Developments and Applications of Cyclic Cell Penetrating
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DEVELOPMENTS AND APPLICATIONS OF CYCLIC CELL PENETRATING PEPTIDES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ziqing Qian Graduate Program in Chemistry The Ohio State University 2014 Dissertation Committee: Professor Dehua Pei, Advisor Professor Venkat Gopalan Professor Dennis Bong Copyright by Ziqing Qian 2014 ABSTRACT Cell penetrating peptides (CPP) have been featured as a powerful delivery vector for the intracellular delivery of membrane-impermeable cargoes. This dissertation primarily focuses on the development, characterization, and application of a new class of CPP: cyclic cell penetrating peptides. Prompted by long-standing interests of the Pei group in developing cyclic peptides as biological tools and potential therapeutics, I describe our exploration of the permeability properties of the cyclic peptides, leading to the discovery that cyclic peptides with a short sequence motif rich in arginine and hydrophobic residues display significantly higher cellular permeability than their linear counterparts. The resulting cyclic CPP also translocated into the mammalian cell interior at significantly higher efficiencies than canonical arginine-rich linear CPPs. Subsequent internalization mechanistic investigations involving model lipid vesicles and pharmacological inhibitors, as well as genetic mutations, which perturb individual internalization steps, have shown conclusively that cyclic CPPs enter cells through endocytosis and are capable of escaping from early endosomes. To explore the utilities of cyclic CPP as cytoplasmic delivery vehicles, we developed endocyclic, exocyclic, and bicyclic delivery methods to deliver monocyclic peptides, bicyclic peptides, and proteins into cells. Biologically active cargoes, such as fluorogenic phosphatase substrates, phosphatase inhibitors, green ii fluorescent protein, and protein tyrosine phosphatase 1B were efficiently delivered by cyclic CPP. Additionally, we explored a potentially general strategy to deliver linear peptidyl ligands into mammalian cells through reversible, disulfide bond mediated cyclization. Cell permeable fluorogenic caspase substrates and cell-permeable CAL-PDZ domain inhibitors were developed using this method. The last chapter focuses on the structure-based development of a peptidyl ligand for selectively disrupting calcineurin/NFAT protein-protein interaction (PPI). Through relatively minor structural modifications, we were able to improve the calcineurin- binding affinity of peptide VIVIT by ~200-fold. The resulting ligand ranks among the most potent calcineurin inhibitors reported to date, positioning it for future development as an efficient – and less toxic – therapeutic alternative to cyclosporin A and FK506. iii DEDICATION Dedicated To My Family iv ACKNOWLEDGMENTS My advisor, Dr. Dehua Pei, has been an excellent mentor. He is full of knowledge, wisdom, enthusiasm, and kindness. Trained as an organic chemist, I am indebted to him for his encouragement, excellent guidance and mentoring in conducting this interdisciplinary research. His dedication and philosophy to science has greatly shaped my own growth as a scientist along the way. I am especially grateful for the opportunity that I was given to develop my own individuality. For everything you have done for me, Dr. Pei, I thank you. Dr. Tao Liu was also extremely helpful when I joined the Pei group. He is very knowledgeable and selfless, and I thank him for all of his assistance and guidance in getting my graduate career started and for our ongoing friendship. I also want to thank Dr. Qing Xiao, Dr. Punit Upadhyaya, Dr. Thi Trinh, Dr. Ryan Hard, Wenlong Lian, Bisheng Jiang, Patrick Dougherty, Yujing Zhai and all other current and former Pei group members for their help and support. I have been incredibly fortunate to spend my time among so many talented and diligent labmates with a strong team spirit. Additionally, I have been very blessed to have the opportunities in collaborating and working with many extraordinary professors and scientists over the country. I am indebted to Dr. Jonathan LaRochelle, Dr. Estelle Cormet-Boyaka, Dr. Chris Hadad, Dr. Dennis Bong, Dr. Venkat Gopalan, Dr. Dmitri Kudrysshov, Dr. Dean Madden, Dr. Amy Barrios, Dr. Patrick Hogan, v Dr. Shawn Li and all other personnel involved for their generous guidance and input in my research. I thank my committee members, Dr. Dennis Bong and Dr. Venkat Gopalan for their instrumental assistance during my graduate life at OSU. I also want to thank Dr. Jian Zhou for providing me a solid synthetic chemistry training. I want to acknowledge friends, scientists and staff I have gotten to know and work with. I have always appreciated all your help and generous support in research, classes and extracurricular activities. Finally, and most importantly, I would like to thank my family for their unconditional support. My parents, Zhengyi and Yongke, have always been inspiring and supporting of my endeavors. I really appreciate all the effort they have made in educating and caring for me. I would like to thank my brilliant and beautiful wife, Yisha Yao, for her faith in me and for her unwavering love and encouragement. I also wish to thank Yisha’s parents, Qi and Yinxiang. They are both simply amazing individuals and provide me with unending encouragement and support. vi VITA June 2009 .................................................. B.S. Chemistry, East China Normal University 2009 to present ......................................... Graduate Teaching and Research Associate, Department of Chemistry and Biochemistry, The Ohio State University Publication 1. Wenlong Lian, Bisheng Jiang, Ziqing Qian, and Dehua Pei. “Cell-permeable bicyclic peptide inhibitors against intracellular proteins.” J. Am. Chem. Soc. [Online early access]. DOI: 10.1021/ja503710n. 2. Punit Upadhyaya, Ziqing Qian, Nurlaila A. A. Habir, and Dehua Pei. “Direct Ras inhibitor identified from a structurally rigidified bicyclic peptide library” Tetrahedron [Online early access]. DOI: 10.1016/j.tet.2014.05.113. 3. Ziqing Qian, Jonathan R. LaRochelle, Bisheng Jiang, Wenlong Lian, Ryan L. Hard, Nicholas G. Selner, Rinrada Luechapanichkul, Amy M. Barrios, and Dehua Pei. “Early endosomal escape of a cyclic cell-penetrating peptide enables effective cytosolic cargo delivery.” Biochemistry 2014, 53(24), 4034-4046. 4. Anamitra Ghosh, Hariharan Saminathan, Arthi Kanthasamy, Vellareddy Anantharam, Huajun Jin, Gautam Sondarva, Dilshan S. Harischandra, Ziqing Qian, Ajay Rana, and Anumantha G. Kanthasamy. “The Peptidyl-prolyl isomerase Pin1 upregulation and proapoptotic function in dopaminergic neurons.” J. Biol. Chem. 2013, 288(30), 2494–2501 vii 5. Ziqing Qian, Tao Liu, Yu-Yu Liu, Roger Briesewitz, Amy M. Barrios, Sissy M. Jhiang, and Dehua Pei. “Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs.” ACS Chem. Biol. 2013, 8(2), 423-431 6. Varun Dewan, Tao Liu, Kuan-Ming Chen, Ziqing Qian, Yong Xiao, Lawrence Kleiman, Kiran V. Mahasenan, Chenglong Li, Hiroshi Matsuo, Dehua Pei, and Karin Musier-Forsyth. “Cyclic peptide inhibitor of HIV-1 capsid-human lysyl-tRNA synthetase interaction” ACS Chem Biol. 2012, 7(4), 761-769 7. Tao Liu, Ziqing Qian, Qing Xiao, and Dehua Pei. “High-throughput screening of one-bead-one-compound libraries: identification of cyclic peptidyl inhibitors against calcineurin/NFAT interaction.” ACS Comb. Sci. 2004, 15(2), 300-306. 8. Miao Ding, Feng Zhou, Zi-Qing Qian, and Jian Zhou. “Organocatalytic Michael addition of unprotected 3-substituted oxindoles to nitroolefins.” Org. Biomol. Chem. 2010, 8(13), 2912-2914. 9. Zi-Qing Qian, Feng Zhou, Tai-Ping Du, Bo-Lun Wang, Miao Ding, Xiao-Li Zhao, and Jian Zhou. “Asymmetric construction of quaternary stereocenters by direct organocatalytic amination of 3-substitued oxindoles” Chem. Commun. 2009, 28(44), 6753-6755. Fields of Study Major Field: Chemistry viii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DEDICATION ................................................................................................................... iv ACKNOWLEDGMENTS .................................................................................................. v VITA vii TABLE OF CONTENTS ................................................................................................... ix LIST OF TABLES ........................................................................................................... xiii LIST OF FIGURES ......................................................................................................... xiv LIST OF ABBREVIATIONS ......................................................................................... xvii Chapter 1: Introduction ....................................................................................................... 1 1.1 Cell Penetrating Peptides ...................................................................................... 1 1.2 Classification of Cell Penetrating Peptides ........................................................... 2 1.2.1 Cationic Cell Penetrating Peptides.................................................................... 3 1.2.2 Amphipathic Cell Penetrating Peptide .............................................................. 4 1.2.3 Hydrophobic Cell Penetrating Peptides ...........................................................