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Characterization of a Bicyclic Peptide Neuropilin-1 (NP-1) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-1 Binding and Role of NP-1 in KDR Signaling

2006; Elsevier BV; Volume: 281; Issue: 19 Linguagem: Inglês

10.1074/jbc.m512121200

ISSN

1083-351X

Autores

Haiyan Jia, Azadeh Bagherzadeh, Basil Hartzoulakis, Ashley Jarvis, Marianne Löhr, Shaheda Shaikh, Rehan Aqil, Lili Cheng, Michelle Tickner, Diego Esposito, Richard Harris, Paul C. Driscoll, David L. Selwood, Ian Zachary,

Tópico(s)

Lymphatic System and Diseases

Resumo

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (Ki 1.2μm) and effectively (>95% inhibition at 100 μm) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-γ tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1. Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (Ki 1.2μm) and effectively (>95% inhibition at 100 μm) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-γ tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1. Vascular endothelial growth factor A (VEGF-A) 2The abbreviations used are: VEGF-A, vascular endothelial growth factor A; ERK1/2, extracellular signal-regulated kinases 1 and 2; HUVEC, human umbilical vein endothelial cells; KDR, kinase insert domain-containing receptor; NMR, nuclear magnetic resonance; NP, neuropilin; PAE/KDR, porcine aortic endothelial cells expressing KDR; PAE/NP-1, porcine aortic endothelial cells expressing NP-1; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; FBS, fetal bovine serum; HPLC, high pressure liquid chromatography; EGFR, epidermal growth factor receptor. 2The abbreviations used are: VEGF-A, vascular endothelial growth factor A; ERK1/2, extracellular signal-regulated kinases 1 and 2; HUVEC, human umbilical vein endothelial cells; KDR, kinase insert domain-containing receptor; NMR, nuclear magnetic resonance; NP, neuropilin; PAE/KDR, porcine aortic endothelial cells expressing KDR; PAE/NP-1, porcine aortic endothelial cells expressing NP-1; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; FBS, fetal bovine serum; HPLC, high pressure liquid chromatography; EGFR, epidermal growth factor receptor. is an essential mediator of vasculogenesis and angiogenesis during embryonic development and plays a central role in pathophysiological neovascularization in human disease (1Ferrara N. Amer J. Physiol. Cell Physiol. 2001; 280: C1358-C1366Crossref PubMed Google Scholar, 2Carmeliet P. Jain R. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7354) Google Scholar). Five isoforms of VEGF-A have been identified, VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189, and VEGF-A206, generated by alternative mRNA splicing of a single VEGF gene transcript containing 8 exons (3Houck K.A. Ferrara N. Winer J. Cachianes G. Li B. Leung D.W. Mol. Endocrinol. 1991; 5: 1806-1814Crossref PubMed Scopus (1230) Google Scholar, 4Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar, 5Poltorak Z. Cohen T. Sivan R. Kandelis Y. Spira G. Vlodavsky I. Keshet E. Neufeld G. J. Biol. Chem. 1997; 272: 7151-7158Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar), of which VEGF-A165 is the most abundant and biologically active.VEGF-A165 exerts its biological effects through high affinity binding to two tyrosine kinase receptors, Flt-1 (VEGFR1) and KDR (VEGFR2), which are expressed in most vascular endothelial cells (6Zachary I. Biochem. Soc. Trans. 2003; 31: 1171-1177Crossref PubMed Google Scholar). KDR binds VEGF-A165 with lower affinity than Flt-1, and is also recognized by VEGF-C, VEGF-D, and VEGF-E (7Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 8Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1011) Google Scholar, 9Ogawa S. Oku A. Sawano A. Yamaguchi S. Yazaki Y. Shibuya M. J. Biol. Chem. 1998; 273: 31273-31282Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 10Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (229) Google Scholar, 11Meyer M. Clauss M. Lepple Wienhues A. Waltenberger J. Augustin H.G. Ziche M. Lanz C. Buttner M. Rziha H.J. Dehio C. EMBO J. 1999; 18: 363-374Crossref PubMed Scopus (409) Google Scholar), whereas Flt-1 is also a receptor for PlGF and VEGF-B (12Clauss M. Weich H. Breier G. Knies U. Rockl W. Waltenberger J. Risau W. J. Biol. Chem. 1996; 271: 17629-17634Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, 13Olofsson B. Korpelainen E. Pepper M.S. Mandriota S.J. Aase K. Kumar V. Gunji Y. Jeltsch M.M. Shibuya M. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11709-11714Crossref PubMed Scopus (443) Google Scholar). After binding and activation of KDR, VEGF-A165 stimulates ERK activation and an array of other early signaling events followed by short and long term cellular biological effects including production of prostacyclin (PGI2) and nitric oxide, increased cell survival, cell migration, proliferation, and angiogenesis (14Zachary I. Gliki G. Cardiovasc. Res. 2001; 49: 568-581Crossref PubMed Scopus (567) Google Scholar, 15Waltenberger J. Claesson Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 16Laitinen M. Zachary I. Breier G. Pakkanen T. Hakkinen T. Luoma J. Abedi H. Risau W. Soma M. Laakso M. Martin J.F. Yla Herttuala S. Hum. Gene Ther. 1997; 8: 1737-1744Crossref PubMed Scopus (180) Google Scholar, 17Wheeler-Jones C. Abu-Ghazaleh R. Cospedal R. Houliston R.A. Martin J. Zachary I. FEBS Lett. 1997; 420: 28-32Crossref PubMed Scopus (235) Google Scholar, 18Jia H. Bagherzadeh A. Bicknell R. Duchen M.R. Liu D. Zachary I. J. Biol. Chem. 2004; 279: 36148-36157Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Gerber H.P. McMurtrey A. Kowalski J. Yan M. Keyt B.A. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 30336-30343Abstract Full Text Full Text PDF PubMed Scopus (1731) Google Scholar, 20Takahashi T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (594) Google Scholar, 21Gliki G. Abu-Ghazaleh R. Jezequel S. Wheeler-Jones C. Zachary I. Biochem. J. 2001; 353: 503-512Crossref PubMed Scopus (86) Google Scholar, 22Abedi H. Zachary I. J. Biol. Chem. 1997; 272: 15442-15451Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The function of Flt-1 in the endothelium is unclear, but it is thought to regulate the activity of VEGF-A partly by acting as a decoy receptor, and in part through direct regulatory effects on KDR (23Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Neuropilin-1 (NP-1) has recently been identified as a non-tyrosine kinase receptor for VEGF-A165, the heparin-binding PlGF-2 isoform, VEGF-B, and VEGF-E (24Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 25Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 26Makinen T. Olofsson B. Karpanen T. Hellman U. Soker S. Klagsbrun M. Eriksson U. Alitalo K. J. Biol. Chem. 1999; 274: 21217-21222Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). NP-1 was first identified as a receptor for semaphorin 3A (Sema3A), a member of a family of proteins involved in axonal guidance (27He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 28Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar), and is expressed in endothelial cells, several tumor cell types, and in certain classes of neuron including cells of the dorsal root ganglion (DRG), olfactory, and optic nerves (24Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 29Fujisawa H. Kitsukawa T. Kawakami A. Takagi S. Shimizu M. Hirata T. Cell Tissue Res. 1997; 290: 465-470Crossref PubMed Scopus (77) Google Scholar). NP-2 has a similar domain structure to NP-1 with 44% amino acid identity, and exhibits a distinct expression pattern in the developing nervous system (28Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 30Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Sema3A induces neuronal growth cone collapse specifically through NP-1, whereas sema 3B, 3C, 3E, and 3F recognize both neuropilins, acting as NP-1 antagonists and NP-2 agonists (30Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 31Chen H. He Z. Bagri A. Tessier-Lavigne M. Neuron. 1998; 21: 1283-1290Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 32Giger R.J. Urquhart E.R. Gillespie S.K. Levengood D.V. Ginty D.D. Kolodkin A.L. Neuron. 1998; 21: 1079-1092Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 33Takahashi T. Nakamura F. Jin Z. Kalb R.G. Strittmatter S.M. Nat. Neurosci. 1998; 1: 487-493Crossref PubMed Scopus (186) Google Scholar). A growing body of evidence also indicates a role for neuropilins in angiogenesis. Overexpression of NP-1 in mice results in increased capillary formation, vasodilatation, and malformation of the heart (34Kitsukawa T. Shimono A. Kawakami A. Kondoh H. Fujisawa H. Development. 1995; 121: 4309-4318Crossref PubMed Google Scholar), whereas mice deficient in NP-1 exhibit defects in embryonic axonal patterning and an array of vascular abnormalities including defective development of large vessels and impaired neural and yolk sac vascularization (35Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar). Inactivation of both NP-1 and NP-2 causes a more severe failure of embryonic vascularization resulting in death at E8.5 (36Takashima S. Kitakaze M. Asakura M. Asanuma H. Sanada S. Tashiro F. Niwa H. Miyazaki J. Hirota S. Kitamura Y. Kitsukawa T. Fujisawa H. Klagsbrun M. Hori M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3657-3662Crossref PubMed Scopus (320) Google Scholar). Despite the strong evidence that NP-1 is essential for normal vascular development, the underlying mechanisms remain obscure, and the role of NP-1 in the biological functions of VEGF-A are not fully understood.We recently reported that a specific bicyclic peptide based on the C-terminal NP binding domain of VEGF-A165 (37Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) is an antagonist of VEGF binding to NP-1 and inhibits the anti-chemorepulsive effect of VEGF-A165 in DRG neuronal explants (38Cheng L. Jia H. Löhr M. Bagherzadeh A. Holmes D. Selwood D. Zachary I. J. Biol. Chem. 2004; 279: 30654-30661Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here, we have identified the key features of EG3287 responsible for its antagonistic properties through a detailed structure-function analysis, and investigated its biological effects in vascular endothelial cells. Our findings show that the C-terminal six amino acid domain encoded by exon 8 plays a crucial role in VEGF-A165 binding to NP-1. Evaluation of EG3287 in endothelial cells demonstrates that VEGF-A165 binding to NP-1 is required for stable binding to KDR, full activation of KDR and downstream signaling and biological responses. This antagonist should be a valuable tool for probing the biological role of NP-1 in diverse cell types, and will also be useful for designing improved neuropilin antagonists.EXPERIMENTAL PROCEDURESPeptide Synthesis—Amino acids were purchased from Calbiochem Novabiochem, Alexis (both Nottingham, UK) or Bachem AG (Bubendorf, Switzerland). Linear, monocyclic, and bicyclic peptides based on residues 111–165 in VEGF-A165 were synthesized by Fmoc solid-phase synthesis using either Wang or Rink amide linkers. Side chain protections were: Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Cys(Acm), Gln-(Trt), Glu(OtBu), Lys(Boc), Ser(tBu), Thr(tBu), and Tyr(tBu); additional linear peptides (peptides 12–19) were synthesized by Pepceuticals Ltd; additional bicyclic peptides (peptides 3–8) were synthesized by Bachem (UK) Ltd.Linear peptides were synthesized by an automated multiple solid phase approach using the Fmoc-Arg(Pbf)-p-alkoxybenzyl alcohol resin (0.59 mmol/g loading) or Fmoc-Rink Amide MBHA resin (0.59 mmol/g or 0.68 mmol/g loading). Amino acids were attached by Fmoc strategy on a 25- or 50-μmol scale with a basis coupling time of 30 min followed by a recoupling step. Each amino acid was sequentially coupled to the growing peptide chain from the C to the N termini applying benzotriazol-1-yloxy-Tris-pyrrolidino-phosphonium hexafluoro-phosphate and N-methyl morpholine as coupling reagents via the active ester method. Removal of the N-Fmoc protecting group was carried out with 20% piperidine in DMF followed by sequential washes with DMF and DCM. The coupling reagent, Pybop, NMM, and all amino acid derivatives were dissolved in DMF (0.7 m, 4-fold excess). All solvents used were of HPLC grade quality.The peptides were cleaved from the resin with simultaneous deprotection using 90% trifluoroacetic acid at room temperature for 3 h, either in the presence of 5% thioanisole, 2.5% water, and 2.5% ethanedithiol or with 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane. The cleavage mixture was filtered and precipitated in ice-cold methyl tert-butyl ether. The remaining resin was washed once with the cleavage reagent, filtered, and combined with the previous fractions. The precipitates were collected after centrifugation, washed three times with ice-cold methyl tert-butyl ether, and allowed to dry overnight at room temperature. The crude peptides were dissolved in 15% aqueous acetic acid and lyophilized for 2 days (40 °C, 6 mbar).General Method for Bicyclic Peptide Synthesis—The crude linear precursor, prepared as above, was dissolved in the minimum trifluoroacetic acid and diluted to 2 liter/0.25 mmol with water. The first disulfide bridge was formed between unprotected Cys residues using K3Fe(CN)6. The peptide solution was adjusted to pH 7.5 with aqueous ammonium hydroxide. To this solution, 0.01 m K3Fe(CN)6 was added dropwise to excess, until a slight yellow color remained. The completion of the reaction was confirmed by HPLC sampling after acidification. The pH of the solution was adjusted to 4 using 50% aqueous acetic acid. The crude reaction mixture was stirred with Bio-Rex 70 weak cation-exchange resin (Bio-Rad) overnight and packed into a glass column. After thorough washing with water, the peptide was eluted using 50% aqueous acetic acid and detected by TLC using ninhydrin. Ninhydrin-positive fractions were pooled and lyophilized. Crude material was purified via reverse-phase HPLC (Gilson) using a preparative C-8 column (see conditions for linear peptides). The purified fractions were collected, combined, and lyophilized. The second disulfide bridge was formed via I2-oxidation between Cys(Acm) protected residues. A solution of the peptide (5 mg/ml) in 10% aqueous trifluoroacetic acid was mixed vigorously with 8 equivalents of iodine, and the resulting suspension stirred for 1.5 h.The progress of the cyclization reactions was monitored by analytical reverse-phase LC-MS. At completion of the reaction the excess iodine was quenched using 1 m ascorbic acid. The reaction mixture was diluted ×2 with 0.1% trifluoroacetic acid/water, filtered through a 0.45-μm disposable filter and purified directly via preparative reverse-phase HPLC (Gilson) using a preparative C-8 column (see conditions for linear peptides). The relevant fractions were collected, evaporated, lyophilized, and stored at 4 °C. Confirmation of the structure was performed by analytical reverse-phase LC-MS and/or MALDI mass spectroscopy and amino acid analysis.Alternative Cleavage Procedure for Bicyclic Peptides—The peptides were cleaved from the resin with simultaneous deprotection using 82.5% trifluoroacetic acid at room temperature for 3 h in the presence of 5% thioanisole, 5% water, 2.5% ethanedithiol, and 6% (w/v) phenol. In the case of Cys-containing peptides, the cleavage mixture was filtered and precipitated in ice-cold diethyl ether. The remaining resin was washed once with trifluoroacetic acid, filtered, and combined with the previous fractions. The precipitates were stored at 4°C overnight and were collected by filtration, washed with ice-cold diethyl ether, and allowed to dry at room temperature. For non-Cys-containing peptides the cleavage mixture was filtered, the resin washed with trifluoroacetic acid then dichloromethane, and the filtrates combined and concentrated under vacuum to oil. The peptide was precipitated in ice-cold diethyl ether, collected by filtration and allowed to dry at room temperature. The crude peptides were dissolved in trifluoroacetic acid/acetonitrile/water and lyophilized overnight (-50 °C, 6 mbar). Crude peptides were characterized by reverse-phase HPLC (Gilson) using an analytical C-18 column (Vydac 218TP54, 250 × 4.6 mm, 5-μm particle size, and 300 Å pore size) and a linear AB gradient of 0–100% for B over 40 min at a flow rate of 1 ml/min, where eluent A was 0.1% trifluoroacetic acid /water and eluent B was 0.1% trifluoroacetic acid in 60% CH3CN/water. Mass was confirmed using MALDI-MS, and Ellman's color test confirmed the presence of free sulfhydryl groups where applicable.NMR Spectroscopy and Three-dimensional Structure Determination— For NMR studies, 15 mg of EG3287 (Ser138-Arg165, VEGF-A165) was prepared in 90% H2O, 10% D2O, with the resulting solution at pH 2.3. NMR spectra were acquired at 293 K and 298 K on a Varian INOVA spectrometer (operating at nominal 1H frequency of 600 MHz) equipped with triple resonance probe including Z-axis pulse field gradients. All two-dimensional data were recorded in phase-sensitive mode using the States-TPPI method for quadrature detection, and data were apodized using shifted cosine-bell squared functions, followed by zero-filling once in each dimension. Water suppression was achieved using WATERGATE (39Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3501) Google Scholar). Sequence-specific resonance assignments were obtained from two-dimensional 1H TOCSY (80-ms mixing time), 1H NOESY (100-ms mixing time), [1H,15N] HSQC, and [1H,13C] HSQC spectra (at natural isotopic abundance). All spectra were processed using NMRpipe/NMRDraw (40Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11388) Google Scholar) and analyzed using ANSIG for openGL v1.0.3 (41Kraulis J. Clore G.M. Nilges M. Jones T.A. Pettersson G. Knowles J. Gronenborn A.M. Biochemistry. 1989; 28: 7241-7257Crossref PubMed Scopus (487) Google Scholar). 1H, 13C, and 15N chemical shifts were referenced indirectly to DSS, using absolute frequency ratios (42Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2053) Google Scholar). Interproton distance restraints were derived from two-dimensional 1H NOESY spectra with a mixing time of 250 ms. Cross-peaks were grouped into four categories according to their relative peak intensities: strong, medium, weak, and very weak, and were designated with interproton distance restraint bounds of 1.8–2.5 Å, 1.8–3.0 Å, 1.8–3.5 Å, and 1.8–4.5 Å, respectively. 0.5 Å was added for distances that involved methyl groups. The structure calculations were carried out using the PARALLHDGv5.1 parameter, with the PROLSQ non-bonded energy function (43Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar) within the CNS program (44Wakefield R.I. Smith B.O. Nan X. Free A. Soteriou A. Uhrin D. Bird A.P. Barlow P.N. J. Mol. Biol. 1999; 291: 1055-1065Crossref PubMed Scopus (166) Google Scholar), modified to allow floating stereochemistry, and to include active swapping, of prochiral centers (45Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar). 229 interproton distance restraints (138 intraresidue, 56 sequential, 9 short range, 17 long range, and 9 ambiguous) were applied in restrained molecular dynamics simulated-annealing calculations.Measurement of EG3287 Stability—EG3287 was recovered from tissue culture medium by a 1:1 addition of 20% trifluoroacetic acid and centrifugation of the precipitate. This method resulted in ∼90% recovery. Quantitation of EG3287 was performed by Inpharmatica Ltd (Cambridge, UK) using a liquid chromatography mass spectrometric (LC-MS/MS) assay on a Micromass Quattro Micro.Computational Chemistry—Molecules were analyzed using SYBYL® 7.0 (Tripos Inc., St. Louis, MO). The Biopolymer tools in Sybyl were used to build some of the peptides, check structures for errors, correct atom types, and add/remove hydrogens as necessary. For energy minimizations or molecular dynamics simulations we used starting conformations as described by Fairbrother et al. (46Fairbrother W.J. Champe M.A. Christinger H.W. Keyt B.A. Starovasnik M.A. Structure. 1998; 6: 637-648Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) (VEGF-A55, Protein Data Bank code: 2vgh, Ala111-Arg155, VEGF-A165) or EG3287 VEGF-A55 (Protein Data Bank code: 1KMX, Ser138-Arg155 VEGF-A165). The terminal amino acids and polar side chains were charged, and the peptides were solvated in a box of water molecules (Tripos explicit box solvation algorithm). Electrostatic charges were calculated with the method of Gasteiger and Marsili (47Gasteiger J. Marsili M. Tetrahedron. 1980; 36: 3219-3228Crossref Scopus (3545) Google Scholar). Structures were minimized using the Tripos force field with a steepest descent gradient of 100 iterations followed by a conjugate gradient of 0.01 kcal/mol or a maximum of 10,000K iterations as termination criteria. During minimizations, a 12-Å non-bonded cut-off was applied, and when solvent was not present and ionic strength was set to 4.00.For molecular dynamics simulations, all starting structures were solvated in a cubic box of explicit water molecules (edge 40 Å) using the XFIT solvation algorithm. This led to approximately ∼1700 water molecules depending on the solute starting conformation. All simulations were performed using periodic boundary conditions and the SHAKE algorithm. Constant temperature simulations (NTV, 300°K) were run at a time step of 0.1 ps for a total period of 2000 ps. The Lennard-Jones interactions were evaluated with an 8.0-Å cutoff value. The non-bonded pair list was updated every 100 steps. Generated structures were stored in trajectory files every 5 ps, providing 400 conformers for each run. The collected structural data were analyzed with the graphic tools of the SYBYL® 7.0. All calculations were performed on the dual Hewlett-Packard work station XW6000, 2 × 2.8 GHz CPUs running REDHAT Enterprise Linux WS4.Cell Culture—Human umbilical vein endothelial cells (HUVECs) were obtained from TCS CellWorks (Buckingham, UK) and cultured in endothelial cell basal medium (EBM) supplemented with 10% fetal bovine serum (FBS), 10 ng/ml human epidermal growth factor, 12 μg/ml bovine brain extract, 50 μg/ml gentamicin sulfate, and 50 ng/ml amphotericin-8. Porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) (16Laitinen M. Zachary I. Breier G. Pakkanen T. Hakkinen T. Luoma J. Abedi H. Risau W. Soma M. Laakso M. Martin J.F. Yla Herttuala S. Hum. Gene Ther. 1997; 8: 1737-1744Crossref PubMed Scopus (180) Google Scholar) were provided by Dr. Shay Soker and grown in Ham's F12 medium containing 10% FBS and 25 μg/ml hygromycin B. PAE cells expressing either KDR (PAE/KDR) (36Takashima S. Kitakaze M. Asakura M. Asanuma H. Sanada S. Tashiro F. Niwa H. Miyazaki J. Hirota S. Kitamura Y. Kitsukawa T. Fujisawa H. Klagsbrun M. Hori M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3657-3662Crossref PubMed Scopus (320) Google Scholar) or Flt-1 (PAE/Flt-1) were provided by Professor Lena Claesson-Welsh (Uppsala University, Sweden) and grown in Ham's F12 medium containing 10% FBS and 250 μg/ml Gentamicin G418. MDA-MB-231 breast carcinoma cells (gift of Professor Mike O'Hare) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS.Radiolabeled Ligand Binding—Confluent cells in 24-well plates were washed twice with phosphate-buffered saline. At 4 °C various concentrations of peptides diluted in binding medium (Dulbecco's modified Eagle's medium, 25 mm HEPES pH 7.3 containing 0.1% bovine serum albumin) were added, followed by addition of the indicated concentration of 125I-VEGF-A165 (1200–1800 Ci/mmol, GE Healthcare Plc) or 125I-EGF. After 2 h of incubation at 4 °C (or the indicated time at 37 °C), the medium was aspirated, and washed four times with cold phosphate-buffered saline. The cells were lysed with 0.25 m NaOH, 0.5% SDS solution, and the bound radioactivity of the lysates was measured. Nonspecific binding was determined in the presence of 100-fold excess unlabeled VEGF-A165 or EGF (R & D Systems). Equilibrium dissociation constants (Ki) for peptides were calculated from IC50 values and the Kd of VEGF-A165 for NP-1 (0.3 nm) from Soker et al. (24Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar), using the formula Ki = IC50/1 + ([125I-VEGF-A165]/Kd).Cross-linking—Confluent cells were bound with 125I-VEGF-A165 at 4 °C for 2 h as described above and then washed three times with phosphate-buffered saline. The bound 125I-VEGF-A165 was cross-linked to the cells by incubation with 1.5 mm disuccinimidyl suberate (DSS) for 20 min at room temperature. After three washes with phosphate-buffered saline at 4 °C, the cells were solubilized in lysis buffer (64 mm Tris-HCl, pH 6.8, 0.2 mm Na3VO4, 2% SDS, 10% glycerol, 0.1 mm AEBSF, 5 μg/ml leupeptin) and scraped off the plates. After centrifugation at 16,000 × g for 20 min at 4 °C, cross-linked 125I-VEGF-A165-receptor complexes were subjected to 7.5% SDS-PAGE. Gels were dried and exposed to x-ray film.Immunoblotting—Cells were pretreated with peptides for 15 min followed by treatment with growth factors for 10 min, and cells were immediately extracted by lysis buffer (64 mm Tris-HCl, pH 6.8, 0.2 mm Na3VO4, 2% SDS, 10% glycerol, 0.1 mm AEBSF, 5 μg/ml leupeptin). Activation of KDR, epidermal growth factor receptor (EGFR), PLC-γ, Akt, and ERKs1/2 was determined by immunoblotting cell extracts with a

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