Antibiotics Targeting Tuberculosis: Biosynthesis of A-102395 and Discovery of Novel Actinomycins
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University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2015 ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A-102395 AND DISCOVERY OF NOVEL ACTINOMYCINS Wenlong Cai University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Cai, Wenlong, "ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A-102395 AND DISCOVERY OF NOVEL ACTINOMYCINS" (2015). Theses and Dissertations--Pharmacy. 52. https://uknowledge.uky.edu/pharmacy_etds/52 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. 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Wenlong Cai, Student Dr. Steven Van Lanen, Major Professor Dr. Jim Pauly, Director of Graduate Studies ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A- 102395 AND DISCOVERY OF NOVEL ACTINOMYCINS DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Wenlong Cai Lexington, Kentucky Director: Dr. Steven Van Lanen，Professor of Pharmaceutical Science Lexington, Kentucky 2015 Copyright © Wenlong Cai 2015 ABSTRACT OF DISSERTATION ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A- 102395 AND DISCOVERY OF NOVEL ACTINOMYCINS The increase in antibiotic resistance of many bacterial strains including multidrug- resistant tuberculosis (MDR-TB) due to over- and misuse of antibiotics is a serious medical and economical problem. Therefore discovery and development of new antibiotics are urgently needed. Two projects were undertaken to address the need for new anti- tuberculosis antibiotics. 1. Discovery of new chemical entities. A-102395, a new nucleoside inhibitor of bacterial MraY (translocase I, EC 2.7.8.13) that is essential for bacterial survival, was isolated from the culture broth of Amycolatopsis sp. SANK 60206 in 2007. Although A-102395 is a potent inhibitor of translocase I with IC50 of 11 nM, it contradictingly does not have any antibiotic activity. A-102395 is a derivative of capuramycin with a unique aromatic side chain. A semisynthetic derivative of capuramycin is currently in clinical trials as an anti- tuberculosis antibiotic, suggesting potential for using A-102395 as a starting point for antibiotic discovery. The biosynthetic gene cluster of A-102395 was identified, including 35 putative open reading frames responsible for biosynthesis and resistance. A series of gene inactivation abolished the A-102395 production, indicating those genes within the cluster are essential for A-102395 biosynthesis. Functional characterization of Cpr17, which has sequence similarity to aminoglycoside phosphotransferases, revealed that it functions as a phosphotransferase conferring self-resistance by using GTP as phosphate donor. Furthermore the enzyme is characterized by low substrate specificity, as Cpr17 was capable of modifying a large series of natural or semi-synthesized analogues of capuramycins. A series of organism-specific high-throughput screening models for potential antibacterial agents targeting on bacterial cell wall synthesis have been established, including Escherichia coli and Mycobacterium tuberculosis. For this screen ten enzymes were successfully used to reconstitute cell wall biosynthesis in vitro. This screening is expected to allow us to identify the targets of novel antibiotics rapidly and in a cost- efficient manner 2. Rediscovering old antibiotics. As part of our long term goal of discovering and developing novel anti-tuberculosis antibiotics, four novel actinomycins were isolated from the scale-up fermentation of Streptomyces sp. Gö-GS12, and their structures were characterized using mass spectrometry and 1D and 2D NMR. Their antibacterial activity against Gram-positive and Gram- negative strains were determined, as well as their cytotoxicity. Key words: capuramycins, peptidoglycan cell wall, phosphotransferase, actinomycins. Wenlong Cai Student’s Signiture 9/22/2015 Date ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A- 102395 AND DISCOVERY OF NOVEL ACTINOMYCINS by Wenlong Cai Steven Van Lanen Director of Dissertation Jim Pauly Director of Graduate Studies 9/22/2015 To Ling and Lucy ACKNOWLEDGMENTS I would like to take this moment to express my gratitude to my mentor, Dr. Steven Van Lanen, for his guidance, support, and inspiration throughout my study in his group. His expertise and insightful discussions have benefited me greatly. I would also like to thank my committee members, Dr. Jürgen Rohr, Dr. Kyung-Bo Kim, and Dr. Joe Chappell, for their invaluable advice and comments over the years, and I would like to sincerely thank Dr. Aardra Kachroo for being the external- department examiner for my dissertation defense. Special thanks to all current and previous members from the Van Lanen group: Dr. Zhaoyong Yang, Dr. Sandra Barnard, Dr. Xiuling Chi, Dr. Anwesha Goswami, Dr. Xiaodong Liu, Zheng Cui, Matthew McErlean, Ashley Arlinghaus, and Tyler Bucci. It was a great honor to work with and learn from you all! And last but not least, I would like to acknowledge my family and friends for their constant support and encouragement. iii TABLE OF CONTENT ACKNOWLEDGMENTS .................................................................................................................. iii TABLE OF CONTENTS .................................................................................................................. iv LIST OF FIGURES ......................................................................................................................... vii LIST OF SCHEMES ........................................................................................................................ x LIST OF TABLES ............................................................................................................................ xi LIST OF ABBREVIATIONS ............................................................................................................. xii Chapter 1: Introduction and Background ......................................................................................... 1 1.1 Need for new anti-tuberculosis antibiotics ......................................................................................... 1 1.2 Significance of natural products in antibiotic discovery ................................................................... 3 1.3 Current antibacterial targets ................................................................................................................ 4 1.4 Biosynthesis of peptidoglycan cell wall .............................................................................................. 6 1.5 MraY: structure and inhibitors ............................................................................................................. 9 1.6 Targeting bacterial MraY: discovery of capuramycins .................................................................. 13 1.7 Binding to DNA: a brief history of actinomycins ............................................................................. 16 1.8. Aims of this study ............................................................................................................................... 20 Chapter 2: Characterization of the biosynthetic gene cluster for A-102395 and reconstituting bacterial cell wall biosynthesis ...................................................................................................... 21 2.1 Background .......................................................................................................................................... 21 2.2 Materials and methods ....................................................................................................................... 27 2.2.1 Instrumentation,