of a RGD peptide-fluorescein conjugate in a fluorescent polarization assay has recently been reported (26), a technique that is often applied for high-throughput
10.1021/bc900155j CCC: $40.75 2009 American Chemical Society Published on Web 07/22/2009
1612 Bioconjugate Chem., Vol. 20, No. 8, 2009
screening in the drug discovery process. During the preparation of this paper, the work of the Li group has been published on a photostable fluorescent probe for imaging of tumor cells possessing integrin Rvβ3 (27). We report here the conjugation of cyclic RGD compounds containing the conformationally constrained homoSer-Pro dipeptide unit with the fluorescein probe, the study of their binding properties, and the biological effects of these compounds on human cell cultures. We demonstrate that such fluorescent compounds could be useful for Rvβ3 integrin imaging, thus suggesting their possible use in diagnostic techniques.
MATERIALS AND METHODS General. All chemicals and solvents were of reagent grade and were used without further purification. Solvents were dried by standard procedures and reactions requiring anhydrous conditions were performed under nitrogen or argon atmosphere. 1 H and 13C NMR spectra were recorded at 300 K on a Bruker AVANCE-400 and 600 MHz. Chemical shifts δ are expressed in ppm relative to internal Me4Si as standard. Mass spectra were obtained with an ESI apparatus Bruker Esquire 3000 plus. Column chromatography was carried out with the Biotage SP1 or SP4 systems using the Biotage cartridges. Compound 11. A solution of c-RGD-N3 7 (498 mg, 0.56 mmol) in MeOH (25 mL) containing a catalytic amount of Pd/C 10% was stirred overnight under hydrogen atmosphere. After reaction completion (monitored by TLC: CH2Cl2/MeOH 9:1), the mixture was filtered through a pad of Celite and washed with MeOH. The collected organic phase was evaporated under reduced pressure. The crude was dissolved in dry DMF (5 mL) and mixed, under N2 atmosphere and at room temperature, with a solution of spacer 10 (253 mg, 0.742 mmol), HATU (282 mg, 0.742 mmol), HOAt (94 mg, 0.689 mmol), and DIPEA (0.37 mL, 2.12 mmol) in dry DMF (5 mL). The reaction mixture was stirred overnight, and after reaction completion, the solvent was evaporated under reduced pressure. The pure product was obtained after reverse-phase column chromatography on a Biotage C18 25+M cartridge eluting with water/acetonitrile (95:5 f 10:90). Yield: 85% (white solid). 1H NMR (400 MHz, acetone-d6): δ 1.31 (s, 6H, C(CH3)2 Pmc), 1.36 (m, 1H, H-5), 1.43 (s, 9H, C(CH3)3), 1.43-1.55 (m, 2H, Hγ Arg, Hβ Arg), 1.55-1.61 (m, 2H, Hγ Arg, H-7), 1.84 (t, 2H, J ) 6.8 Hz, CH2CH2Ar Pmc), 1.99 (m, 1H, H-8), 2.07-2.25 (m, 4H, CH3 Pmc, Hβ Arg), 2.33-2.48 (m, 3H, H-5, H-7, H-8), 2.52-2.64 (m, 7H, Hβ Asp, 2 CH3 Pmc), 2.67 (t, 2H, J ) 6.6 Hz, CH2CH2Ar Pmc), 2.80 (m, 1H, H-4), 3.03 (dd, 1H, J ) 16.8 Hz, J ) 6.8 Hz, Hβ Asp), 3.12-3.29 (m, 4H, 2 Hδ Arg, NHCH2), 3.29-3.40 (m, 2H, NCH2CH2O), 3.45 (d, 1H, J ) 13.6 Hz, HR Gly), 3.57 (m, 2H, NCH2CH2O), 3.60-3.80 (m, 8H, 4 OCH2), 3.90 (s, 2H, OCH2CO), 4.16 (m, 1H, H-6), 4.19-4.36 (m, 3H, HR Gly, H-9, H-3), 4.50-4.62 (m, 2H, HR Arg, HR Asp), 5.09 (s, 2H, CH2Ph), 6.20-6.75 (m, 4H, (NH)2CdNH, NHCbz), 7.23 (m, 1H, NHCH2), 7.28-7.45 (m, 6H, 5 Aromatic protons, NH bicycle.), 7.51 (m, 1H, NH Arg), 7.60 (m, 1H, NH Gly), 8.40 (m, 1H, NH Asp). 13C NMR (100.6 MHz, acetone-d6): δ 173.2, 171.5, 171.2, 170.3, 170.2, 170.0, 169.8, 156.4, 152.9, 137.6, 135.6, 135.0, 134.6, 128.3, 127.8, 127.7, 123.1, 117.8, 80.0, 73.4, 70.8, 70.2, 70.1, 70.0, 69.6, 65.6, 62.5, 55.7, 53.5, 51.3, 42.7, 40.7, 40.5, 39.8, 36.3, 35.0, 33.3, 32.6, 30.1, 28.2, 27.4, 26.1, 25.9, 21.1, 17.9, 16.8, 11.4. MS (ESI+) m/z: 1183.7 (M+H)+. Compound 12. A solution of compound 11 (260 mg, 0.22 mmol) in MeOH (13 mL) containing a catalytic amount of Pd/C 10% was stirred for 3 h under hydrogen atmosphere. After reaction completion (monitored by TLC: CH2Cl2/MeOH 9:1), the mixture was filtered through a pad of Celite and washed with MeOH. The collected organic phase was evaporated under
Arosio et al.
reduced pressure and submitted to the following deprotection step without any further purification. A solution of protected compound (0.040 mmol) in TFA/ thioanisole/1,2-ethanedithiol/anisole 90:5:3:2 (2 mL) was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and then the crude was dissolved in H2O and washed with iPr2O. The aqueous phase was evaporated under reduced pressure and the crude purified by HPLC (Atlantis C18 OBD 5 µm 19 mm × 10 cm, flow 20 mL/min, gradient 100% A for 1 min, from 100% A to 85% A over 8 min, from 85% A to 0% A over 8 min. A H2O + 0.1% TFA, B CH3CN + 0.1% TFA). Yield: 70%. White foam. 1H NMR (400 MHz, D2O): δ 1.23 (m, 1H, H-5), 1.44-1.70 (m, 4H, 2 Hγ Arg, Hβ Arg, H-7), 1.85 (m, 1H, H-8), 2.04 (m, 1H, Hβ Arg), 2.36-2.50 (m, 3H, H-8, H-7, H-5), 2.71 (dd, 1H, J ) 7.1 Hz, J ) 17.0 Hz, Hβ Asp), 2.39 (m, 1H, H-4), 3.07 (dd, 1H, J ) 7.5 Hz, J ) 17.0 Hz, Hβ Asp), 3.10-3.24 (m, 6H, 2 Hδ Arg, NH2CH2CH2O, CH2NH), 3.48 (d, 1H, J ) 14.6 Hz, HR Gly), 3.63-3.75 (m, 10H, 4 CH2O, NH2CH2CH2O), 3.90-4.06 (m, 3H, H-6, OCH2CO), 4.20-4.30 (m, 2H, H-9, HR Gly), 4.38 (m, 1H, H-3), 4.48-4.60 (m. 2H, HR Asp, HR Arg). 13C NMR (100.6 MHz, D2O): 174.8, 174.5, 174.1, 173.1, 172.6, 171.6, 170.1, 156.8, 70.3, 69.7, 69.6, 69.5, 69.4, 66.4, 62.2, 56.0, 53.1, 51.7, 51.4, 42.6, 40.5, 40.2, 39.2, 35.7, 33.3, 32.9, 32.6, 27.1, 24.5. MS (ESI+) m/z: 727.1 (M+H)+, 364.0 (M+2H)2+. Compound 8. A solution of protected compound 7 (324 mg, 0.365 mmol) in TFA/thioanisole/1,2-ethanedithiol/anisole 90: 5:3:2 (15 mL) was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and then the crude was dissolved in H2O and washed with iPr2O. The aqueous phase was evaporated under reduced pressure. The crude was purified by HPLC (Atlantis C18 OBD 5 µm 19 mm × 10 cm, flow 20 mL/min, gradient from 5% A to 15% A over 10 min, A CH3CN + 0.1% HCOOH, B H2O + 0.1% HCOOH). Yield: 97%. White foam. 1H NMR (400 MHz, D2O): δ 1.32 (m, 1H, H-5), 1.43-1.61 (m, 3H, 2 Hγ Arg, Hβ Arg), 1.66 (m, 1H, H-7), 1.83 (m, 1H, H-8), 2.03 (m, 1H, Hβ Arg), 2.34-2.47 (m, 3H, H-8, H-7, H-5), 2.69 (dd, 1H, J ) 7.1 Hz, J ) 16.9 Hz, Hβ Asp), 2.78 (m, 1H, H-4), 3.05 (dd, 1H, J ) 7.5 Hz, J ) 16.9 Hz, Hβ Asp), 3.09-3.20 (m, 2H, 2 Hδ Arg), 3.32-3.42 (m, 2H, CH2N3), 3.47 (d, 1H, J ) 14.7 Hz, HR Gly), 3.96 (m, 1H, H-6), 4.20-4.32 (m, 3H, HR Gly, H-9, H-3), 4.48-4.58 (m. 2H, HR Asp, HR Arg). 13C NMR (100.6 MHz, D2O): 175.0, 174.8, 174.1, 173.0, 171.6, 170.6, 156.7, 62.2, 56.0, 52.8, 52.0, 51.6, 51.3, 42.4, 40.4, 35.5, 33.2, 32.7, 30.1, 27.0, 24.4. MS (ESI+) m/z: 564.1 (M+H)+. Compound 13. FITC (3.0 mg, 0.0075 mmol) dissolved in DMSO (0.4 mL) was mixed with compound 12 (6.0 mg, 0.0063 mmol) dissolved in sodium borate buffer pH ) 9 (0.4 mL) in the dark and at room temperature. After stirring for ca. 18 h, the reaction was complete. The solvent was removed and the crude was purified by HPLC (Ascentis RPAMIDE 150 × 21.1 mm column, guard cartridge, RPAMIDE 20 × 4 mm; 12 mL/ min flow; gradient 100% A for 2 min, from 100% A to 70% A over 3 min, from 70% A to 50% A over 20 min, from 50% A to 20% A over 5 min, then from 20% A to 0% A over 2 min A, H2O + 0.2% TFA; B, CH3CN + 0.2% TFA). Yield: 70%. Yellow foam. 1H NMR (600 MHz, D2O): δ 1.00 (m, 1H, H-5), 1.24-1.50 (m, 4H, 2 Hγ Arg, Hβ Arg, H-7), 1.65 (m, 1H, H-8), 1.87 (m, 1H, Hβ Arg), 2.13-2.28 (m, 3H, H-8, H-7, H-5), 2.48-2.62 (m, 2H, Hβ Asp, H-4), 2.77-2.91 (m, 3H, Hβ Asp, CH2NH), 2.91-3.02 (m, 2H, 2 Hδ Arg), 3.25 (d, 1H, J ) 15.6 Hz, HR Gly), 3.42-3.69 (m, 12H, 5 CH2O, NH2CH2CH2O), 3.69-3.83 (m, 3H, OCH2CO, H-6), 4.03-4.11 (m, 2H, H-9, HR Gly), 4.18 (d, 1H, J ) 8.4 Hz, H-3), 4.28 (m, 1H, HR Asp), 4.36 (m. 1H, HR Arg), 6.63. (m, 2H, fluorescein protons), 6.77 (m, 2H, fluorescein protons), 8.87-7.16 (m, 3H, fluorescein
Rvβ3 Integrin Receptor Imaging
protons), 7.56 (m, 1H, fluorescein proton), 7.95 (m, 1H, fluorescein proton). 13C NMR HETCOR (600 MHz, D2O): 130.7, 129.9, 122.8, 116.2, 102.5, 70.2, 69.4, 69.1, 61.8, 55.7, 52.7, 51.4, 51.3, 44.0, 42.1, 40.2, 39.9, 35.5, 33.0, 32.5, 29.8, 26.8, 24.3. MS (ESI+) m/z: 1116.4 (M+H)+, 558.8 (M+2H)2+. General Procedure for Click Reaction. To a solution of compound 8 (0.02 mmol) and the opportune alkynyl derivative (14 or 1-hexyne) (0.02 mmol) in H2O/t-BuOH 1:1 (500 µL.), a 0.9 M solution of sodium ascorbate (9 µL, 0.008 mmol, 0.4 mol equiv) and a 0.3 M solution of Cu(OAc)2 (13 µL, 0.004 mmol, 0.2 mol eq) were added. The reaction was stirred at r.t. for ca. 18 h. After reaction completion, compounds 15 or 16 were purified by HPLC (Waters X-Terra PR 18 19 × 50 mm, 5 µm column; 3.5 mL/min flow; gradient from 20% B to 35% B over 11 min, then washed 60% B for 1 min; A, 97% H2O/3% CH3CN + 0.1% TFA; B, CH3CN + 0.1% TFA). Compound 15. Yield: 60%. White foam. 1H NMR (600 MHz, D2O): δ 1.10 (m, 1H, H-5), 1.41 (m, 2H, Hγ Arg), 1.44 (m, 1H, H-7), 1.46 (m, 1H Hβ Arg), 1.49 (m, 2H, CH2 spacer), 1.58 (m, 2H, CH2 spacer), 1.69 (m, 2H, CH2 spacer), 1.72 (m, 1H, H-8), 1.85 (m, 2H, CH2 spacer), 1.95 (m, 1H, Hβ Arg), 2.19 (m, 1H, H-5), 2.23 (m, 1H, H-7), 2.31 (m, 1H, H-8), 2.56 (m, 1H, Hβ Asp), 2.91 (m, 1H, Hβ Asp), 3.03 (m, 1H, Hδ Arg), 3.07 (m, 1H, H-4), 3.09 (m, 1H, Hδ Arg), 3.24-3.55 (m, 8H, OCH2 spacer), 3.29 (m, 1H, NHCH spacer), 3.36 (m, 1H, HR Gly), 3.44 (m, 1H, NHCH spacer), 3.84 (m, 1H, H6), 3.91 (m, 1H, COCH2O), 3.99 (m, 1H, COCH2O), 3.95 (m, 1H, HCHN), 4.07 (m, 1H, H-9), 4.11 (m, 1H, NHCH spacer), 4.15 (m, 1H, HR Gly), 4.28 (m, 1H, H-3), 4.30 (m, 1H, HR Asp), 4.32 (m, 2H, NCH2d), 4.46 (m, 1H, HR Arg). 13C NMR HETCOR (400 MHz, DMSO-d6): 69.7, 68.9, 62.0, 55.5, 52.6, 51.1, 42.2, 40.1, 37.9, 35.9, 33.8, 33.3, 32.5, 32.2, 29.9, 28.0, 27.0, 24.3. δ MS (ESI+) m/z: 1295.6 (M+H)+, 648.3 (M+2H)2+. Compound 16. Yield: 80%. White foam. 1H NMR (400 MHz, D2O): δ 0.82 (m, 3H, CH3CH2CH2CH2Cd), 1.17-1.33 (m, 3H, H-5, CH3CH2CH2CH2Cd), 1.41-1.66 (m, 6H, 2 Hγ Arg, Hβ Arg, CH3CH2CH2CH2Cd, H-7), 1.82 (m, 1H, H-8), 2.00 (m, 1H, Hβ Arg), 2.32-2.46 (m, 3H, H-8, H-7, H-5), 2.60-2.71 (m, 3H, Hβ Asp, CH3CH2CH2CH2Cd), 2.99 (m, 1H, Hβ Asp), 3.06-3.19 (m, 2H, 2 Hδ Arg), 3.25 (m, 1H, H-4), 3.46 (m, 1H, HR Gly), 3.99 (m, 1H, H-6), 4.10-4.30 (m, 3H, H-9, HCHN, HR Gly), 4.31-4.43 (m, 3H, HR Asp, HCHN, H-3), 4.54 (m. 1H, HR Arg), 7.80 (s, 1H, H triazole). 13C NMR (100.6 MHz, D2O): 174.6, 174.0, 173.0, 171.6, 170.8, 156.7, 148.1, 124.4, 62.2, 55.8, 52.9, 51.8, 51.6, 51.2, 42.4, 40.4, 36.1, 33.2, 32.7, 32.2, 30.6, 30.0, 27.0, 24.4, 23.7, 21.4, 13.0. MS (ESI+) m/z: 646.2 (M+H)+. Solid-Phase Receptor-Binding Assay. Purified Rvβ3 and Rvβ5 receptors (Chemicon International, Inc., Temecula, CA) were diluted to 0.5 µg/mL in coating buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L MnCl2, 2 mmol/L CaCl2, and 1 mmol/L MgCl2. An aliquot of diluted receptors (100 µL/well) was added to 96-well microtiter plates (NUNC MW 96F MEDISORP STRAIGHT) and incubated overnight at 4 °C. The plates were then incubated with blocking solution (coating buffer plus 1% bovine serum albumin (Sigma) for additional 2 h at room temperature to block nonspecific binding followed by 3 h incubation at room temperature with various concentrations (10-10-10-5 M) of test compounds in the presence of 1 µg/mL Vitronectin biotinylated using EZ-Link Sulfo-NHS-Biotinylation kit (Pierce, Rockford, IL). After washing, the plates were incubated for 1 h at room temperature with streptavidin-biotinylated peroxidase complex (Amersham Biosciences, Uppsala, Sweden) followed by 30 min incubation with 100 µL Substrate Reagent Solution (R&D Systems, Minneapolis, MN) before stopping the reaction by
Bioconjugate Chem., Vol. 20, No. 8, 2009 1613
addition of 50 µL of 1 N H2SO4. Absorbance at 415 nm was read in a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.). Each data point is the result of the average of triplicate wells and was analyzed by nonlinear regression analysis with the Prism GraphPad program. Reagents for Cellular Experiments. All reagents were purchased from Sigma-Aldrich (unless otherwise indicated). For all our tests, phosphate buffer saline supplemented with calcium and magnesium (Euroclone) was used. Cell Lines and Culture. Human umbilical vein vascular endothelial cells (HUVECs) were obtained from Promocell GmbH (cat. no C-12200; Heidelberg, Germany) and were routinely cultured on tissue flasks (BD Biosciences, Franklin Lakes, NJ, USA) coated with 0.1% gelatin (Gibco) in medium M199 supplemented with 20% fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL), 100 µg/mL porcine heparin, and 10 ng/mL endothelial cells growth factor (ECGF). HUVEC cells were used until 5-6 passages. ECV-304 human bladder carcinoma cells were kindly provided by Prof. M. L. Villa (University of Milan, Italy). ECV304 cells were grown on tissue flasks in medium M199 supplemented with 10% FCS, 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). MDA-MB-231 human breast adenocarcinoma cells, T98G human glioblastoma multiforme cells, and PC3 human prostate cancer cells were purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia (cat. nos BS TCL 223, BS TCL 175, and BS TCL 197, respectively; Brescia, Italy) and were grown on tissue flasks in medium RPMI supplemented with 10% FCS, 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). Caki-1 human renal clear cell carcinoma cells were purchased from the American type Culture Collection (ATCC) (cat. no HTB-46) and were grown on tissue flasks in medium RPMI supplemented with 10% FCS, 2 mM Lglutamine, penicillin (100 U/mL), and streptomycin (100 µg/ mL). All cell lines were maintained at 37 °C in an atmosphere of 95% air and 5% CO2. Immunofluorescence on Adherent Cells. For Rvβ3 staining, cells were washed in PBS and fixed in 4% paraformaldehyde (Fluka) for 10 min at room temperature. Cells were then washed and permeabilized with 0.1% Triton-X100 (Fluka) for 3 min at 4 °C. Blocking was performed in PBS with 5% bovine serum albumin (BSA) for 1 h at room temperature. We incubated cells with anti-RVβ3 antibody (clone LM609, Chemicon, Temecula, California) diluted in PBS + 5% BSA at a final concentration of 10 µg/mL, overnight at 4 °C. Cells were then extensively washed in PBS + 5% BSA and incubated with 3.75 µg/mL donkey-antimouse cy3-conjugated secondary antibody (Jackson Immunoresearch Laboratories Inc., UK) diluted in PBS + 5% BSA, for 1 h at RT in the dark. Nuclei were counterstained for 5 min at room temperature with 0.25 µg/mL 4′,6-diamidino-2phenylindole (DAPI) diluted in PBS. Cells were finally mounted with Vectashield mounting medium and visualized with Zeiss Axio Observer A1 microscope (Carl Zeiss Inc., Chester, VA, USA). All fuorescence microscopy scans were acquired with Zeiss AxioCam MRm (Carl Zeiss Inc., Chester, VA, USA). Treatment of Cells with Compounds and Analysis. Cells (1.6 × 104/cm2) were seeded and allowed to adhere overnight in complete medium on glass slides. Cells were treated 4 h at 37 °C with selected compounds diluted in fresh medium supplemented with 0.1% FCS. After incubation, cells were extensively washed in PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Nuclei were then counterstained with 0.25 µg/mL DAPI diluted in PBS for 5 min at room
1614 Bioconjugate Chem., Vol. 20, No. 8, 2009
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Scheme 1. Synthesis of the RGD Derivatives 8 and 12a
a Reagents: i. H2, Pd/C, MeOH; ii. HATU, HOAt, DIPEA, DMF, 85% over two steps; iii. TFA, thioanisole, 1,2-ethanedithiol, anisole, 90:5:3:2, (8) 97%, (12) 70% over two steps.
Scheme 2. FITC Conjugation
temperature. Cells were finally mounted and visualized as previously described. Experiments were repeated in triplicate.
RESULTS Chemistry. Among a small collection of functionalized cyclic RGD (5, Figure 1) (23), we chose compound 5a (Scheme 1) that showed the highest affinity for Rvβ3 integrin in an in Vitro binding assay (IC50 of 53.7 ( 17.3 nM) as a good candidate for fluorescein conjugation. The fluorescent dye can be directly conjugated to the suitably fuctionalized RGD derivative, but, in order to avoid a decrease in the affinity due to steric hindrance, a spacer was introduced. We decided to use two different synthetic strategies for conjugation. The first one involves the functionalization of the RGD cyclopeptide with a short poly(ethylene glycol) spacer armed with a terminal amine that can be reacted with fluoresceine isothiocyanate (FITC). The second approach involves the preparation of a fluorescein spacer derivative that can be directly
reacted with an azide bearing RGD peptide through the coppercatalyzed click reaction (28, 29). The hydroxyl group of the pseudopentapeptide 6 was easily transformed into the azide using a reported procedure (23). The standard hydrogenation procedure was used to reduce the azide into amine 9. After the coupling with spacer 10, carried out using standard coupling procedure (HATU, HOAt, DIPEA), the Cbz was removed by hydrogenation, while the side-chain protecting groups (Pmc and tBu) were cleaved by TFA in the presence of ion scavengers. Compounds 8 and 12 were obtained, after HPLC purification, in good yields. Fluoresceine 5(6)-isothiocyanate (FITC) was used for direct conjugation to the amine derivative 12. The reaction was performed dissolving compound 12 in sodium borate buffer (pH ) 9) and adding FITC dissolved in DMSO. The obtained compound 13 was purified by HPLC. The fluorescein-spacer derivative 14 was prepared by adding commercially available 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester to a basic solution of the spacer (24). The 1,3dipolar cycloaddition between compounds 8 and 14 was performed using Cu(OAc)2 and Na ascorbate as catalysts in t-BuOH/H2O 1:1 (Scheme 3). The reaction proceeded overnight at room temperature, and the desired product 15 was isolated after HPLC purification in good yield. Compound 16 was synthesized in an analogous way to that reported above in order to evaluate the influence of the triazole moiety on the biological activity. Receptor Binding Assay. The derivatives 13, 15, and 16 were examined in Vitro for their abilities to inhibit the binding of biotinylated vitronectin to the isolated, immobilized Rvβ3 integrin (Table 1). Affinities of commercially available compound c(RGDfV) and our compound ST1646 were determined as references, in the same assays. The functionalized cyclic pentapeptide 5a displayed a lower binding affinity toward Rvβ3 integrin when compared to the low nanomolar reference ligand ST1646. Nevertheless, the affinities in the nanomolar range toward Rvβ3 integrin suggested that the functionalization of the scaffold does not influence the binding activity and it could be a suitable platform for targeted imaging. In order to evaluate the influence of a triazole moiety near the RGD sequence, we have
Rvβ3 Integrin Receptor Imaging
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Scheme 3. Synthetic Pathway for Conjugate RGD Derivative with Click Reaction
Table 1. Compound-Mediated Inhibition of Vitronectin Binding to rvβ3 Integrina entry
compound
IC50 (nM) ( SD for Rvβ3
1 2 3 4 5 6 7
Vitronectin c(RGDfV) ST1646 5a 13 15 16
47.1 ( 10.0 3.2 ( 1.3 1.0 ( 0.5 53.7 ( 17.3 142 ( 6 207 ( 85 465 ( 297
a IC50 values were calculated as the concentration of compound required for 50% inhibition of biotinylated vitronectin binding as estimated by Prism GraphPad program. All values are the mean ((standard deviation) of triplicate determinations.
synthesized compound 16, which showed a slightly lower affinity to Rvβ3 compared with the compound 5a, probably due the increased steric hindrance of the triazole. However, the affinity in the nanomolar range toward integrin receptors suggested that the “click reaction” could be a suitable method for the conjugation of molecules of biological interest. Compound 13 compared with 15 showed a slightly better binding affinity for the receptor; this could be due to the presence of a less hindered amide bond instead of triazole and also to a different length of the spacer between the RGD sequence and the fluoresceine moiety. Fluorescein Detection in Human Cell Lines. The above results demonstrate that compounds 13 and 15 have a high affinity for the Rvβ3 integrin; this prompted us to test their possible use as tracers of cells highly expressing RVβ3 integrin. For this purpose, we tested each compound on cells highly expressing Rvβ3 integrin, namely, human umbilical vein endothelial cells (HUVECs), and on a panel of cell lines deriving from solid tumors in which Rvβ3 overexpression is reported (ECV304 bladder cancer cells, T98G glioblastoma multiforme cells, PC3 prostate cancer cells, Caki-1 renal clear cell carcinoma
cells). We included also a cell line derived from human breast adenocarcinoma (MDA-MB-231 cells) as a control, because this cell line is known to express very low amounts of Rvβ3 integrin (31). We first verified and confirmed by immunofluorescence that HUVECs and all tumor-derived cells highly expressed Rvβ3 integrin, apart from MDA-MB-231 cells with very little expressed Rvβ3 integrin (Figure 2). All cell lines were exposed to scalar concentrations of compounds 13 and 15 for different incubation times. We found that a 4 h incubation with 10 µM of both compounds determined a good detection threshold for the fluorescein signal in vascular endothelial cells and all tumor-derived cells expressing Rvβ3 integrin (Figure 3). In particular, compound 13 gave a signal stronger than that of compound 15. Each compound displayed a fluorescence distributed both at the cell surface and in putative cytosolic vesicles, suggesting that both compounds were internalized upon binding with Rvβ3 integrin. Moreover, the morphological analysis of cells showed no signs of toxicity or stress after 4 or 24 h incubation with 10 µM of either compound 13 or 15 (data not shown). Notably, breast adenocarcinoma cells subjected to the treatment previously described did not show any sign of fluorescence (Figure 3), suggesting that both compounds were highly specific for Rvβ3 integrin.
DISCUSSION The diagnostic approaches based on modern imaging techniques are important tools to prevent and cure lethal pathologies such as cardiovascular diseases and cancer. In this paper, we describe the characterization, the synthesis, and the biological activity of RGD-containing peptides conjugated with the fluorescent probe fluorescein as tracers for Rvβ3 integrin expression on cells. Using the solid-phase receptor binding assay, we demonstrated that compounds 13 and 15 were able to efficiently inhibit the binding of biotinylated vitronectin to immobilized Rvβ3
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Figure 2. Expression of the Rvβ3 integrin in the cell panel. HUVEC (A), ECV-304 (B), T98G (C), PC3 (D), Caki-1 (E), and MDA-MB-231 (F) cells were subjected to immunofluorescence staining for Rvβ3 integrin (red). Nuclei were counterstained with DAPI (blue). (A,B,C,D,E) HUVEC, ECV-304, T98G, PC3, and Caki-1 cells showed high amounts of the Rvβ3 integrin, which was predominantly clustered at focal contacts (arrows). (F) MDA-MB-231 cells expressed lower amounts of Rvβ3 integrin, which was mainly diffused on the cell surface. Original magnification 63×.
several human cell lines derived from solid tumors (bladder cancer, glioblastoma multiforme, prostate cancer, and renal clear cell carcinoma). According to the results of the solid-phase receptor binding assay, compound 13 gave a fluorescent signal stronger than that of compound 15 on cells. In both cases, the pattern of the detected fluorescence also suggested that the two compounds were dynamically internalized by cells. Notably, cancer cells expressing very faint levels of Rvβ3 integrin (such as breast carcinoma cells) could not be visualized with these compounds. Moreover, treated cells retained their viability, making both molecules attractive as tools for the imaging of both angiogenic vessels and tumor masses specifically and highly expressing Rvβ3 integrin. In summary, the described fluorescent probes are able to efficiently target and “visualize” the Rvβ3 integrin on human cells in Vitro, suggesting their possible future use for a noninvasive imaging of the Rvβ3 integrin expression also in ViVo.
ACKNOWLEDGMENT The authors thank CNR and MUR (FIRB RBNE03LF7X and PRIN 2006030449 research projects) for financial support. Elena M.V. Araldi was supported by a fellowship of the Doctorate School of Molecular Medicine, University of Milan.
LITERATURE CITED
Figure 3. HUVEC, ECV-304, T98G, Caki-1, PC3, and MDA-MB231 cells were incubated 4 h with treatment medium alone (left column), 10 µM compound 15 (middle column, green), or 13 (right column, green). Nuclei were counterstained with DAPI (blue). HUVEC, ECV304, T98G, Caki-1, and PC3 cells were positively stained after treatment with either compound 13 or 15, even if the signal provided by compound 13 is stronger. In particular, the signal was highly detectable and located both at the cell surface and in putative cytosolic vescicles (arrows). Differently, MDA-MB-231 cells did not display any staining after treatment with either compound 13 or compound 15. All three cell lines incubated with treatment medium alone displayed no fluorescence at all. Original magnification 63×.
integrin. Although we found that the binding affinity of both compounds for this integrin was less potent than the low nanomolar values of ST1646, their affinities in the nanomolar range prompted us to explore the possibility to use them as suitable platforms for Rvβ3-targeted imaging. Therefore, we generated compounds 13 and 15 conjugated with the fluorescent probe fluorescein. We demonstrated that, after in Vitro treatment with these two conjugated compounds, the fluorescein probe was detectable in a panel of cell lines highly expressing the Rvβ3 integrin, namely, primary endothelial cells (HUVEC) and
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