Binding of Small Mono- and Oligomeric Integrin Ligands to Membrane

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Anal. Chem. 2006, 78, 4524-4533

Binding of Small Mono- and Oligomeric Integrin Ligands to Membrane-Embedded Integrins Monitored by Surface Plasmon-Enhanced Fluorescence Spectroscopy Daniela Lo 1 ssner,† Horst Kessler,‡ Georgette Thumshirn,‡ Claudia Dahmen,‡ Birgit Wiltschi,§ Motomu Tanaka,| Wolfgang Knoll,⊥ Eva-Kathrin Sinner,⊥ and Ute Reuning*,†

Klinische Forschergruppe der Frauenklinik der Technischen Universita¨t Mu¨nchen (TUM), 81675 Mu¨nchen, Germany, Institut fu¨r Organische Chemie & Biochemie (TUM), 85748 Garching, Germany, Max-Planck Institut fu¨r Biochemie, 82152 Martinsried, Germany, Institut fu¨r Physikalische Chemie, University of Heidelberg, 85748 Garching, Germany, and Max-Planck Institut fu¨r Polymerforschung, 55128 Mainz, Germany

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins rvβ3 and rvβ5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward rvβ3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with rvβ3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins. The study of the interactions of transmembrane receptors with their respective ligands is a prerequisite for the understanding of * Corresponding author. Tel.: +49-89-4140 7407. Fax: +49-89-4140 7410. E-mail: [email protected]. † Klinische Forschergruppe der Frauenklinik der Technischen Universita¨t Mu ¨ nchen. ‡ Institut fu ¨ r Organische Chemie & Biochemie. § Max-Planck Institut fu ¨ r Biochemie. | University of Heidelberg. ⊥ Max-Planck Institut fu ¨ r Polymerforschung.

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complex biological pathways involving cell membranes. Knowledge of the nature and requirements for receptor/ligand associations will also further facilitate the design and development of synthetic ligands/antagonists. One important group of membrane-spanning cell surface receptors is the integrin superfamily. Integrins by mediating cell adhesion to the extracellular matrix (ECM) and transducing intracellular signals are crucially implicated in many physiological as well as pathophysiological events and thus represent important therapeutical target structures. Integrins are composed of noncovalently associated R- and β-subunits, their heterodimeric composition determining specificity and selectivity toward certain ECM ligands. About half of all integrins bind to ECM proteins via the sequence motif Arg-Gly-Asp (RGD).1,2 Among those is the major vitronectin receptor Rvβ3, which is involved in tumor metastasis and tumor-induced angiogenesis.1-3 Based on accumulated knowledge of the impact of the structure and conformation of the RGD moiety on binding to different integrin subtypes, RGD-based peptides have been developed within the past decade. In fact, the first synthetic, highly active Rvβ3-selective cyclic peptide containing a conformationally restrained RGD sequence, cyclo(-RGDfV-), was developed and synthesized in our laboratories.4-6 Systematic derivatization of this peptide resulted in the N-alkylated peptide c(-RGDf[NMe]V-) (Cilengitide),7 which has already entered clinical phase II studies as an angiogenesis inhibitor.8-10 Meanwhile, multimeric cyclic RGD (1) Felding-Habermann, B. Clin. Exp. Metastasis 2003, 20, 203-213. (2) Arnaout, M. A.; Mahalingam, B.; Xiong, J.-P. Annu. Rev. Cell Dev. Biol. 2005, 21, 381-410. (3) Stupack, D. G.; Cheresh, D. A. Curr. Top. Dev. Biol. 2004, 64, 207-238. (4) Aumailley, M.; Gurrath, M.; Mu ¨ ller, G.; Calvete, J.; Timpl, R.; Kessler, H. FEBS Lett. 1991, 291, 50-54. (5) Pfaff, M.; Tangemann, K.; Mu ¨ ller, B.; Gurrath, M.; Mu ¨ ller, G.; Kessler, H.; Timpl, R.; Engel, J. J. Biol. Chem. 1994, 269, 20233-20238. (6) Haubner, R.; Gratias, R.; Diefenbach, B.; Goodman, S. L.; Jonczyk, A.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 7461-7472. (7) Dechantsreiter, M. A.; Planker, E.; Matha¨, B.; Lohof, E.; Ho¨lzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. J. Med. Chem. 1999, 42, 30333040. (8) Eskens, F. A.; Dumez, H.; Hoekstra, R.; Perschl, A.; Brindley, C.; Bottcher, S.; Wynendaele, W.; Drevs, J.; Verweij, J.; van Oosterom, A. T. Eur. J. Cancer 2003, 39, 917-926. 10.1021/ac052078+ CCC: $33.50

© 2006 American Chemical Society Published on Web 05/17/2006

peptides have been developed with the goal to achieve enhanced integrin targeting.10-12 However, so far, the influence of spatial arrangement and separation of RGD moieties for optimal cellsurface integrin targeting is not well understood. Thus, cyclic RGD peptides linked to aminohexanoic acid (Ahx) spacer of different lengths were synthesized and chemically assembled to multimers, which may allow varying flexibility and presentation of several integrin-binding RGD motifs at a time. Since spacers may affect the biological activity of compounds, in addition, RGD monomers with gradually increasing Ahx spacer lengths were synthesized and tested for integrin-binding capacity.10,11,13 Although the cyclic peptides are metabolically stable, there is interest in improved bioavailability. Thus, we and others have developed selective nonpeptidic Rvβ3 antagonists by transforming the structure of the lead peptide c(-RGDfV-) into a peptidomimetic.14-18 To determine binding specificity and selectivity of natural as well as synthetic ligands to transmembrane receptors, sensitive and reproducible in vitro binding assays are needed. In the case of integrins, for presentation in their native conformation, in vitro cell culture models have been widely used as a source of integrins. However, the cell assay-based experimental results are often difficult to interpret, since integrins are highly abundant and ubiquitous on natural cell membranes, displaying frequently overlapping ECM recognition epitopes. Consequently, in addition, cell-free binding tests have been employed in recent years, in which, for example, purified integrins are immobilized onto plastic devices implying, however, the shortcoming that they are presented in a nonordered fashion without preserving the native conformation.19 Moreover, this assay format neglects membrane insertion of integrins, which may crucially determine their ECM ligand-binding properties.20 To solve these intrinsic experimental drawbacks and to allow for reliable screening of selective receptor/ligand interactions, we recently developed an alternative cellfree binding test. For this, we employed integrin-functionalized artificial phospholipid bilayers, which are peptide-tethered to biosensor surfaces in order to perform surface plasmon resonance (9) Raguse, J. D.; Gath, H. J.; Bier, J.; Riess, H.; Oettle, H. Oral Oncol. 2004, 40, 228-230. (10) Arndt, T.; Arndt, U.; Reuning, U.; Kessler, H. Integrins in angiogenesis: implications for tumor therapy. In Cancer Therapy: Molecular Targets in Tumor-Host Interactions; Weber, G. F., Ed.; Horizon Bioscience: Norfolk, U.K., 2005; pp 93-141. (11) Thumshirn, G.; Hersel, U.; Goodman, S. L.; Kessler, H. Chem. Eur. J. 2003, 9, 2717-2725. (12) Poethko, T.; Schottelius, M.; Thumshirn, G.; Herz, M.; Haubner, R.; Henriksen, G.; Kessler, H.; Schwaiger, M.; Wester, H.-J. Radiochim. Acta 2004, 92, 317-327. (13) Hersel, U.; Dahmen, C.; Kessler, H. Biomaterials 2003, 24, 4385-4415. (14) Gibson, C.; Goodman, S. L.; Hahn, D.; Ho ¨lzemann, G.; Kessler, H. J. Org. Chem. 1999, 64, 7388-7394. (15) Duggan, M. E.; Duong, L. T.; Fisher, J. E.; Hamill, T. G.; Hoffman, W. F.; Huff, J. R.; Ihle, N. C.; Leu, C.-T.; Nagy, R. M.; Perkins, J. J.; Rodan, S. B.; Wesolowski, G.; Whitman, D. B.; Zartman, A. E.; Rodan, G. A.; Hartman, G. D. J. Med. Chem. 2000, 43, 3736-3745. (16) Sulyok, G. A. G.; Gibson, C.; Goodman, S. L.; Ho ¨lzemann, G.; Wiesner, M.; Kessler, H. J. Med. Chem. 2001, 44, 1938-1950. (17) Gibson, C.; Sulyok, G. A. G.; Hahn, D.; Goodman, S. L.; Ho¨lzemann, G.; Kessler, H. Angew. Chem., Int. Ed. 2001, 40, 165-169. (18) Yasuda, N.; Hsiao, Y.; Jensen, M. S.; Rivera, N. R.; Yang, C.; Wells, K. M.; Yau, J.; Palucki, M.; Tan, L.; Dormer, P. G.; Volante, R. P.; Hughes, D. L.; Reider, P. J. J. Org. Chem. 2004, 69, 1959-1966. (19) Goodman, S. L.; Ho ¨lzemann, G.; Sulyok, G. A. G.; Kessler, H. J. Med. Chem. 2002, 45, 1045-1051. (20) Shimaoka, M.; Takagi, J.; Springer T. A. Annu. Rev. Biophys. Biomol. Struct. 2002, 31, 485-516.

spectroscopy (SPS).21-26 At resonant plasmon excitation, the high electromagnetic field intensity can be used to strongly enhance the emission from fluorescence-labeled, surface-bound molecules, thus allowing performance of surface plasmon-enhanced fluorescence spectroscopy (SPFS). Herewith, sensitive recording of the association of even very small ligands is enabled, which is not possible using conventional SPS. In the present study, we used SPFS as proof of principle in order to measure binding of a series of synthetic RGD-containing peptides and an RGD-based peptidomimetic to membrane-embedded integrins Rvβ3 and Rvβ5, and, as control, to the platelet integrin RIIbβ3. In biological assays, the capacity of the synthetic ligands to interfere with Rvβ3-mediated human ovarian cancer cell adhesion to vitronectin was evaluated in in vitro cell adhesion tests. EXPERIMENTAL SECTION Materials. Purified integrins Rvβ3, Rvβ5 as well as monoclonal antibodies (MAB) directed to Rvβ3 (clone LM609) and Rvβ5 (clone P1F6), respectively, were purchased from Chemicon Inc. (Temecula, CA). Platelet integrin RIIbβ3 was purified by Tanaka27 (Institute of Biophysics, Technische Universita¨t Mu¨nchen, Garching, Germany). The MAB directed to the integrin subunit RIIb was obtained from Beckman Coulter GmbH (Krefeld, Germany). Vitronectin was obtained from Promega (Madison, WI). Phospholipid molecules (dimyristoylphosphatidylethanolamine), fibrinogen, collagen type I, the 19-mer R-laminin-derived peptide CSRARKQAASIKVAVSADR (P19), N-ethyl-N-(dimethylaminopropyl) carbodiimide, phosphatidylcholine from soybean, and Nhydroxysuccinimide were bought from Sigma-Aldrich (Deisenhofen, Germany). The monofunctional cyanine dye Cy5-labeling kit was purchased from Amersham Biosciences (Freiburg, Germany). Synthetic Integrin Ligands. Mono- and multimeric RGD peptides designed to target integrins Rvβ3 and Rvβ5 were developed as previously published by us using solid-phase peptide synthesis with Fmoc strategy28 as linear precursors and cyclization performed after cleavage from the resin.4,6,11,29 Since an exchange of valine within the lead cyclic pentapeptide c(-RGDfV-)4 for almost any other amino acid does not influence integrin-binding activity and selectivity,6 the RGD di- and tetramers used in the present study were constructed based on the cyclic pentapeptide, cyclo(-ArgGlyAsp-D-PheGlu-) (c(-RGDfE-), and all other compounds are based on the peptide cyclo(-ArgGlyAsp-D-PheLys-) (21) Schmidt, E. K.; Liebermann, T.; Kreiter, M.; Jonczyk, A.; Naumann, R.; Offenha¨usser, A.; Neumann, E.; Kukol, A.; Maelicke, A.; Knoll, W. Biosens. Bioelectron. 1998, 13, 585-591. (22) Liebermann, T.; Knoll, W. Colloids Surf., A 2000, 171, 115-130. (23) Sinner, E. K.; Knoll, W. Curr. Opin. Chem. Biol. 2002, 5, 705-711. (24) Sinner, E. K.; Reuning, U.; Sacca`, B.; Moroder, L.; Knoll, K.; Oesterhelt, D. Anal. Biochem. 2004, 333, 216-224. (25) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Gra¨ber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 6188-6194. (26) Naumann, R.; Schmidt, E. K.; Jonczyk, A.; Fendler, K.; Kadenbach, B.; Liebermann, T.; Offenha¨usser, A.; Knoll, W. Biosens. Bioelectron. 1999, 14, 651-662. (27) Kouns, W. C.; Kirchhofer, D.; Hadvary, P.; Edenhofer, A.; Weller, T.; Pfenninger, G.; Baumgartner, H. R.; Jennings, L. K.; Steiner, B. Blood 1992, 80, 2539-2547. (28) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (29) Gurrath, M.; Mu ¨ller, G.; Kessler, H.; Aumailley, M.; Timpl, R. Eur. J. Biochem. 1992, 210, 911-921.

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Figure 1. Structure of RGD-based synthetic integrin ligands. The structures of RGD-containing mono-, di-, and tetrameric peptides and of the RGD-based peptidomimetic used in the present study are depicted. The monomers display Ahx spacers of different lengths (Ahx, (Ahx)2, (Ahx)3) and are based on the functional integrin-binding group c(-RGDfK-). The RGD di- and tetramers were constructed with single Ahx spacers carrying the pentapeptide c(-RGDfE-). The RGD-based peptidomimetic was developed according to previous publications.15,27 To generate a site for fluorescence labeling (one fluorochrome per molecule), oxime ligation was chosen to chemically link unprotected aminooxy-functionalized peptidic fragments to marker molecules such as Cy5.

(c(-RGDfK-).6,30 The compounds were coupled to Ahx spacers of different lengths (length of one unit Ahx, 7.6 Å), which are nontoxic and unreactive. The RGD-based peptidomimetic was developed and synthesized according to previous publications.15,31 The formulas of the different compounds used in the present study are depicted in Figure 1. As controls served respective RADcontaining peptidic or peptidomimetic compounds. Fluorescence Labeling of Synthetic Integrin Ligands, Vitronectin, Fibrinogen, and Integrin-Specific Antibodies. (a) Synthetic Integrin Ligands. To generate a moiety for fluorescence labeling (one fluorochrome per molecule in order to allow comparison of binding signals), oxime ligation (a chemoselective reaction of an aldehyde group with a hydroxyl amino group) was chosen,32 representing an elegant way to link unprotected aminooxy-functionalized peptidic fragments to various marker molecules.11 The amino-functionalized peptides and the peptidomi(30) Auernheimer, J.; Zukowski, D.; Dahmen, C.; Kantlehner, M.; Enderle, A.; Goodman, S. L.; Kessler, H. ChemBioChem. 2005, 6, 2034-2040. (31) Dahmen, C.; Auernheimer, J.; Meyer, A.; Enderle, A.; Goodman, S. L.; Kessler, H. Angew. Chem., Int. Ed. 2004, 43, 6649-6652. (32) Wahl, F.; Mutter, M. Tetrahedron Lett. 1996, 37, 6861-6864.

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metic were labeled by using the monofunctional cyanine dye Cy5 (exc at 649 nm, em at 670 nm) at a final concentration of 30 nmol, according to the manufacturers’ recommendations. (b) Labeling of Vitronectin, Fibrinogen, and IntegrinSpecific MABs. Vitronectin and fibrinogen were labeled at a final concentration of 0.3 mg/mL; the MABs directed to either Rvβ3, Rvβ5, or RIIbβ3 at 0.1 mg/mL. Each integrin ligand was dissolved in phosphate-buffered saline (PBS) and mixed with lyophilized Cy5 dye in a final volume of 100 µL and the labeling procedure conducted according to the manufacturers’ instructions for 1 h at room temperature. Thereafter, the reaction was terminated by adding 2 µL of 1 M ethanolamine-HCl, pH 8.5. All Cy5-labeled compounds were stored at -20 °C until use. Surface Plasmon Resonance Spectroscopy and Surface Plasmon-Enhanced Fluorescence Spectroscopy. SPS/SPFS measurements were performed in a home-built apparatus as previously described.23,24 Preparation and spreading of integrinfunctionalized vesicles on peptide-lipid layers were also conducted as published earlier23,24 with some minor modifications: 50 µL of a solution of 1 mg/mL phosphatidylcholine in chloroform

was pipetted into a 5-mL glass vial. A thin phospholipid film was generated by evaporating the solvent under nitrogen flow in order to prevent oxidation. Detergent-solubilized Rvβ3 or Rvβ5 (2.5 µg/ chip) was each added in 2.5 mL of binding buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 2 mM MgCl2‚6H2O, and 1 mM MnCl2‚ 2H2O) and vigorously vortexed to form spontaneously integrinfunctionalized multilamellar vesicles. A vesicle extruder (LiposoFast, Avestin, Ottawa, Canada) equipped with a polycarbonate filter (pore size, 100 nm) was used to prepare unilamellar vesicles from the protein/lipid mixture, which were immediately thereafter applied onto the biosensor surface in order to increase spreading ability and to avoid intervesicular fusion. Addition of vesicles to the peptide-lipid layer resulted in spontaneous formation of integrin-functionalized phospholipid bilayers.23,24 Ligand-Binding Assays by SPS/SPFS. Prior to the adsorption procedure, a SPS/SPFS scan was performed for determining the optical properties of the gold film and the level of background fluorescence. Binding assays were conducted by adding Cy5labeled RGD peptides and the peptidomimetic at a final concentration of 6 nmol for 30 min at room temperature as formerly described.24 Cy5-labeled vitronectin and fibrinogen were added at a final concentration of 0.2 nmol, the integrin-specific MABs at a final concentration of 15 nmol. The fluorescence signal, detected at the angular position of maximal field intensity of the surface plasmon, was detected and does represent surface specific binding. Interference phenomena resulted in additional fluorescence peaks not in the angular regime of plasmon enhancement and thus do not represent binding signals.22 (a) Competition of Peptide Binding to Integrin rvβ3 by Vitronectin and Vice Versa. After successful binding of Cy5labeled peptides, Rvβ3-functionalized phospholipid bilayers were incubated for 30 min with unlabeled vitronectin in order to compete for the binding of the synthetic ligand to membraneinserted integrin as proof of binding specificity and affinity. Prior to the next SPFS scan, replaced Cy5-labeled ligands were eliminated from the biosensor surface by washing in binding buffer. Vice versa, Cy5-labeled vitronectin was first bound and displacement followed by the addition of unlabeled peptides. (b) Dissociation of Integrin/Ligand Interaction by EDTA. To reuse the integrin-functionalized phospholipid bilayers as binding surface on the biosensor, bound ligand was dissociated by adding 0.5 M EDTA, pH 8.0. Prior to repeated binding tests, the surface was equilibrated in binding buffer to reestablish the dielectrical properties of the interface. (c) Treatment of Membrane-Embedded Integrins with Proteinase K. Cy5-labeled synthetic ligands were bound to immobilized integrins and binding was recorded by SPFS. Thereafter, integrin-functionalized phospholipid bilayers were incubated with proteinase K at a final concentration of 0.4 µg/µL in 10 mM Tris-HCl, pH 8, 5 mM EDTA, and 0.5% (w/v) sodium dodecyl sulfate for 40 min at room temperature. Phospholipid bilayers were then washed in PBS and binding buffer prior to repeated binding analyses. Cell Culture. The origin and cultivation of the human ovarian cancer cell line OV-MZ-6 as well as its characterization with respect to integrin expression and adhesion properties were described previously.33-36

In Vitro Cell/ECM Adhesion Assay. The adhesive capacity of the human ovarian cancer cell line OV-MZ-6 was assessed by means of a chromogenic assay as described earlier34-36 with some minor modifications: 96-well cell culture plates were pretreated for 2 h at room temperature with vitronectin or collagen type I at a concentration of 5 µg/mL PBS. After two rinsing steps in PBS, cell culture plates were blocked for 1 h at room temperature in PBS containing 2% (w/v) bovine serum albumin (BSA) followed by another two washes in PBS. Thereafter, 2.5 × 104 cells were added to the microtiter plates together with the synthetic ligands in DMEM, 0.5% (w/v) BSA, and 20 mM HEPES. Integrin ligands were used at a concentration ranging between 0.625 and 40 µM. After an adhesion time of 90 min at 37 °C, nonadherent cells were eliminated by washing in PBS, and then 10 mM chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide was added in 0.5% (v/v) Triton X-100, 100 mM sodium citrate, pH 5, for 90 min to detect N-acetyl-β-D-hexoaminidase activity. The reaction was stopped by 0.2 M NaOH, 5 mM EDTA, and the optical density recorded at 405 nm as a measure for adherent cell numbers as previously described.34-36 RESULTS The aim of the present study was to screen newly developed synthetic integrin ligands for their capacity to specifically and selectively bind to Rvβ3 and Rvβ5 and, as control, to the platelet integrin RIIbβ3, incorporated into artificial phospholipid bilayers by our recently established cell-free SPS/SPFS-binding test. Assembly of Integrin rvβ3-, rvβ5-, and rIIbβ3-Functionalized, Peptide-Supported Artificial Phospholipid Bilayers. Reconstitution of purified integrins into unilamellar lipid vesicles was performed directly prior to vesicle spreading. Since the amount of protein in lipid vesicles has an impact on their spreading behavior, by varying the integrin/detergent/lipid ratio and the vesicle size, the procedure for vesicle functionalization was optimized with respect to maximal integrin-binding activity.24 Addition of vesicles to the peptide-tethered phospholipid monolayers led to spontaneous formation of phospholipid bilayers, carrying incorporated integrin molecules. Even under identical technical conditions for layer assembly, there is no possibility to reliably predict the total amount of membrane proteins incorporated into vesicles. However, as prerequisite for comparable measurements of integrin/ligand associations, the thickness of integrin-functionalized phospholipid bilayers has to be within a similar range, reflecting a comparable amount of integrin molecules available on the biosensor surface for binding experiments. Thus, as a measure for the extent of integrin membrane insertion, the thickness of integrin-functionalized phospholipid bilayers was monitored by SPS. A “protein-free” membrane was employed as reference. Multiple SPS scans on a series of assembled integrin Rvβ3- and Rvβ5-functionalized phospholipid bilayers, respectively, revealed a variation of optical thickness ranging from 18 to 24%. (33) Mo ¨bus, V.; Gerharz, C. D.; Press, U.; Moll, R.; Beck, T.; Mellin, W.; Pollow, K.; Knapstein, P. G.; Kreienberg, R. Int. J. Cancer 1992, 52, 76-84. (34) Hapke, S.; Kessler, H.; Arroyo de Prada, N.; Benge, A.; Schmitt, M.; Lengyel, E.; Reuning, U. J. Biol. Chem. 2001, 276, 26340-26348. (35) Hapke, S.; Gawaz, M.; Dehne, K.; Ko¨hler, J.; Marshall, J. F.; Graeff, H.; Schmitt, M.; Reuning, U.; Lengyel, E. Mol. Cell. Biol. 2001, 21, 21182132. (36) Hapke, S.; Kessler, H.; Luber, B.; Benge, A.; Hutzler, P.; Ho ¨fler, H.; Schmitt, M.; Reuning, U. J. Biol. Chem. 2003, 384, 1073-1083.

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Figure 2. Binding of ECM protein ligands to integrins Rvβ3, Rvβ5, and RIIbβ3 incorporated into peptide-tethered phospholipid bilayers. Binding of natural ECM proteins was measured by SPFS in order to confirm functional incorporation of integrins into phospholipid bilayers. For this, membrane-embedded Rvβ3 (A) and Rvβ5 (B), respectively, were incubated with Cy5-labeled vitronectin and in the case of RIIbβ3 (C) with Cy5-labeled fibrinogen (9). In parallel, data are depicted from binding assays on integrin-free phospholipid bilayers spread onto the biosensor surface (b). The open symbols depict the respective SPFS scans after addition of ECM proteins to integrin-functionalized (0) and integrin-free (O) phospholipid bilayers.

Proof of Orientation and Functionality of Integrin rvβ3, rvβ5, or rIIbβ3, Incorporated into Peptide-Tethered Phospholipid Bilayers. (a) Binding of Integrin-Specific MABs Directed to the ECM-Binding Region of rvβ3, rvβ5, or rIIbβ3. Spreading and functionalization of lipid vesicles cannot be controlled with respect to orientation of incorporated transmembrane receptor proteins. Thus, to prove that integrins were inserted with the heterodimeric extracellular domain extending the headgroup region from the lipid bilayer into the aqueous phase, we performed binding experiments using MABs directed to the ECM protein-binding domains of Rvβ3 and Rvβ5, respectively. Functionally active platelet integrin RIIbβ3 was detected using a MAB recognizing the RIIb in complex with the β3 integrin subunit. In all cases, strong binding signals were recorded, implying that a sufficient amount of integrin molecules had been incorporated into the lipid bilayers with the ECM recognition site pointing into the aqueous phase (data not shown24). Control measurements showed that the MABs did not associate with integrin-free lipid surfaces; neither did an irrelevant, nonintegrin MAB bind to either integrin-functionalized artificial membrane (data published in ref 24). 4528

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(b) Binding of Naturally Occurring ECM Protein Ligands. Functional incorporation of integrins into the peptide-supported artificial lipid membranes was further confirmed by binding of the respective ECM protein ligands. As binding partner for Rvβ3 or Rvβ5, the ECM component vitronectin was added; for the platelet integrin RIIbβ3, we used fibrinogen as preferred binding substrate. On both, Rvβ3- and Rvβ5-functionalized phospholipid bilayers, respectively, incubation with Cy5-labeled vitronectin resulted in specific binding signals, indicating that both integrins were membrane incorporated in an active ligand-binding conformation and orientation, at least to an extent sufficient for the performance of reliable binding experiments (Figure 2 A and B). Most interestingly, the specific binding of vitronectin to Rvβ3 and Rvβ5, respectively, resulted in an increase of the angle of incidence of ∼1°, indicating that the minimum of reflectivity of the SP scan corresponds to the binding of a large multimeric ligand complex. As controls, binding experiments were conducted on integrin-free phospholipid bilayers, which did in neither case demonstrate binding of vitronectin or fibrinogen (Figure 2). To prove an even distribution of incorporated integrins on the phospholipid surface, association of fluorescence labeled ECM proteins was measured

Figure 3. Binding of synthetic peptides as a function of multimerization of integrin-binding RGD motifs. Mono- (9), di- (b), or tetrameric (2) RGD peptides linked to a single Ahx spacer were incubated with Rvβ3 (A) or Rvβ5 (B) functionalized phospholipid bilayers as described under Experimental Section. The different ligands were all measured at the same position of identical integrin-functionalized phospholipid bilayers in order to allow direct comparison of binding signals. The solid lines depict the corresponding SPFS scan after addition of Cy5-labeled compounds in order to demonstrate the minimum of SP reflectivity corresponding to maximal field intensity for fluorochrome excitation.

Figure 4. Competition of peptide binding to integrin Rvβ3 by unlabeled vitronectin and vice versa. (A) Binding of Cy5-labeled RGD monomerAhx (9) to an Rvβ3-functionalized phospholipid bilayer was competed for in the presence of unlabeled vitronectin at a concentration of 0.2 (O) or 0.8 nmol (4) for 30 min. (B) Displacement of Cy5-labeled vitronectin (9) from membrane-inserted Rvβ3 in the presence of equimolar concentrations (0.2 nmol) of unlabeled RGD monomer-Ahx (O) and RGD dimer-Ahx (4).

at five different positions of each lipid bilayer. By calculating and comparing those binding data, we evaluated a variation in binding signal intensity between five different spots of up to 20% (data not shown). Binding of Synthetic RGD-Based Integrin Ligands to Membrane-Incorporated Integrins. (a) Binding of Multimeric RGD Peptides. For a direct comparison of binding events, associations of RGD peptides containing either one, two, or four integrin-binding RGD motifs linked to a single Ahx spacer, were monitored on identical Rvβ3- (Figure 3A) or Rvβ5-functionalized artificial lipid membranes (Figure 3B). It became obvious that the RGD dimer exhibited highest, and the RGD monomer lowest binding capacity toward both Rv integrins. Most interestingly, even having the highest number of integrin-binding RGD groups, the tetramer was not as effective in binding to Rvβ3 or Rvβ5 as the dimer, the latter revealing a slightly higher selectivity toward Rvβ3. (b) Competition of Peptide Binding to Integrin rvβ3 by Vitronectin and Vice Versa. Binding of Cy5-labeled RGD

peptides was competed for by the addition of the unlabeled natural ECM ligand vitronectin (Figure 4 A). A concentration of as low as 0.2 nmol of vitronectin (when compared to 6 nmol of added peptide monomersAhx) resulted in an ∼60% peptide displacement from membrane-incorporated Rvβ3; an >90% competition was achieved by adding 0.8 nmol of vitronectin (Figure 4A). By performing the competition experiments the other way around, binding of Cy5-labeled vitronectin and displacement by unlabeled peptide monomer-Ahx, binding of vitronectin was already totally abrogated by adding equimolar concentrations of the RGD monomer within 30 min (Figure 4B). Displacement of vitronectin by the RGD dimer present at equimolar concentration competed binding by up to 83% (Figure 4B). (c) Binding of Monomeric RGD Peptides. Monomeric RGDcontaining compounds were synthesized as controls, linked to Ahx spacers of gradually increasing length, and incubated with integrinfunctionalized phospholipid bilayers. The binding data revealed that the RGD monomer-(Ahx)2 tended to be most effective in Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

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Figure 5. Binding of RGD peptide monomers to integrin Rvβ3-, Rvβ5-, and RIIbβ3-functionalized phospholipid bilayers. Three Cy5-labeled RGD peptide monomers coupled to Ahx spacers of different lengths (Ahx (9), (Ahx)2 (b), and (Ahx)3 (2)) were subsequently incubated with Rvβ3- (A), Rvβ5- (B), and RIIbβ3-functionalized phospholipid bilayers (C), respectively, on identical biosensor surfaces after dissociation of each ligand by EDTA treatment as described under Experimental Section. The solid lines illustrate the respective SPFS scans after addition of Cy5labeled RGD ligands in order to specify the angular position of the SP-enhanced evanescent field.

binding to Rvβ3 (Figure 5 A) as well as to Rvβ5 (Figure 5 B), whereas the RGD monomer-(Ahx)3 showed lowest binding capacity toward both integrins (Figure 5A and B); however, the differences were within the experimental error. All different peptidic monomers displayed comparable selectivity toward the two Rv integrins. None of the three Rvβ3/Rvβ5-selective RGD monomers, with either Ahx spacer, showed binding to lipid surfaces exposing RIIbβ3 (Figure 5C). RAD-based peptides did not bind to either membrane-inserted integrin (data not shown). In neither case, were binding events of any compound recordable on unfunctionalized, integrin-free artificial membranes. (d) Binding of a Synthetic RGD-Based Peptidomimetic. As an alternative to RGD peptides, we used an RGD-based peptidomimetic linked to an (Ahx)3 spacer as integrin ligand within SPFS-binding studies. By comparing its efficiency to bind to Rvβ3 with the respective RGD peptide monomer-(Ahx)3, we observed almost overlapping binding curves for both compounds. The respective RAD peptidomimetic did not bind (Figure 6A). Next, we evaluated the binding efficiency of the peptidomimetic with respect to its selectivity toward either Rv integrin. For this, we performed binding experiments on Rvβ3- or Rvβ5-functionalized phospholipid bilayers with almost identical layer thickness (3 nm for Rvβ3 versus 3.1 nm for Rvβ5) in order to ensure a similar 4530 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

extent of integrin incorporation. The peptidomimetic showed lower binding capacity to Rvβ5 than to Rvβ3 (Figure 6 B); no binding signal was obtained when incubating the peptidomimetic on RIIbβ3-functionalized phospholipid bilayers (data not shown). In general, for statistical analyses of SPFS measurements, we conducted repetitions of binding experiments using the same compound on an identical substrate and position spot on identical biosensor chips carrying phospholipid bilayers. By this, we determined an intraassay variation ranging between 15 and 18%. Dissociation of Integrin/Ligand Interactions by EDTA. RGD-based binding of natural as well as synthetic ligands to integrins depends on the presence of divalent cations, such as Ca2+, Mg2+, and Mn2+.2 Removing these cations upon complexation by EDTA, integrin/ligand complexes may be dissociated. To reuse integrin-functionalized phospholipid bilayers as a binding surface, we added 0.5 M EDTA, pH 8, after a certain ligand had been bound and observed a gradual decrease of ligand binding back to baseline levels (data not shown). Prior to repeated binding tests, the lipid surface was equilibrated in binding buffer in order to reestablish the dielectrical properties of the interface. Integrinfunctionalized phospholipid surfaces can thus be regenerated for repeated screening of ligands at the same spot on identical

Figure 6. Comparison of binding of synthetic peptidic and peptidomimetic RGD-based monomers to phospholipid-incorporated Rvβ3. (A) The binding signals for the RGD peptide monomer-(Ahx)3 (9) and the RGD-based peptidomimetic-(Ahx)3 (0) to Rvβ3 are given. As control for specific binding served the respective RAD monomer-(Ahx)3 (b) and the RAD-based peptidomimetic-(Ahx)3 (O). (B) Binding of the peptidomimetic (Ahx)3 to artificial membranes functionalized either with Rvβ3 (9) or with Rvβ5 (b). For direct comparison of SPFS-binding data, we performed binding experiments on Rvβ3- or Rvβ5-functionalized artificial lipid membranes, which exerted identical layer thickness (3 nm for Rvβ3 versus 3.1 nm for Rvβ5) in order to ensure a similar extent of integrin incorporation. The SPFS scans are given by solid lines after addition of the Cy5-labeled peptidomimetic-(Ahx)3.

Figure 7. Treatment of lipid-embedded Rvβ3 by proteinase K. The RGD monomer-(Ahx)3 was incubated on Rvβ3-functionalized phospholipid bilayers as described under Experimental Section prior to (0) and after (3) treatment with proteinase K. The solid symbols indicate SPS measurements before (9) and after (1) proteinase K treatment. The shift of the minimum of reflectivity of the SPFS scan to a lower angle of incidence after proteinase K treatment is indicative of integrin degradation.

integrin-functionalized phospholipid bilayers, thereby allowing direct comparison of ligand affinity and selectivity under the same experimental conditions. Treatment of Integrin-Functionalized Phospholipid Bilayers by Proteinase K. As a further control for functional integrin targeting, first, Rvβ3-functionalized phospholipid bilayers were incubated with a respective Cy5-labeled ligand (Figure 7; here shown for the RGD monomer-(Ahx)3) and binding recorded within the SPFS mode. Thereafter, Rvβ3-functionalized lipid membranes were treated with proteinase K for 40 min at room temperature and binding experiments repeated. Proteinase K treatment completely abrogated ligand binding to incorporated integrins. Also, the downward shift in the minimum of reflectivity

of ∼1° indicated decreasing thickness of the lipid bilayer caused by the degradation of membrane-inserted integrins. Effect of Mono-, Di-, and Tetrameric RGD Peptides and the RGD-Based Peptidomimetic on rvβ3-Mediated Ovarian Cancer Cell Adhesion. In in vitro cell adhesion assays, we investigated the impact of the synthetic integrin ligands on the adhesive capacity of human ovarian cancer cells. By using the Rvβ3-selective peptide c(-RGDfV-) as competitor, we have demonstrated before that the human ovarian cancer cell line OVMZ-6 almost exclusively adheres to vitronectin via Rvβ3.34,36 Thus, for adhesion assays, microtiter cell culture plates were pretreated with vitronectin and, as control, with collagen type I, which is addressed by other integrins, mostly by members of the β1 subfamily. (a) Monomeric RGD Peptides and the Peptidomimetic. In principle, all RGD monomers markedly reduced OV-MZ-6 cell adhesion to vitronectin (Figure 8). The capacity of cells to adhere to collagen type I was unimpaired (data not shown). RGD monomers with different Ahx spacer lengths showed very similar IC50 values, the RGD monomer-(Ahx)2 tending toward the strongest effect on cell detachment from vitronectin (IC50 ) 2.6 ( 1.1 µM), followed by the monomer-Ahx (IC50 ) 3.2 ( 0.7 µM), and subsequently, the monomer-(Ahx)3 (IC50 ) 5.2 ( 2.5 µM). Best cell detachment from vitronectin was achieved by the monomeric RGD-based peptidomimetic (IC50 ) 1.2 ( 0.8 µM) (Figure 8). The RAD monomer-(Ahx)3 did not influence cell adhesion to either ECM protein. (b) Multimeric RGD Peptides. As shown for RGD peptide monomers, neither the RGD dimer nor the RGD tetramer altered OV-MZ-6 cell adhesion to collagen type I. By evaluating the effects of RGD multimerization on cell adhesion to vitronectin, we found almost comparable IC50 values for the RGD dimer (IC50 ) 8.4 ( 0.2 µM) and the RGD tetramer (IC50 ) 7.7 ( 0.4 µM) (Figure 8). Even having the highest number of integrin-binding RGD groups, the RGD di- and tetramers displayed higher IC50 values than the Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

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Figure 8. Effect of mono-, di-, and tetrameric RGD peptides and the RGD-based peptidomimetic on Rvβ3-mediated human ovarian cancer cell adhesion to vitronectin. Impact of synthetic RGD monomers as a function of Ahx spacer length: In in vitro cell adhesion assays, increasing concentrations of RGD monomers coupled to Ahx spacers of different lengths (Ahx (]), (Ahx)2 (0), and (Ahx)3 (4)) as well as the RGD-based peptidomimetic (Ahx)3 ([) were incubated with human OV-MZ-6 cells attached to vitronectin as described under Experimental Section. The OD values obtained by the chromogenic adhesion assays were transformed into numbers of adherent cells based on cell calibration curves. Numbers of untreated cells adherent to vitronectin were set to 100%; mean values of four determinations are given ((SD). Effect of multimerization of RGD motifs within synthetic integrin ligands: Increasing concentrations of mono- (]), di- (9), or tetrameric (2) synthetic RGD peptides linked to a single Ahx spacer were incubated with human OV-MZ-6 cells attached to vitronectin as described under Experimental Section. As control, the RAD monomer-(Ahx)3 (b) is depicted, which was not effective as adhesion competitor.

respective RGD monomer. An alternatively used RGD dimer with a hexaoxaeicosanoic acid spacer of a length comparable to that of the (Ahx)3 spacer provoked even less efficient cell detachment from vitronectin (IC50 ∼ 16 µM), indicating that a longer spacer in multimers might mask or sterically hinder integrin binding of RGD functional groups (data not shown). DISCUSSION Adhesive interactions of cells with the ECM are crucially implicated in many physiological and pathophysiological processes. Two integrins, Rvβ3 and Rvβ5, have received considerable attention due to their implication in pathologies as diverse as osteoporosis, restenosis, acute renal failure, and ocular diseases. Moreover, these integrins have been shown to be upregulated in tumor cells during cancer progression and in endothelial cells during tumor angiogenesis.3,10 Within the past decade, RGD-based peptides have been developed that antagonize Rvβ3/Rvβ5-mediated cell adhesion and, consequently, intracellular signal transduction. In fact, the first synthetic Rvβ3-selective cyclic peptide c(-RGDfV-) was designed and synthesized by us.4-6 Meanwhile, in an effort to develop improved peptide antagonists with enhanced integrin avidity, multimeric RGD peptides have also been synthesized.11,12 To screen newly developed synthetic ligands with respect to their specificity and selectivity toward different integrin subspecies, sensitive and reliable in vitro binding tests are required. Thus, we recently established a cell-free in vitro binding test system by employing purified native integrins incorporated into artificial 4532 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

phospholipid bilayers. The method preserves structural integrity of transmembrane receptors such as integrins and, via an optimized hydrophilic spacer peptide, provides access space and flexibility of, for example, integrin cytoplasmic domains.23,24 Ligand association to membrane-embedded integrins on the biosensor device was followed by SPS/SPFS, allowing the detection of binding events using even very small ligands. In the case of the synthetic RGD-based ligands used in the present study, the molecular masses range from approximately 700 to 4500 Da. In contrast, in conventional SPS settings, ligand sizes should not fall below a molecular mass of ∼10 000 Da. Herewith, only indirect testing of small ligands is possible, e.g., by determining their capacity to act as soluble competitors for larger ligands, such as ECM proteins like vitronectin. As prerequisite for SPFS, we first followed integrin binding of purified ECM proteins since correct protein incorporation and spreading of lipid vesicles cannot be controlled with respect to orientation of transmembrane receptors within lipid bilayers. In the case of Rvβ3 or Rvβ5, we used vitronectin; for RIIbβ3, fibrinogen was taken as preferred ECM substrate. Binding of vitronectin resulted in a prominent angular shift of the minimum of the SPS scan, indicating high molecular mass binding. In fact, it is well known that vitronectin forms multimers still exerting integrin-binding capacity.37 In parallel, orientation of integrins within artificial phospholipid membranes was controlled by using specific MABs directed to the ECM-binding domains of Rvβ3, Rvβ5, and RIIbβ3, respectively. In all cases, we proved by SPFS that a sufficient portion of integrin molecules had been phospholipid-incorporated with the ECM-binding domain facing the aqueous phase and thus allowing performance of reliable binding experiments. Binding of Cy5-labeled RGD peptides was competed for by the addition of the unlabeled natural ECM ligand vitronectin (0.2 versus 6 nmol of added RGD peptide) by ∼60% from membrane-inserted Rvβ3; an over 90% competition was achieved by adding 0.8 nmol of vitronectin; thus, even without having molar excess of added natural competitor present in the binding assay, optimal peptide displacement was recordable. Also, Cy5-labeled vitronectin was displacable by unlabeled peptide monomer-Ahx; by adding equimolar concentrations of the RGD monomer, binding of vitronectin was totally abrogated. Displacement of vitronectin by an equimolar concentration of the RGD dimer was slightly less efficient than that provoked by the RGD monomer, resulting in ∼80% vitronectin displacement. Next, we evaluated and compared the integrin-binding capacity of a series of synthetic monomeric RGD peptides linked to Ahx spacers of different lengths. The RGD monomer exhibiting a medium spacer length of ∼15 Å ((Ahx)2) was most effective in binding to Rvβ3 as well as to Rvβ5, followed by the RGD monomer-Ahx, and, last, the RGD monomer-(Ahx)3. All RGD monomers did not exert differences regarding their selectivity toward either one of the two Rv integrins. According to the recently published crystal structure of the extracellular domain of Rvβ3, the RGD-binding site is located between the surface region of the globular head of the two subunits of integrin Rvβ3, only a few angstroms deep. Therefore, one would not assume a requirement for long spacer moieties.38,39 Still, spacers (37) Schvartz, I.; Seger, D.; Shaltiel, S. Int. J. Biochem. Cell Biol. 1999, 31, 539544. (38) Xiong, J. P.; Stehle, T.; Diefenbach, B.; Zhang, R.; Dunker, R.; Scott, D. L.; Joachimiak, A.; Goodman, S. L.; Arnaout, M. A. Science 2001, 294, 339345.

might be necessary to compensate for cell surface roughness and, under some circumstances, for binding of RGD peptides within surface clefts of the cell membrane. To achieve enhanced integrin targeting due to polyvalency effects, we also analyzed binding of multimeric RGD peptides. The exact spatial arrangement of RGD moieties within multimers for optimal integrin targeting on cell surfaces is not well characterized yet. For the compounds used in the present study, we synthesized lysine trees within the compounds and connected the different RGD moieties via Ahx units. This resulted in large macromolecules in which integrin recognition motifs are distributed in a more complex manner.11,40 For direct comparison of binding signals, associations of mono-, di-, or tetrameric RGD peptides were monitored on identical membranes having either Rvβ3 or Rvβ5 incorporated. Theoretically, multimerization of RGD motifs should enhance avidity of synthetic compounds by simultaneous integrin targeting.11,41,42 Indeed, SPFS measurements revealed that the RGD dimer exhibited a higher binding capacity toward both Rv integrins than the RGD monomer. To our surprise, the tetramer was not as effective in binding as the dimer, a finding that is in contrast to data by others, who observed increased affinity of RGD oligomers due to multivalent integrin interactions.43,44 One explanation for our findings on the artificial phospholipid bilayers could be that the dimer may allow binding of two individual integrin molecules at a time, whereas the tetramersbecause of the large size of the integrin headgroups will not permit targeting of more than two integrin molecules due to sterical hindrance. Moreover, the spacing between integrins is important for their activity. It has even been observed that by reducing the distance between the surface and the RGD ligands coated onto biomaterials, binding of cells (e.g., osteoblasts) can be reduced or abolished.45 We also evaluated integrin targeting by the different compounds in cell biological assays by perfoming in vitro cell adhesion tests. For the human ovarian cancer cell line OV-MZ-6, we have demonstrated before that Rvβ3 is predominantly engaged by the ECM protein vitronectin.34,36 Consequently, in OV-MZ-6 cells, we found that all RGD peptide monomers drastically reduced Rvβ3(39) Xiong, J. P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.; Goodman, S. L.; Arnaout, M. A. Science 2002, 296, 151-155. (40) Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M. Science 2001, 294, 1708-1712. (41) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2755-2794. (42) Maynard, H. D.; Okada, S. Y.; Grubbs, R. H. J. Am. Chem. Soc. 2001, 123, 1275-1279. (43) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2001, 113, 1677-1686. (44) Kok, R. J.; Schraa, A. J.; Bos, E. J.; Moorlag, H. E.; Asgeirsdottir, S. A.; Everts, M.; Meijer, K. K. F.; Molema, G. Bioconjugate Chem. 2002, 13, 128-135. (45) Auernheimer, J.; Dahmen, C.; Hersel, U.; Bausch, A.; Kessler, H. J. Am. Chem. Soc. 2005, 127, 16107-16110. (46) Kantlehner, M.; Schaffner, P.; Finsinger, D.; Meyer, J.; Jonczyk, A.; Diefenbach, B.; Nies, B.; Ho¨lzemann, G.; Goodman, S. L.; Kessler, H. ChemBioChem. 2000, 1, 107-114. (47) Jeschke, B.; Meyer, J.; Jonczyk, A.; Kessler, H.; Adamietz, P.; Meenen, N. M.; Kantlehner, M.; Goepfert, C.; Nies, B. Biomaterials 2002, 23, 34553463. (48) Mann, B. K.; West, J. L. J. Biomed. Mater. Res. 2002, 60, 86-93. (49) Brandley, B. K.; Schnaar, R. L. Dev. Biol. 1989, 135, 74-86. (50) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blu ¨ mmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. Chem. Phys. Chem. 2004, 5, 383388.

mediated cell adhesion to vitronectin, butsas expectedsdid not influence cell attachment to collagen type I, which is addressed by other integrins (e.g., R2β1). The RGD monomers with different Ahx spacer lengths showed very similar IC50 values, the RGD monomer-(Ahx)2 tending toward the strongest effect on cell detachment from vitronectin, followed by the monomer-Ahx, and, subsequently, the monomer-(Ahx)3. Our observations are in accordance with the general notion that flexible spacers may allow arrangement of RGD peptides in spatial cellular microdomains; however, in many cases, reduced integrin targeting was noticed before, when the spacer moiety was too long. This might be due to increasing entropy of longer flexible spacers opposing stronger binding.41 Since no general conclusion can be drawn from publications so far, the need for a certain spacer length has to be tested empirically in each individual cell system. Cell/ECM adhesion influences cellular behavior not only by the presence of RGD motifs but also by their distribution and density within the cellular microenvironment. On the molecular level, we are only beginning to understand how a certain spatial RGD arrangement affects cellular responses such as adhesion, migration, and growth. Thus, in in vitro cell adhesion assays, we compared RGD multimers with respect to their capacity to inhibit Rvβ3-dependent cell adhesion. In fact, we did not find prominent differences between the IC50 values of the RGD dimer and tetramer, which were higher than those of the respective monomers. These results were not in line with the SPFS-binding data and can best be explained by the differences in the binding surface architecture presented within cell-based assays when compared to the artificial membrane exposed on the SPFS biosensor. They are certainly also due to differences in the spatial arrangement of integrins on natural cell membranes versus that on integrinfunctionalized phospholipid bilayers. By immobilizing RGD peptides as cell adhesion-supporting substrates, it has been found by others that RGD surface density is clearly related to the extent of cell adhesion, focal contact formation, spreading, migration, proliferation, and survival.46-48 Also, the direction of cell migration seems to be governed by RGD surface distribution.49 However, exact measurements on nanodesigned surfaces proved that a maximal distance of 65 nm is allowed to form focal adhesions.49 Since synthetic integrin antagonists are planned to be used as therapeutic tools for systemic treatment of patients, to prevent platelet activation and aggregation, it is strongly demanded that these compounds do not associate with the platelet integrin RIIbβ3. Indeed, the presented SPS/SPFS measurements clearly revealed that none of the Rvβ3/Rvβ5-selective RGD-based integrin ligands bound to RIIbβ3. In summary, the present study further underlines the suitability of the novel SPS/SPFS-binding assay platform for real-time measurements of small ligand associations with transmembrane receptors such as integrins, in a lipid microenvironment with low background signals and high precision. ACKNOWLEDGMENT We thank the Deutsche Forschungsgemeinschaft (DFG) for funding this project. Received for review November 24, 2005. Accepted April 19, 2006. AC052078+ Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

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