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Evaluation of Vegf Peptide Mimics As Inhibitors of Angiogenesis

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EVALUATION OF VEGF PEPTIDE MIMICS AS INHIBITORS OF ANGIOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Daniele Vicari, M.S. * * * * * The Ohio State University 2008 Dissertation Committee: Dr.Pravin T.P. Kaumaya, Adviser Approved by Dr. Neil R. Baker Dr. Clay B. Marsh Dr. Marshall V. Williams Adviser Microbiology Graduate Program ABSTRACT Rationally designed peptide mimics that can substitute for protein biological activity are good candidates for a new generation of therapeutic drugs. Peptides have higher tissue penetration, relatively low cost production and can be easily modified for improved bioavailability. The formation of new blood vessels, angiogenesis, is required for cancer tumor growth and metastasis. Since vascular endothelium growth factor (VEGF) and VEGFR-2 is the most significant interaction for angiogenesis activation; we designed conformational peptides that can mimic the VEGF binding site. The inhibitory potential of peptide mimic was evaluated in several angiogenesis model assays such as HUVEC proliferation, tube formation in Matrigel and migration. The VEGF conformational peptide mimic, VEGF-P3(CYC), was synthesized with two extra cysteine residues at positions 80 and 92 , which upon cyclization constrain the peptide in a loop conformation, in order to better mimic the anti-parallel twisted structure in the VEGF native protein structure. This constrained peptide demonstrated the best angiogenic inhibitory effects in in vitro assays and significantly delayed tumor growth in VEGF-/+/Neu2-5-/+ transgenic animal model when compared to VEGF-P3(CYC), non- cyclic peptide, and VEGF 102-122, natural sequence peptide. ii Peptides in which the amino acid sequence and the chirality are reversed are termed retro-inverso peptide. We synthesized and tested the angiogenesis inhibitory effects of retro-inverso peptide VEGF-RI-P4(CYC) which demonstrated some degree of anti-angiogenesis effects in vitro. Chimeric VEGF peptide mimics incorporating promiscuous T-cell epitope were synthesized and used to elicit antibodies in rabbits. The conformational peptide VEGF- P3(CYC) showed to be the best mimitope of VEGF, generating antibodies with tighter affinity to VEGF. Preliminary results showed that the anti-MVF-VEGF 102-122 delayed tumor growth in VEGF-/+/Neu2-5-/+ transgenic mice. However, anti-MVF-VEGF- P3(CYC) was the best inhibitor in angiogenesis in vitro assays likely due to the blockade of VEGF-VEGFR-2 interaction by binding to VEGF binding site. iii Dedicated to people that I love, particularly my husband, Sandro Ataide and my parents, Nelson and Maria Sirlei Vicari iv ACKNOWLEDGMENTS I wish to thank my advisor, Dr. Pravin Kaumaya, for the opportunity to work in his Lab as well as for his support and encouragement. His enthusiasm and interest have been essential for the success of this project. I am also grateful to my committee members, Drs. Neil R. Baker, Paula Bryant, Dr. Clay B. Marsh and Dr. Williams for their time and guidance. I thank Dr. Sharad Rawale for his assistance with the synthesis of peptides and for his helpful suggestions. I am grateful to all members of the Kaumaya lab past and present, particularly Dr. Joan Garrett, Dr. Stephanie Allen, Dr. Marcus Lynch, Dr. Bing Wang, Danielle Carbin, and Eric Liotta. Additionally, I thank those who helped me throughout graduate school, specially Dr. Corinne Hausmann, Dr. Aurea Sousa, Dr. Kimberly Roth, and Dr. Daniela Oliveira. I would like to thank God, my family and friends. I thank my parents for their unconditional love, inspiration, and encouragement to pursue my interests. I thank my brothers and sister for their friendship. I am grateful to all my friends who shared wonderful times with me, maintaining my sanity throughout this process. Finally, I thank my beloved husband, Sandro, for all the love and support. He has always believed on me, giving me persistence to be better in all areas of my life. v VITA January 10, 1976...................................... Born – Sao Paulo, SP, Brazil 1999… …………………………………B.S. Chemistry, University of Campinas (UNICAMP), Brazil 2002…………………………………… M.S.Biotechnology, University of São Paulo (USP), Brazil, 2003 – present………………………… Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication • Vicari, D.; Rawale, S.; Liotta, E; Kaumaya, P. (2008)” Engineered Conformation- dependent VEGF Peptide Mimics are Effective in inhibiting VEGF signaling pathways” in preparation • Vicari, D., and I. Artsimovitch. 2004. Virulence regulators RfaH and YaeQ do not operate in the same pathway. Mol Genet Genomics 272:489. FIELDS OF STUDY Major Field: Microbiology vi TABLE OF CONTENTS Page Abstract………………………………………...………………………………………….ii Dedication……………………………………………………………………………...…iv Acknowledgments…………………………………………………………………..…….v Vita…………………………………………………………………………………….....vi List of Tables……………………………………………………………………………..x List of Figures……………………………………………………………………………xi Abbreviations……………………………………………………………………………xiv Chapters: 1. Introduction…………………………………………………………………………….1 1.1 Angiogenesis…………………………………………..…………………...….1 1.2 VEGF …………..……………………………………………………………..5 1.3 Peptides……….……………………………………………………...………13 1.4 Hypothesis and Overview of Chapters 2-4.………………………………….15 1.5 Figures……….……………………...………………………………………..18 vii 2. VEGF peptide mimics Demonstrate in Vitro and In Vivo Antitumor Inhibition of VEGF Dependent pathways……………………..………………....………..………22 2.1 Introduction…………………………………………………………………..22 2.2 Materials and Methods……………………………………………………….25 2.3 Results………………………………………………………………………..31 2.4 Discussion………………..…………………………………………………..40 2.5 Tables and Figures……….…………………………………………………..45 3. Retro-inverso VEGF peptide mimics and their Effects on VEGF Dependent Pathways………………………………………………………….……………….…63 3.1 Introduction…………………………………………………………………..63 3.2 Materials and Methods……………………………………………………….65 3.3 Results………………………………………………………………………..70 3.4 Discussion………………..…………………………………………………..75 3.5 Tables and Figures……….……………………………………………..……79 4. VEGF Peptide Mimics are Immunogenic and Antigenic…...……………………..…87 4.1 Introduction…………………………………………………………………..87 4.2 Materials and Methods……………………………………………………….90 4.3 Results………………………………………………………………………..95 4.4 Discussion………………..……………………………………...………….100 4.5 Tables and Figures……….…………………………………………..…..…105 viii 5. Summary and Future Perspectives…………………………………...……………...117 Bibliography…………………………………………………………………………....119 ix LIST OF TABLES Table Page 2.1 Amino acid sequences and MW of VEGF peptide mimics …..………………..……45 2.2 Kinetic parameters of VEGF peptide mimics binding to VEGFR-2..….……………46 4.1 Kinetics parameters of anti-VEGF peptide mimics binding to rhVEGF ..………....105 x LIST OF FIGURES Figure Page 1.1 Representation of angiogenesis-dependent tumor growth metastasis……………….18 1.2 VEGF and VEGF receptors ………………………………..…………………….….19 1.3 Representation of the strategy to inhibit angiogenesis blocking VEGF-VEGFR-2 interaction ……………………………………………………………………..…….20 1.4 VEGF binding sites …………………………………….…….……………….…..…21 2.1 Representation of the region selected for VEGF peptide mimic …..………….…….47 2.2 Schematic representation VEGF peptide mimic design …………………….………48 2.3 Schematic representation of VEGFR-2 activation upon VEGF binding ……..……..49 2.4 Secondary structure analysis of VEGF-P3 peptides constructs .…………….………50 2.5 Surface Plasmon Resonance Binding assay …………………………………………51 2.6 Surface Plasmon Resonance Competition assay …………….………………………53 2.7 Inhibition of KDR Phosphorylation in HUVEC...……………………….…………..54 2.8 Inhibition of KDR Phosphorylation in 293/KDR cell line.………………………….55 2.9 Inhibition of p44/42 MAPK Phosphorylation in HUVEC.………………………….56 2.10 HUVEC proliferation assay using peptide as inhibitors …………………………...57 2.11 Tube formation in Matrigel assay using peptides as inhibitors….…………………58 2.12 HUVEC migration in scratch wound assay using peptides as inhibitors…………...60 xi 2.13 Passive treatment using peptides as inhibitors of mammary tumor development in mouse VEGF-/+/ Neu2-5-/+ ………………………………………………………..62 3.1 Schematic representation of the effects of retro-inverso peptides……………….…..79 3.2 CD spectra of retro-inverso VEGF peptide mimic.…………………………..….…..80 3.3 Passive treatment using retro-inverso peptide VEGF-P4(CYC) as inhibitors of mammary tumor development in mouse VEGF-/+/ Neu2-5-/+....................................81 3.4 Inhibition of KDR Phosphorylation in 293/KDR cell line……………..………...….82 3.5 Inhibition of p42/44 MAPK Phosphorylation in HUVEC ……………………….....83 3.6 Tube formation in Matrigel assay using peptides as inhibitors …….…………….....84 3.7 HUVEC proliferation assay using peptide as inhibitors …………...………………..86 4.1 Antibody responses elicited by peptide vaccines in outbred rabbits………...……..106 4.2 VEGF ELISA…………………………………………. …………………….……..107 4.3 Anti-MVF-VEGF-102-122 competitive inhibition ELISA ………………………..108 4.4 Anti-MVF-VEGF-P3(NC) competitive inhibition ELISA ….……………………..109 4.5 Anti-MVF-VEGF-P3(CYC) competitive inhibition ELISA ..……………………..110 4.6 Binding kinetics of anti-peptides to VEGF ………………………………………..111 4.7 Inhibition of KDR Phosphorylation in 293/KDR cell line .………………………..113 4.8 Tube formation in Matrigel assay using peptides as inhibitors…………………….114 4.9 Proliferation assay using antibodies as inhibitors………………………………….115 xii 4.10 Passive treatment using anti-peptides as inhibitors of mammary tumor development in mouse VEGF-/+/ Neu2-5-/+……………………………………………………116
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