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Investigation Into Catalytic Metallodrugs That Target Hepatitis C IRES RNA

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Investigation Into Catalytic Metallodrugs That Target Hepatitis C IRES RNA Investigation into Catalytic Metallodrugs that Target Hepatitis C IRES RNA: Development, Characterization, and Mechanism DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Martin James Ross B.A. Chemistry Graduate Program in Chemistry The Ohio State University 2015 Dissertation Committee: James A. Cowan (Advisor) A. Douglas Kinghorn Hannah Shafaat Claudia Turro Copyright by Martin James Ross 2015 Abstract Metals have been used for therapeutic purposes since the dawn of civilization including the ancient Egyptians using copper jars to sterilize their water. This use of metal and others were commonplace until the discovery of penicillin. With the discovery of penicillin, small molecules with a rational and target approach became the standard of the drug industry. Traditional drugs to this today mostly consist of organic compounds, composed primarily of carbon, hydrogen, nitrogen, oxygen, chlorine, and fluorine. The discovery of cis-platin and the advancement of our understanding of how the body works, including how our bodies handle metals, and advance techniques have created the environment for the renaissance in interest and development of bioinorganic compounds for therapeutic use. Metal complexes offer unique opportunities and properties that traditional small molecules lack. One such approach is through the development of catalytic metallodrugs which by design are able to recognize and target multiple of the same therapeutic target. This ability enable these compounds to be dose at lower dosage, sub-stoichiometric equivalents, which will lead to fewer off-targeting and side-effects. Limited preliminary studies have applied this approach towards ribonucleic acids, such as Hepatitis C IRES RNA. ii Hepatitis C Virus (HCV) affects over 200 million people globally which unchecked can lead to cirrhosis or liver cancer. Unfortunately, there is not a vaccine available for HCV like hepatitis A or hepatitis B. The current approach towards treatment involve cocktails, mixtures of several compounds each with a different therapeutic target. Initial reports have demonstrated the activity of the Cu-GGHYrFK, copper peptide complex, in recognition of stem-loop IIb of the HCV IRES RNA. This research starts by understanding the products and binding of Cu-GGHYrFK to stem-loop IIb (SLIIb). With the lead compound, pathways and mechanism for oxidative degradation of RNA were developed. Derivatives including the all D-configuration and all L-configuration of this peptide were synthesized to examine the importance of stereochemistry on reactivity. After this, a structure activity relationship study based upon the all L configuration was preformed to evaluate the role of each of the targeting domain amino acids on binding, reactivity, and cellular uptake. This was then continued to the first position after the Cu- GGH domain to examine catalytic properties. A series of different metal ions, Ni2+, Co3+, Pd2+, Pt2+, Au3+, were incorporated into GGHYrFK, the lead compound, to investigate the importance of the metal ion with reactivity. A further more in-depth mechanistic study was carried out with Cu-GGHYrFK 18 with the use of heavy water, H2 O to determine the source of oxygen into products of RNA degradation. The novel 5’-product, 5’-geminal diol was reported. iii Finally, several catalytic drugs based on reported peptides that bind stem-loop IV of the HCV IRES RNA were developed and characterized. These compounds represent a paradigm shift in therapeutic approach for the treatment of hepatitis c virus. iv Dedication This dissertation is dedicated to Marty and Sue Ross, my parents, who always supported every goal, taught me to dream, and instilled the passion of learning and to Keisha Neidrich for her patience, support, and love. v Acknowledgments This endeavor would not have been possible without many talented and wonderful individuals. At this time I would like to recognize a few. I would initially would like to thank Dr. James Cowan, my advisor, for recognizing my potential, allowing the freedom to explore concepts and ideas while fostering the chemist in me. To the Department of Chemistry and Biochemistry at the Ohio State for their continued financial support and access to some of the most talented researchers, educators, and instruments in the world. Thank you is such an understatement for all the assistance from the former and current Cowan lab members that aided in this research; between the lively discussions to the social gatherings. Dr. Seth Bradford, thank you for being the one person that listened to all the ideas I had; whether they were crazy or far-fetch. Further, thank you for the opportunity to continue the work on the project you conceived and started. Dr. Jeff Joyner, thank you for the development of the MassDaddy and our heated debates in interpretation of data. Also thank you for your entertaining group meetings which have come to be vi measured in Joyners. To Insiya Fidai for the countless hours of trouble-shooting, sorting, and eventually producing informative computation data. In addition to all the CD and ITC data in assistance in completing this. vii Vita 2009 to Current ..............................................The Ohio State University, Department of Chemistry and Biochemistry 2009................................................................B.A. Chemistry, Hiram College 2005................................................................Graduated from Meadville Area Senior High 1986................................................................Born in Meadville, PA Publications Bradford S; Ross, M. J.; Fidai, I.; Cowan, J.A. “Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop IIb of hepatitis C IRES RNA.” ChemMedChem. 2014, 9, 1275-1285. Fields of Study Major Field: Chemistry viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii List of Tables ................................................................................................................... xvi List of Figures ................................................................................................................... xx – Introduction ....................................................................................................... 1 – History of Metals in Medicine from Serendipity to Design .............................. 3 1.3 – Cisplatin and derivatives ................................................................................... 7 1.4 – US FDA ........................................................................................................... 12 1.5 – Clinical and FDA-approved metallodrugs ...................................................... 14 1.5.1 First Row Transition Metals ....................................................................... 14 1.5.2 Second and Third Row Transition Metals .................................................. 16 ix 1.5.3 Other notable elements ............................................................................... 20 1.6 – Novel Approaches – Catalytic Metallodrugs .................................................. 22 1.6.1 Therapeutic Approach ................................................................................. 22 1.6.2 Design and Double Filter Effect ................................................................. 25 1.6.3 Amino Terminal Copper Nickel Motif (ATCUN) ...................................... 28 1.6.4 Previous applications of the Cu-GGH motif ............................................... 29 Chapter 2: Insights into the Recognition, Binding and Reactivity of Catalytic Metallodrugs Targeting Stem Loop IIb of Hepatitis C IRES RNA .................................. 31 2.1 – Introduction ..................................................................................................... 31 2.2 – Materials .......................................................................................................... 33 2.3 – Methods ........................................................................................................... 34 2.3.1 Binding Constant Determination ................................................................ 34 2.3.2 Melting Temperatures ................................................................................. 35 2.3.3 Molecular Dynamics ................................................................................... 35 2.3.4 Reaction Kinetics via Fluorescence. ........................................................... 36 2.3.5 Mass Spectrometry...................................................................................... 37 2.3.6 HCV Cellular Replicon Assay. ................................................................... 38 x 2.3.7 RT-PCR....................................................................................................... 39 2.4 – Results ............................................................................................................
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