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An Ephrin Mimetic Peptide That Selectively Targets the EphA2 Receptor

2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês

10.1074/jbc.m208495200

1083-351X

Mitchell Koolpe, Monique Dail, Elena B. Pasquale,

Angiogenesis and VEGF in Cancer

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologicversus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolarK D values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration. Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologicversus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolarK D values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration. Originally identified as regulators of neural development, the Eph family of receptor tyrosine kinases and their ephrin ligands are also critical for vascular development and pathological forms of angiogenesis (1Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Google Scholar, 2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar, 3Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Google Scholar). For example, the EphA2 receptor and ephrin-A1, a ligand for EphA2, are coordinately expressed in the vasculature of human tumors and mouse xenograft tumors grown from human cancer cells (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar). The EphA2 receptor plays a critical role in tumor necrosis factor α (TNFα) 1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein -induced neovascularization because TNFα up-regulates ephrin-A1, which causes receptor activation in blood vessels (5Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Google Scholar). Similarly, the homeobox transcription factor Hox B3 promotes angiogenesis by up-regulating ephrin-A1 (6Myers C. Charboneau A. Boudreau N. J. Cell Biol. 2000; 148: 343-351Google Scholar). Furthermore, EphA2 signaling is required for the formation of endothelial capillary tubes in vitro (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 7Daniel T.O. Stein E. Cerretti D.P. St. John P.L. Robert B. Abrahamson D.R. Kidney Int. Suppl. 1996; 57: S73-S81Google Scholar) and promotes the formation of blood vessel-like structures by melanoma cells (8Hess A.R. Seftor E.A. Gardner L.M. Carles-Kinch K. Schneider G.B. Seftor R.E. Kinch M.S. Hendrix M.J. Cancer Res. 2001; 61: 3250-3255Google Scholar). The expression of EphA2 appears to be restricted to “activated” adult blood vessels as this receptor has not been detected in either embryonic or adult quiescent blood vessels (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 9Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Google Scholar,10Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624Google Scholar). Ephrin-A1 has also not been detected in adult blood vessels, although it is present in the embryonic vasculature (11McBride J.L. Ruiz J.C. Mech. Dev. 1998; 77: 201-204Google Scholar).In addition to being present in tumor endothelial cells, EphA2 and ephrin-A1 are up-regulated in the transformed cells of a wide variety of tumors including breast, prostate, colon, skin, and esophageal cancers (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 12Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306Google Scholar, 13Walker-Daniels J. Coffman K. Azimi M. Rhim J.S. Bostwick D.G. Snyder P. Kerns B.J. Waters D.J. Kinch M.S. Prostate. 1999; 41: 275-280Google Scholar, 14Easty D.J. Herlyn M. Bennett D.C. Int. J. Cancer. 1995; 60: 129-136Google Scholar, 15Nemoto T. Ohashi K. Akashi T. Johnson J.D. Hirokawa K. Pathobiology. 1997; 65: 195-203Google Scholar). Many factors increase EphA2 expression in cancer cells including the H-Ras oncogene, E-cadherin, members of the p53 family of transcriptional regulators, DNA damage, and loss of estrogen receptors and c-Myc (16Andres A.C. Reid H.H. Zurcher G. Blaschke R.J. Albrecht D. Ziemiecki A. Oncogene. 1994; 9: 1461-1467Google Scholar, 17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar, 18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar).Because the tumor vasculature is discontinuous and leaky in nature, it is possible to utilize the up-regulation of EphA2 and ephrin-A1 to deliver cancer-eradicating agents to both blood vessels and tumor cells (19Dvorak H.F. Nagy J.A. Dvorak J.T. Dvorak A.M. Am. J. Pathol. 1988; 133: 95-109Google Scholar). Indeed, systemically administered biological agents can easily penetrate into tumors from the blood circulation (20Essler M. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2252-2257Google Scholar). Selectively targeting EphA2 and ephrin-A1 is a challenging task because these proteins belong to large families of closely related proteins (21Eph Nomenclature Committee Cell. 1997; 90: 403-404Google Scholar). One approach that has been successfully used to identify peptides that exhibit selectivity for their targets is to screen random peptide libraries displayed on the surface of filamentous bacteriophage (22Koivunen E. Wang B. Ruoslahti E. J. Cell Biol. 1994; 124: 373-380Google Scholar, 23Koivunen E. Arap W. Valtanen H. Rainisalo A. Medina O.P. Heikkila P. Kantor C. Gahmberg C.G. Salo T. Konttinen Y.T. Sorsa T. Ruoslahti E. Pasqualini R. Nat. Biotechnol. 1999; 17: 768-774Google Scholar, 24Smith G.P. Science. 1985; 228: 1315-1317Google Scholar, 25Devlin J.J. Panganiban L.C. Devlin P.E. Science. 1990; 249: 404-406Google Scholar). Therefore, we used a phage display approach to search for peptides that bind selectively to the extracellular domains of EphA2 and ephrin-A1. Remarkably the two peptides identified bind selectively to EphA2, but not other Eph receptors, and antagonize ephrin binding. Intriguingly, at least one of the peptides has bioactive properties in that it stimulates EphA2 tyrosine phosphorylation and signaling. In addition, this peptide delivers phage particles to EphA2-expressing cells and thus can have therapeutic value in delivering agents to tissues that express EphA2. Thus, Eph receptor-binding peptides with different agonistic and drug targeting activities can have important therapeutic applications.DISCUSSIONWe have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Google Scholar, 33Olson T.A. Mohanraj D. Roy S. Ramakrishnan S. Int. J. Cancer. 1997; 73: 865-870Google Scholar, 34Ellerby H.M. Arap W. Ellerby L.M. Kain R. Andrusiak R. Rio G.D. Krajewski S. Lombardo C.R. Rao R. Ruoslahti E. Bredesen D.E. Pasqualini R. Nat. Med. 1999; 5: 1032-1038Google Scholar, 35Arap W. Haedicke W. Bernasconi M. Kain R. Rajotte D. Krajewski S. Ellerby H.M. Bredesen D.E. Pasqualini R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1527-1531Google Scholar, 36Gerlag D.M. Borges E. Tak P.P. Ellerby H.M. Bredesen D.E. Pasqualini R. Ruoslahti E. Firestein G.S. Arthritis Res. 2001; 3: 357-361Google Scholar). The YSA peptide has the added benefit in that it stimulates EphA2 activation, which likely mediates internalization of the receptor and the peptide (37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar, 39van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). Therefore, toxic or apoptotic substances could be delivered intracellularly to selectively kill cells (34Ellerby H.M. Arap W. Ellerby L.M. Kain R. Andrusiak R. Rio G.D. Krajewski S. Lombardo C.R. Rao R. Ruoslahti E. Bredesen D.E. Pasqualini R. Nat. Med. 1999; 5: 1032-1038Google Scholar). Furthermore, activation of EphA2 signaling induced by the YSA peptide should reduce proliferation, invasiveness, and metastatic behavior of EphA2-expressing cancer cells (28Miao H. Wei B.R. Peehl D.M. Li Q. Alexandrou T. Schelling J.R. Rhim J.S. Sedor J.R. Burnett E. Wang B.C. Nature Cell Biol. 2001; 3: 527-530Google Scholar, 37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar). This is consistent with the finding that EphA2 activation correlates with decreased malignancy of breast and prostate cancer cells and reverses the transforming effects of EphA2 overexpression (18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar, 37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar). Intriguingly, EphA2 activation could sensitize cells to apoptotic stimuli when the YSA peptide is used to deliver cytotoxic agents (17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar).Phage-displayed peptides isolated by panning on receptors often bind within ligand-binding sites (40Koivunen E. Gay D.A. Ruoslahti E. J. Biol. Chem. 1993; 268: 20205-20210Google Scholar, 41Wang B. Yang H. Liu Y.C. Jelinek T. Zhang L. Ruoslahti E. Fu H. Biochemistry. 1999; 38: 12499-12504Google Scholar, 42Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Google Scholar, 43Rajotte D. Ruoslahti E. J. Biol. Chem. 1999; 274: 11593-11598Google Scholar) and can mimic natural receptor-binding motifs of ligands (42Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Google Scholar, 44Yayon A. Aviezer D. Safran M. Gross J.L. Heldman Y. Cabilly S. Givol D. Katchalski-Katzir E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10643-10647Google Scholar, 45Wright R.M. Gram H. Vattay A. Byme S. Lake P. Dottavio D. Bio-Technology. 1995; 13: 165-169Google Scholar). Our evidence strongly suggests that the YSA and SWL peptides bind to the interface of EphA2 that mediates the initial high affinity heterodimerization with ephrins (31Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Google Scholar). First, the peptides interact with the ligand-binding globular domain of EphA2 and antagonize ephrin binding. Second, at least one of the peptides causes EphA2 phosphorylation and signaling, similar to ephrins. Third, the YSA and SWL peptide, as ephrin-A5, bind to a site whose affinity is regulated by the ephrin-derived A5 peptide (Fig. 5,B and C). This effect of the A5 peptide, which presumably binds to the low affinity tetramerization interface of EphA2 (31Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Google Scholar), also suggests a previously unrecognized allosteric regulation between the two ephrin-binding sites of an Eph receptor.The YSA and SWL peptides bind selectively only to EphA2. Therefore these peptides have features that confer specificity and are not shared by the ephrins and the ephrin-derived peptides. However, the two related YSA and SWL peptides do show similarity to receptor-binding sequences of A-ephrins, including a conserved ΦxxΦ motif (where Φ is an aromatic amino acid and x is a non-conserved amino acid) present in both the dimerization and tetramerization interfaces. Understanding the structural determinants of peptide-binding promiscuityversus specificity and high affinity may allow the rational design of new peptides that bind with high affinity to specific Eph receptors.Conceivably, phage display could also be used to isolate peptides that specifically bind to each of the fifteen known Eph receptors (cbweb.med.harvard.edu/eph-nomenclature). Different Eph receptors have been implicated in various types of cancer and should be investigated as possible therapeutic targets (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Indeed, we have already isolated peptides that bind EphB4 (data not shown), another receptor expressed in blood vessels and up-regulated in tumors (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Eph receptors could also be targets for promoting nerve regeneration, where ephrin mimetic peptides could desensitize regenerating nerves to the repulsive effects of ephrins up-regulated at injury sites (46Hornberger M.R. Dutting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. Tanaka H. Drescher U. Neuron. 1999; 22: 731-742Google Scholar, 47Miranda J.D. White L.A. Marcillo A.E. Willson C.A. Jagid J. Whittemore S.R. Exp. Neurol. 1999; 156: 218-222Google Scholar, 48Rodger J. Lindsey K.A. Leaver S.G. King C.E. Dunlop S.A. Beazley L.D. Eur. J. Neurosci. 2001; 14: 1929-1936Google Scholar).Bioactive peptides that selectively target an Eph receptor also represent unique reagents for developmental studies to interfere with the activity of a specific receptor, a feat not possible by using the naturally occurring ligands. In addition, such peptides will be useful to discern the signal transduction mechanisms of different Eph receptors in cells expressing multiple receptors. Small-molecule ephrin mimetics with exclusive specificity have great potential value as tools to characterize Eph receptor function and as therapeutics to change such function in disease. Originally identified as regulators of neural development, the Eph family of receptor tyrosine kinases and their ephrin ligands are also critical for vascular development and pathological forms of angiogenesis (1Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Google Scholar, 2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar, 3Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Google Scholar). For example, the EphA2 receptor and ephrin-A1, a ligand for EphA2, are coordinately expressed in the vasculature of human tumors and mouse xenograft tumors grown from human cancer cells (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar). The EphA2 receptor plays a critical role in tumor necrosis factor α (TNFα) 1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein -induced neovascularization because TNFα up-regulates ephrin-A1, which causes receptor activation in blood vessels (5Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Google Scholar). Similarly, the homeobox transcription factor Hox B3 promotes angiogenesis by up-regulating ephrin-A1 (6Myers C. Charboneau A. Boudreau N. J. Cell Biol. 2000; 148: 343-351Google Scholar). Furthermore, EphA2 signaling is required for the formation of endothelial capillary tubes in vitro (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 7Daniel T.O. Stein E. Cerretti D.P. St. John P.L. Robert B. Abrahamson D.R. Kidney Int. Suppl. 1996; 57: S73-S81Google Scholar) and promotes the formation of blood vessel-like structures by melanoma cells (8Hess A.R. Seftor E.A. Gardner L.M. Carles-Kinch K. Schneider G.B. Seftor R.E. Kinch M.S. Hendrix M.J. Cancer Res. 2001; 61: 3250-3255Google Scholar). The expression of EphA2 appears to be restricted to “activated” adult blood vessels as this receptor has not been detected in either embryonic or adult quiescent blood vessels (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 9Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Google Scholar,10Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624Google Scholar). Ephrin-A1 has also not been detected in adult blood vessels, although it is present in the embryonic vasculature (11McBride J.L. Ruiz J.C. Mech. Dev. 1998; 77: 201-204Google Scholar). In addition to being present in tumor endothelial cells, EphA2 and ephrin-A1 are up-regulated in the transformed cells of a wide variety of tumors including breast, prostate, colon, skin, and esophageal cancers (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 12Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306Google Scholar, 13Walker-Daniels J. Coffman K. Azimi M. Rhim J.S. Bostwick D.G. Snyder P. Kerns B.J. Waters D.J. Kinch M.S. Prostate. 1999; 41: 275-280Google Scholar, 14Easty D.J. Herlyn M. Bennett D.C. Int. J. Cancer. 1995; 60: 129-136Google Scholar, 15Nemoto T. Ohashi K. Akashi T. Johnson J.D. Hirokawa K. Pathobiology. 1997; 65: 195-203Google Scholar). Many factors increase EphA2 expression in cancer cells including the H-Ras oncogene, E-cadherin, members of the p53 family of transcriptional regulators, DNA damage, and loss of estrogen receptors and c-Myc (16Andres A.C. Reid H.H. Zurcher G. Blaschke R.J. Albrecht D. Ziemiecki A. Oncogene. 1994; 9: 1461-1467Google Scholar, 17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar, 18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar). Because the tumor vasculature is discontinuous and leaky in nature, it is possible to utilize the up-regulation of EphA2 and ephrin-A1 to deliver cancer-eradicating agents to both blood vessels and tumor cells (19Dvorak H.F. Nagy J.A. Dvorak J.T. Dvorak A.M. Am. J. Pathol. 1988; 133: 95-109Google Scholar). Indeed, systemically administered biological agents can easily penetrate into tumors from the blood circulation (20Essler M. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2252-2257Google Scholar). Selectively targeting EphA2 and ephrin-A1 is a challenging task because these proteins belong to large families of closely related proteins (21Eph Nomenclature Committee Cell. 1997; 90: 403-404Google Scholar). One approach that has been successfully used to identify peptides that exhibit selectivity for their targets is to screen random peptide libraries displayed on the surface of filamentous bacteriophage (22Koivunen E. Wang B. Ruoslahti E. J. Cell Biol. 1994; 124: 373-380Google Scholar, 23Koivunen E. Arap W. Valtanen H. Rainisalo A. Medina O.P. Heikkila P. Kantor C. Gahmberg C.G. Salo T. Konttinen Y.T. Sorsa T. Ruoslahti E. Pasqualini R. Nat. Biotechnol. 1999; 17: 768-774Google Scholar, 24Smith G.P. Science. 1985; 228: 1315-1317Google Scholar, 25Devlin J.J. Panganiban L.C. Devlin P.E. Science. 1990; 249: 404-406Google Scholar). Therefore, we used a phage display approach to search for peptides that bind selectively to the extracellular domains of EphA2 and ephrin-A1. Remarkably the two peptides identified bind selectively to EphA2, but not other Eph receptors, and antagonize ephrin binding. Intriguingly, at least one of the peptides has bioactive properties in that it stimulates EphA2 tyrosine phosphorylation and signaling. In addition, this peptide delivers phage particles to EphA2-expressing cells and thus can have therapeutic value in delivering agents to tissues that express EphA2. Thus, Eph receptor-binding peptides with different agonistic and drug targeting activities can have important therapeutic applications. DISCUSSIONWe have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Google Scholar, 33Olson T.A. Mohanraj D. Roy S. Ramakrishnan S. Int. J. Cancer. 1997; 73: 865-870Google Scholar, 34Ellerby H.M. Arap W. Ellerby L.M. Kain R. Andrusiak R. Rio G.D. Krajewski S. Lombardo C.R. Rao R. Ruoslahti E. Bredesen D.E. Pasqualini R. Nat. Med. 1999; 5: 1032-1038Google Scholar, 35Arap W. Haedicke W. Bernasconi M. Kain R. Rajotte D. Krajewski S. Ellerby H.M. Bredesen D.E. Pasqualini R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1527-1531Google Scholar, 36Gerlag D.M. Borges E. Tak P.P. Ellerby H.M. Bredesen D.E. Pasqualini R. Ruoslahti E. Firestein G.S. Arthritis Res. 2001; 3: 357-361Google Scholar). The YSA peptide has the added benefit in that it stimulates EphA2 activation, which likely mediates internalization of the receptor and the peptide (37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar, 39van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). Therefore, toxic or apoptotic substances could be delivered intracellularly to selectively kill cells (34Ellerby H.M. Arap W. Ellerby L.M. Kain R. Andrusiak R. Rio G.D. Krajewski S. Lombardo C.R. Rao R. Ruoslahti E. Bredesen D.E. Pasqualini R. Nat. Med. 1999; 5: 1032-1038Google Scholar). Furthermore, activation of EphA2 signaling induced by the YSA peptide should reduce proliferation, invasiveness, and metastatic behavior of EphA2-expressing cancer cells (28Miao H. Wei B.R. Peehl D.M. Li Q. Alexandrou T. Schelling J.R. Rhim J.S. Sedor J.R. Burnett E. Wang B.C. Nature Cell Biol. 2001; 3: 527-530Google Scholar, 37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar). This is consistent with the finding that EphA2 activation correlates with decreased malignancy of breast and prostate cancer cells and reverses the transforming effects of EphA2 overexpression (18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar, 37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar). Intriguingly, EphA2 activation could sensitize cells to apoptotic stimuli when the YSA peptide is used to deliver cytotoxic agents (17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar).Phage-displayed peptides isolated by panning on receptors often bind within ligand-binding sites (40Koivunen E. Gay D.A. Ruoslahti E. J. Biol. Chem. 1993; 268: 20205-20210Google Scholar, 41Wang B. Yang H. Liu Y.C. Jelinek T. Zhang L. Ruoslahti E. Fu H. Biochemistry. 1999; 38: 12499-12504Google Scholar, 42Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Google Scholar, 43Rajotte D. Ruoslahti E. J. Biol. Chem. 1999; 274: 11593-11598Google Scholar) and can mimic natural receptor-binding motifs of ligands (42Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Google Scholar, 44Yayon A. Aviezer D. Safran M. Gross J.L. Heldman Y. Cabilly S. Givol D. Katchalski-Katzir E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10643-10647Google Scholar, 45Wright R.M. Gram H. Vattay A. Byme S. Lake P. Dottavio D. Bio-Technology. 1995; 13: 165-169Google Scholar). Our evidence strongly suggests that the YSA and SWL peptides bind to the interface of EphA2 that mediates the initial high affinity heterodimerization with ephrins (31Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Google Scholar). First, the peptides interact with the ligand-binding globular domain of EphA2 and antagonize ephrin binding. Second, at least one of the peptides causes EphA2 phosphorylation and signaling, similar to ephrins. Third, the YSA and SWL peptide, as ephrin-A5, bind to a site whose affinity is regulated by the ephrin-derived A5 peptide (Fig. 5,B and C). This effect of the A5 peptide, which presumably binds to the low affinity tetramerization interface of EphA2 (31Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Google Scholar), also suggests a previously unrecognized allosteric regulation between the two ephrin-binding sites of an Eph receptor.The YSA and SWL peptides bind selectively only to EphA2. Therefore these peptides have features that confer specificity and are not shared by the ephrins and the ephrin-derived peptides. However, the two related YSA and SWL peptides do show similarity to receptor-binding sequences of A-ephrins, including a conserved ΦxxΦ motif (where Φ is an aromatic amino acid and x is a non-conserved amino acid) present in both the dimerization and tetramerization interfaces. Understanding the structural determinants of peptide-binding promiscuityversus specificity and high affinity may allow the rational design of new peptides that bind with high affinity to specific Eph receptors.Conceivably, phage display could also be used to isolate peptides that specifically bind to each of the fifteen known Eph receptors (cbweb.med.harvard.edu/eph-nomenclature). Different Eph receptors have been implicated in various types of cancer and should be investigated as possible therapeutic targets (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Indeed, we have already isolated peptides that bind EphB4 (data not shown), another receptor expressed in blood vessels and up-regulated in tumors (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Eph receptors could also be targets for promoting nerve regeneration, where ephrin mimetic peptides could desensitize regenerating nerves to the repulsive effects of ephrins up-regulated at injury sites (46Hornberger M.R. Dutting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. Tanaka H. Drescher U. Neuron. 1999; 22: 731-742Google Scholar, 47Miranda J.D. White L.A. Marcillo A.E. Willson C.A. Jagid J. Whittemore S.R. Exp. Neurol. 1999; 156: 218-222Google Scholar, 48Rodger J. Lindsey K.A. Leaver S.G. King C.E. Dunlop S.A. Beazley L.D. Eur. J. Neurosci. 2001; 14: 1929-1936Google Scholar).Bioactive peptides that selectively target an Eph receptor also represent unique reagents for developmental studies to interfere with the activity of a specific receptor, a feat not possible by using the naturally occurring ligands. In addition, such peptides will be useful to discern the signal transduction mechanisms of different Eph receptors in cells expressing multiple receptors. Small-molecule ephrin mimetics with exclusive specificity have great potential value as tools to characterize Eph receptor function and as therapeutics to change such function in disease. We have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Google Scholar, 33Olson T.A. Mohanraj D. Roy S. Ramakrishnan S. Int. J. Cancer. 1997; 73: 865-870Google Scholar, 34Ellerby H.M. Arap W. Ellerby L.M. Kain R. Andrusiak R. Rio G.D. Krajewski S. Lombardo C.R. Rao R. Ruoslahti E. Bredesen D.E. Pasqualini R. Nat. Med. 1999; 5: 1032-1038Google Scholar, 35Arap W. Haedicke W. Bernasconi M. Kain R. Rajotte D. Krajewski S. Ellerby H.M. Bredesen D.E. Pasqualini R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1527-1531Google Scholar, 36Gerlag D.M. Borges E. Tak P.P. Ellerby H.M. Bredesen D.E. Pasqualini R. Ruoslahti E. Firestein G.S. Arthritis Res. 2001; 3: 357-361Google Scholar). The YSA peptide has the added benefit in that it stimulates EphA2 activation, which likely mediates internalization of the receptor and the peptide (37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar, 39van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). Therefore, toxic or apoptotic substances could be delivered intracellularly to selectively kill cells (34Ellerby H.M. Arap W. Ellerby L.M. Kain R. Andrusiak R. Rio G.D. Krajewski S. Lombardo C.R. Rao R. Ruoslahti E. Bredesen D.E. Pasqualini R. Nat. Med. 1999; 5: 1032-1038Google Scholar). Furthermore, activation of EphA2 signaling induced by the YSA peptide should reduce proliferation, invasiveness, and metastatic behavior of EphA2-expressing cancer cells (28Miao H. Wei B.R. Peehl D.M. Li Q. Alexandrou T. Schelling J.R. Rhim J.S. Sedor J.R. Burnett E. Wang B.C. Nature Cell Biol. 2001; 3: 527-530Google Scholar, 37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar). This is consistent with the finding that EphA2 activation correlates with decreased malignancy of breast and prostate cancer cells and reverses the transforming effects of EphA2 overexpression (18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar, 37Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B.C. Brackenbury R. Kinch M.S. Cell Growth Differ. 1999; 10: 629-638Google Scholar, 38Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2002; 62: 2840-2847Google Scholar). Intriguingly, EphA2 activation could sensitize cells to apoptotic stimuli when the YSA peptide is used to deliver cytotoxic agents (17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar). Phage-displayed peptides isolated by panning on receptors often bind within ligand-binding sites (40Koivunen E. Gay D.A. Ruoslahti E. J. Biol. Chem. 1993; 268: 20205-20210Google Scholar, 41Wang B. Yang H. Liu Y.C. Jelinek T. Zhang L. Ruoslahti E. Fu H. Biochemistry. 1999; 38: 12499-12504Google Scholar, 42Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Google Scholar, 43Rajotte D. Ruoslahti E. J. Biol. Chem. 1999; 274: 11593-11598Google Scholar) and can mimic natural receptor-binding motifs of ligands (42Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Google Scholar, 44Yayon A. Aviezer D. Safran M. Gross J.L. Heldman Y. Cabilly S. Givol D. Katchalski-Katzir E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10643-10647Google Scholar, 45Wright R.M. Gram H. Vattay A. Byme S. Lake P. Dottavio D. Bio-Technology. 1995; 13: 165-169Google Scholar). Our evidence strongly suggests that the YSA and SWL peptides bind to the interface of EphA2 that mediates the initial high affinity heterodimerization with ephrins (31Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Google Scholar). First, the peptides interact with the ligand-binding globular domain of EphA2 and antagonize ephrin binding. Second, at least one of the peptides causes EphA2 phosphorylation and signaling, similar to ephrins. Third, the YSA and SWL peptide, as ephrin-A5, bind to a site whose affinity is regulated by the ephrin-derived A5 peptide (Fig. 5,B and C). This effect of the A5 peptide, which presumably binds to the low affinity tetramerization interface of EphA2 (31Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Google Scholar), also suggests a previously unrecognized allosteric regulation between the two ephrin-binding sites of an Eph receptor. The YSA and SWL peptides bind selectively only to EphA2. Therefore these peptides have features that confer specificity and are not shared by the ephrins and the ephrin-derived peptides. However, the two related YSA and SWL peptides do show similarity to receptor-binding sequences of A-ephrins, including a conserved ΦxxΦ motif (where Φ is an aromatic amino acid and x is a non-conserved amino acid) present in both the dimerization and tetramerization interfaces. Understanding the structural determinants of peptide-binding promiscuityversus specificity and high affinity may allow the rational design of new peptides that bind with high affinity to specific Eph receptors. Conceivably, phage display could also be used to isolate peptides that specifically bind to each of the fifteen known Eph receptors (cbweb.med.harvard.edu/eph-nomenclature). Different Eph receptors have been implicated in various types of cancer and should be investigated as possible therapeutic targets (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Indeed, we have already isolated peptides that bind EphB4 (data not shown), another receptor expressed in blood vessels and up-regulated in tumors (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Eph receptors could also be targets for promoting nerve regeneration, where ephrin mimetic peptides could desensitize regenerating nerves to the repulsive effects of ephrins up-regulated at injury sites (46Hornberger M.R. Dutting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. Tanaka H. Drescher U. Neuron. 1999; 22: 731-742Google Scholar, 47Miranda J.D. White L.A. Marcillo A.E. Willson C.A. Jagid J. Whittemore S.R. Exp. Neurol. 1999; 156: 218-222Google Scholar, 48Rodger J. Lindsey K.A. Leaver S.G. King C.E. Dunlop S.A. Beazley L.D. Eur. J. Neurosci. 2001; 14: 1929-1936Google Scholar). Bioactive peptides that selectively target an Eph receptor also represent unique reagents for developmental studies to interfere with the activity of a specific receptor, a feat not possible by using the naturally occurring ligands. In addition, such peptides will be useful to discern the signal transduction mechanisms of different Eph receptors in cells expressing multiple receptors. Small-molecule ephrin mimetics with exclusive specificity have great potential value as tools to characterize Eph receptor function and as therapeutics to change such function in disease. We thank Fernando Ferrer, Christian Lombardo, Arnold Satterthwait, Fatima Valencia, Steven Kridel, and Jason Hoffman for technical help and advice; John Flanagan for the AP vector; and Keith Murai for comments on the manuscript.