BIOCHEMICAL METHODS FOR PREPARATION AND STUDY OF PEPTIDE NATURAL PRODUCT LIBRARIES Steven Robert Fleming A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences in the Doctoral Program of the UNC Eshelman School of Pharmacy (Division of Chemical Biology & Medicinal Chemistry) Chapel Hill 2020 Approved by: Albert A. Bowers Michael B. Jarstfer Rihe H. Liu Kevin M. Weeks Leslie M. Hicks ©2020 Steven Robert Fleming ALL RIGHTS RESERVED ii ABSTRACT Steven Robert Fleming: Biochemical Methods for Preparation and Study of Peptide Natural Product Libraries (Under the direction of Albert Bowers) mRNA Display is an increasingly popular technique in pharmaceutical sciences to make highly diverse peptide libraries to pan for protein inhibitors. The current state of the art applies Flexizyme codon reprogramming with mRNA display to introduce unnatural amino acids for peptide cyclization and to further increase library diversity. Interestingly, ribosomally synthesized and post-translationally modified peptides (RiPP) are a unique class of natural products that transform linear peptides into highly modified and structurally complex metabolites. By combining RiPP biosynthesis with mRNA display, libraries of increasingly greater diversity can be achieved, and impending selected inhibitors will have natural product-like qualities, which we expect will allow these compounds to have better drug-like properties. Herein, we have developed a platform to measure RiPP enzyme modification of mRNA display libraries to show for the first time that RiPP enzymes can modify RNA linked peptide substrates. The platform may be extrapolated to many different RiPP enzymes and provides useful measurements to determine if a RiPP enzyme is promiscuous and effective to produce highly diversified peptides. Thiopeptides are a specific class of RiPP that we would like to apply to mRNA display, because they have broad activities. Additionally, the structure of thiopeptides is primed for mRNA display protocols, consisting of a highly decorated macrocycle with a C-terminal tail that may readily accept an iii mRNA tag. We present a redesigned chemoenzymatic strategy to make thiopeptides using cell free protein synthesis that is promiscuous, can synthesize different thiopeptide classes, and is mRNA display ready. Finally, bioinformatic analysis of publicly available thiopeptide gene clusters shows that many unknown thiopeptides still exist. We have organized a set of 14 new clusters for which we can apply our chemoenzymatic strategy and characterize these unknown compounds, with the hope of finding the best thiopeptide enzymes for use in mRNA display selections. iv To God v ACKNOWLEDGMENTS I would first like to thank my family. Thank you for prioritizing my education and always encouraging me to work hard and do my best. I would not be here without you. Thank you, Dr. Bowers, for taking me into the group, for training me, and for helping to shape my future. I am very happy and thankful to be a chemical biologist and time spent in your lab has been a delight. A special thanks to all the Bowers’ lab alumni whom I’ve had the pleasure to interact with: Dr. Scott Allen, Dr. Walter Wever, Dr. Rachel Bleich, Dr. Paul Himes, Dr. Swapnil Ghodge, Dr. Jon Bogart, Dr. Kelly Bird, Dr. Nick Kramer, Matt Fleming, Tory Haberman, Bree Iskandar, Matt Bowler, Jarrett Pelton, Braxton Cline, and Sungwan Hwang. Lab has been a great place to be, and that is because of you all. A doubly special thanks to Dr. Paul Himes for being my lab mentor and teaching me many basic research skills. Thank you for all of the hard work you put into the development of mRNA display. I would like to thank those on my doctoral committee whose discussions and advice have been invaluable during my Ph.D. training. Thank you, Dr. Mike Jarstfer, for serving as my chair, providing letters of recommendation, and often reaching out with opportunities to present my work. Thank you, Dr. Rihe Liu, for allowing me to rotate in your lab where my interest in mRNA display began. Thank you, Dr. Kevin Weeks, for teaching me to write an abstract. Lastly, but not least, thank you, Dr. Leslie Hicks, for collaborating with me and giving me access to your mass spectrometers. This was absolutely vital for our first publication. vi TABLE OF CONTENTS LIST OF TABLES …………………………………………………………………………......... xi LIST OF FIGURES …………………………………………………………………………..… xii LIST OF ABBREVIATIONS ……………………………………………………………..…... xiv CHAPTER 1: INTRODUCTION ………………………………………………………..………. 1 1.1 Modification of Genetically Encoded Peptide Libraries and Drug Discovery ………. 1 1.2 mRNA Display Has Specific Advantages Over Other Display Technologies ………. 1 1.3 RiPPs Biosynthesis and Application to mRNA Display ………………………..…… 3 1.4 Thiopeptides are a Good Scaffold for mRNA Display Selections ………………...… 4 1.5 Bioinformatic Expansion of Thiopeptides and Chemoenzymatic Synthesis Could Lead to Optimal Enzymes for mRNA Display of Thiopeptides………………. 7 REFERENCES……………………………………………………………………………….…... 9 CHAPTER 2: EXPLORING THE POST-TRANSLATIONAL ENZYMOLOGY OF PAAA BY MRNA DISPLAY…………………………………………………………….… 13 2.1 Introduction ……………………………………………………………………….... 13 2.2 Results and Discussion ……………………………………………………………… 14 2.3 Conclusion ……………………………..…………………………………………… 20 2.4 Experimental ………………………………………………………………………... 20 2.4.1 Materials and General Methods ………………………………………...… 20 2.4.2 Expression and Purification of PaaA-His6 ……………………………...... 22 2.4.3 DNA Preparation for NEB PURExpress Translations ……………………. 23 vii 2.4.4 DNA Preparation of Transcription Templates …………………………..… 23 2.4.5 Transcription with T7 RNA polymerase and Urea Gel Purification ……… 27 2.4.6 N-Biotin-L-Phenylalanine itRNA Acylation Conditions …………….…… 28 2.4.7 mRNA Display Protocols …………………………………………………. 29 2.4.8 MALDI-TOF Experiments ……………………………………………….. 36 2.4.9 Binding Experiments ………………………………………………….….. 36 2.4.10 Peptide Synthesis ………………………………………………………... 37 REFERENCES ……………………...………………………………………………………….. 41 CHAPTER 3: FLEXIZYME ENABLED BENCHTOP BIOSYNTHESIS OF THIOPEPTIDES …..…………………………………………………………………………… 44 3.1 Introduction ………………………………………………………………………… 44 3.2 Results and Discussion ……………………………………………………………… 47 3.3 Conclusion …………………………………………………..……………………… 53 3.4 Experimental ………………………………………………………………………... 53 3.4.1 General Information ….……………………………………….………...... 53 3.4.2 Protein Cloning ………………………………………………….………... 54 3.4.3 Protein Expression Purification ……….………………………………...... 55 3.4.4 Protein Sequences …………………….…………………………………... 59 3.4.5 DNA and RNA Preparation ……….……………………………………… 62 3.4.6 Flexizyme tRNA Acylation of Se-phenylselenocysteine ……….………… 66 3.4.7 Translations …………………………………………………….……….... 66 3.4.8 Enzyme Reactions and Oxidative Elimination …………….……………… 67 3.4.9 Desalting Protocol and Preparation for LC / MS …………….………….... 68 3.4.10 Synthesis of Se-phenylselenocysteine dinitrobenzyl ester …….………… 68 viii 3.4.11 Liquid Chromatography and Mass Spectrometry …….………………..... 70 REFERENCES………………………………………………………………………………….. 72 CHAPTER 4: BIOINFORMATIC EXPANSION OF THIOPEPTIDES AND PRIORITIZATION FOR CHEMOENZYMATIC DISCOVERY……………………………… 75 4.1 Introduction ……………………………………………………………………….... 75 4.2 Diversity of the Publicly Available Pyridine Synthases from Thiopeptide Biosynthesis ...…………………………………………………….…... 77 4.3 Diversity of Thiopeptide Precursor Peptides ……………………………………….. 80 4.4 Prioritization of Unknown Thiopeptide Biosynthetic Gene Clusters for Chemoenzymatic Discovery …………………………………….. 84 4.5 Diversity of Thiopeptide Binding Receptors ………………………………………... 87 4.6 Conclusion ………………………………………………………………………….. 90 4.7 Experimental ………………………………………………………………………... 91 4.7.1 Collecting pyridine synthases from GenBank and RODEO ……………… 91 4.7.2 Curating Thiopeptide Precursor Peptides ………………………………… 97 4.7.3 Creating Sequence Similarity Networks ………………………………….. 97 4.7.4 WEBLOGOs for Predicted Thiopeptide Cores …………………………… 97 4.7.5 Thiopeptide Predicted Gene Clusters with antiSMASH ………………….. 98 REFERENCES………………………………………………………………………………….. 99 CHAPTER 5. CONCLUSION ………………………………………………………………... 103 5.1 Concluding Remarks ………………………………………………………….…… 103 REFERENCES………………………………………………………………………………… 106 APPENDIX A: SUPPLEMENTARY FIGURES AND TABLES FOR CHAPTER 2 …….…. 108 APPENDIX B: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER 3 ……….. 164 REFERENCES ………………………………………………………………………………....191 ix APPENDIX C: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER 4 …….…. 192 x LIST OF TABLES Table 2.1 - Taq polymerase amplification cycles for translation genes …………………….….. 23 Table 2.2 - Flexizyme and mRNA display primers ………………………………………..…… 24 Table 2.3 - Extension Protocol …………………………………………………………….…… 24 Table 2.4 - PCR1 Protocol …………………………………………………………………..….. 25 Table 2.5 - PCR2 protocol ………………………………………………………………..…….. 25 Table 2.6 - PCR3 protocol …………………………………………………………………..….. 25 Table 2.7 - PCR4 protocol ……………………………………………………………..……….. 25 Table 3.1 - Results of flexizyme-enabled benchtop biosynthesis with mutant leader peptides and cores …………………………………………………………..……. 49 Table 3.2 - Extension Protocol ……………………………………………………………..…… 62 Table 3.3 - PCR1 protocol …………………………………………………………………..….. 62 Table 3.4 - PCR2 protocol ………………………………………………………………..…….. 62 Table 3.5 - dFx and AsnE2 primers ……………………………………………………….……. 63 Table 3.6 - Q5 PCR amplification ………………………………………………………….…... 65 Table 3.7 - Taq PCR amplification ……………………………………………………….….….