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Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and Microbials

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Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and Microbials Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and Microbials Jessica E. Wynn Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemistry Webster L. Santos, Chair Karen J. Brewer Felicia A. Etzkorn Tijana Z. Grove David G.I. Kingston July 14, 2016 Blacksburg, VA Keywords: HIV-1 RRE RNA, Branched Peptide Library, Boron-Acridine, Antimicrobial Peptides, Unnatural Amino Acids Copyright (2016), Jessica E. Wynn Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and Antimicrobials Jessica E. Wynn ABSTRACT The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high- affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability. The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding. A second generation BPBA library that introduced acridinyl lysine as an intercalator (4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic. High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication. iii ACKNOWLEDGEMENTS I preface this acknowledgements section with the observation that yes, this section is long, but rightly so. To all those who have read or will read this dissertation (n=10?), it is important to realize that indeed, no man (or woman) is an island1 – I have not gotten to the end of this journey without a little help from my friends2 (and professors, and colleagues…) On the flip side of things, if you are an advisor, a professor, an instructor, or a colleague to a graduate student, please realize that even small gestures of compassion or acknowledgement are forever remembered, especially during the long bouts of struggle and self-doubt that lurk within a developing young scientist. Thank you all so much for everything you have done to allow me to succeed in my endeavors. Without further ado… First, I would like to thank my advisor, Dr. Webster L. Santos, for giving me the opportunity to conduct research in his laboratory. I appreciate the autonomy and freedom you have allowed me over the years to develop and execute my projects, which has in turn given me the confidence to pursue what will hopefully be a successful career as an independent research scientist. Your tireless efforts and work ethic are an inspiration to me. Thank you for teaching me about synthetic chemistry - your ability to communicate complex concepts and make them interesting is unparalleled, and is one of the reasons I was so interested in joining your lab. I also thank you for your input on preparing and giving presentations, which has greatly improved my ability to communicate science with others. Additionally, I would like to thank you for providing the financial support that has allowed me to conduct my research, and for encouraging and supporting me to attend conferences and present my work. Last but not least, I thank you for forever altering the way I communicate – ‘at the end of the day’, ‘gratifyingly’, ‘indeed’, and ‘taken together’ are permanently etched into my shriveled brain.3 iv I would like to thank my committee members for nurturing my growth as a graduate student and for helping me improve by providing honest evaluations of my performance at each stage of my graduate career. I would like to acknowledge Dr. Felicia A. Etzkorn for her positive influence in my life as an example of a strong, successful female scientist in the department. I am indebted to you as your critique of my literature review has stuck with me and improved my scientific writing. I would like to thank Dr. Tijana Z. Grove for lending her analytical expertise when discussing my research, and for always taking the time to mentor me and offer support, both professionally and personally. You have a way of making every student feel valued and respected, and I wish you nothing but success in your future. I would like to thank Dr. David G.I. Kingston for his valuable critiques of my work over the years, especially with my original research proposal. I am constantly amazed by your humility and accessibility to students, even though you are such a distinguished scientist. Lastly, I would like to thank Dr. Karen J. Brewer. She encouraged me from my very first semester as a graduate student, and provided emotional support when all seemed hopeless and lost. Although she is no longer with us, I carry her in my heart during my final sprint to the finish line and know that she would be proud. I would also like to thank several faculty members within the Chemistry department for their endless support during my tenure in graduate school. A big thank you goes to Dr. Jim Tanko, for many reasons. First, thank you for always being supportive of my endeavors and for always making time to meet with me, even though you are insanely busy as the Chair. Also, thank you for teaching a class that made me appreciate both letters and numbers in an equation – I truly learned a great deal in your Chemical Kinetics class, including discovering a level of perseverance within myself that I have made good use of over the years. I also thank you for forever changing the way I see time – it’s five colon zero zero! I am appreciative of both Dr. v John Morris and Dr. Paul Deck for serving as great resources for me during my time at Virginia Tech, and for always making the student a priority. I would like to thank Dr. Paul Carlier and Dr. Richard Gandour for teaching me a great deal about organic and medicinal chemistry. Both of you have been incredibly supportive of my growth as a chemist, from engaging in my work during poster sessions to allowing me to serve on various committees over the years. I want to thank Dr. Brian Tissue for all of his kind words of encouragement over the years, and for giving me the opportunity to serve on the Safety Committee. A thank you also goes out to all of the professors that have encouraged me before I began my journey in graduate school. Thank you, Dr. Ed Smith, for being the first professor to take a chance on me when I started at Virginia Tech as an undergraduate. Your genuine love and concern for students from all walks of life is appreciated and admired. I would like to thank Dr. Neal and Kay Castagnoli for mentoring me during my undergraduate years as well, and for sparking my love of organic chemistry. I would like to thank Dr. Tim Fuhrer for introducing me to chemistry in high school – who knew we would cross paths again many years later in graduate school?! The world is a small place sometimes. There have also been several teachers outside the realm of chemistry that I would like to thank for encouraging me to think critically and for embracing my curious nature: Carolyn Paulette, Catherine Neale, Suzy Bolton, and Dr. Karl Precoda all come to mind. Thank you for the immeasurable impact you have had on my life. I also want to thank many people on the administrative and chemistry support staff that have helped me navigate the waters of paperwork, broken instruments, busted computers, missing packages, NMR questions, and stress: Emillie Shephard, Angie Miller, Angie Kritak, Patty Angus, Roberta Gilbert, Wanda Hensley, Kristen Cox, Melba Edwards, Anna Hawthorne, Tom Bell, Donald Neel, Larry Jackson, Steve Breeding, John Burleson, Tom Wertalik, Sharelle vi Carbaugh, Geno Iannaccone, and Murthy Shanaiah.
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