Towards the elucidation of the mechanism of the antibiotic activity of tamoxifen A Dissertation Presented to The Academic Faculty by Nathanael Simeon Levinson In Partial Fulfillment of the Requirements for the Degree MS in Chemistry in the College of Sciences Georgia Institute of Technology May 2017 COPYRIGHT © 2017 BY NATHANAEL SIMEON LEVINSON Towards the elucidation of the mechanism of theantibiotic activity of tamoxifen Approved by: Dr. Adegboyega Oyelere, Advisor Dr. Stefan France School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. M.G. Finn, Committee Chair Dr. Thomas DiChristina School of Chemistry and Biochemistry School of Biology Georgia Institute of Technology Georgia Institute of Technology Dr. Raquel Lieberman School of Chemistry and Biochemistry Georgia Institute of Technology Date Approved: [April 20, 2017] ACKNOWLEDGEMENTS My deepest gratitude goes to my mother, Valerie, and my father, Jeffrey, who supported me emotionally, financially, intellectually, and spiritually through all the trials and tribulations I have faced not only during my graduate studies but my entire life. My undying thanks to them, without whom I would not be the man I am today. I extend heartfelt thanks to my advisor, Dr. Adegboyega Oyelere who graciously opened his lab to me and did not give up on me in the face of failing projects. I give this gratitude also to Dr. M.G. Finn, without whose support I would not have been able to complete my studies at Georgia Tech. I thank Dr. Maureen Rouhi for taking me under her wing and allowing me to write and hone my craft under her guidance. Finally, I would like to thank the administrative staff of the College of Sciences for their help and understanding during my transitionary periods at Georgia Tech, especially Dr. Cam Tyson and Dr. Kenyatta Johnson. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS iii LIST OF TABLES vii LIST OF FIGURES viii SUMMARY xi CHAPTER 1. ANTIBIOTICS 1 1.1 The discovery and development of antibiotics 1 1.2 Major Antibiotic Classes 2 1.2.1 Antibiotics which target or affect the bacterial cell wall 2 1.2.1.1 Lipopeptides 2 1.2.1.2 Polymyxins 4 1.2.1.3 Glycopeptides 5 1.2.1.4 β-lactams 6 1.2.1.4.1 Penicillins 8 1.2.1.4.2 Cephalosporins 8 1.2.1.4.3 Cephamycins 9 1.2.1.4.4 Carbapenems 10 1.2.1.4.5 Monobactams 10 1.2.1.4.6 Trinems 11 1.2.1.4.7 β-lactam inhibitors 11 1.2.2 Antibiotics which target the ribosome 11 1.2.2.1 Aminoglycosides 12 1.2.2.2 Tetracyclines 13 1.2.2.3 Amphenicols 15 1.2.2.4 Macrolides 17 1.2.2.5 Streptogramins 19 1.2.2.6 Oxazolidinones 20 iv 1.2.2.7 Lincosamides 21 1.2.3 Antibiotics which have other internal targets 21 1.2.3.1 Quinolones 22 1.2.3.2 Sulfa drugs 23 CHAPTER 2. ASSAYS AND TECHNIQUES 24 2.1 Compound screening 24 2.2 Whole cell and blotting assays 29 2.2.1 Blotting to assess compound target 29 2.2.2 Minimum inhibitory concentration (MIC) 31 2.3 Cell fraction/lysate assays 32 2.3.1 RNA Footprinting 32 2.3.2 Affinity Chromatography 32 2.3.3 Thermal Shift 35 2.3.4 Radiolabeling 36 2.4 Imaging 36 2.4.1 Light microscopy 37 2.4.2 Electron microscopy 39 2.4.3 X-ray crystallography 40 CHAPTER 3. TAMOXIFEN 42 3.1 Tamoxifen: discovery, development and usage 42 3.1.1 History of tamoxifen 42 3.1.2 Tamoxifen as an antifungal 43 3.1.3 Tamoxifen as an antileishmanial agent 44 3.1.4 Tamoxifen as an antibiotic 44 CHAPTER 4. STRUCTURE-ACTIVITY RELATIONSHIP OF TAMOXIFEN 48 4.1 Purpose of study 48 4.2 Significance 48 4.3 Synthesis of tamoxifen derivatives 49 v 4.3.1 Preparation of N-desmethyl tamoxifen 49 4.3.2 Preparation of extended-alkyl derivatives 49 4.3.3 N-acetyl tamoxifen 49 4.3.4 Glycine derivative 50 4.3.5 Alanine derivative 50 4.3.6 Phenylalanine derivative 50 4.3.7 Lysine derivative 51 4.3.8 Guanidine derivative 51 4.3.9 Didesmethyl tamoxifen and 4-hydroxy tamoxifen 51 4.4 Compound Assessment 51 4.5 Results 52 4.6 Discussion 54 APPENDIX A. COLLABORATION WITH DR. DEV ARYA 57 A.1 Methods 57 A.2 Results 59 APPENDIX B. NMR AND MASS SPECTROSCOPY DATA 62 B.1 NMR 62 B.2 Mass Spectroscopy 71 APPENDIX C. MIC LINE GRAPHS 79 REFERENCES 91 vi LIST OF TABLES Table 1 – MIC results 52 Table A1 – Cell-free IC50 results 59 vii LIST OF FIGURES Figure 1 – Structure of daptomycin 3 Figure 2 – Mechanism of daptomycin 3 Figure 3 – Structure of colistin 4 Figure 4 – Mechanism of polymyxins 5 Figure 5 – Structure of vancomycin 6 Figure 6 – β-lactam cores 7 Figure 7 – Structure of streptomycin 13 Figure 8 – Structure of tetracycline 14 Figure 9 – Mechanism of tetracycline 14 Figure 10 – Structure of chloramphenicol 15 Figure 11 – Chloramphenicol interaction with peptidyl transferase cavity 16 Figure 12 – Structure of erythromycin/telithromycin 17 Figure 13 – Binding of macrolides in the ribosome 18 Figure 14 – Structure of quinupristin/dalfopristin 19 Figure 15 – Structure of linezolid 20 Figure 16 – Structure of lincomycin 21 Figure 17 – Quinolone core structure 23 Figure 18 – Sulfonamide basic structure 23 Figure 19 – The iChip 25 Figure 20 – FRET diagram 26 Figure 21 – ELISA diagram 27 Figure 22 – blotting diagram 30 Figure 23 – RNA footprinting 33 Figure 24 – Affinity chromatography 34 Figure 25 – Thermal shift diagram 35 Figure 26 – Confocal Microscope 35 viii Figure 27 – X-ray crystallography 41 Figure B1 – NMR of NL-I-43 61 Figure B2 – NMR of NL-I-44 62 Figure B3 – NMR of NL-I-45 63 Figure B4 – NMR of NL-I-69 64 Figure B5 – NMR of NL-I-70 65 Figure B6 – NMR of NL-I-71 66 Figure B7 – NMR of NL-I-72 67 Figure B8 – NMR of NL-I-97 68 Figure B9 – NMR of desmethyl tamoxifen 69 Figure B10 – Mass spec of NL-I-43 70 Figure B11 – Mass spec of NL-I-44 71 Figure B12 – Mass spec of NL-I-45 72 Figure B13 – Mass spec of NL-I-69 73 Figure B14 – Mass spec of NL-I-70 74 Figure B15 – Mass spec of NL-I-71 75 Figure B16 – Mass spec of NL-I-72 76 Figure B17 – Mass Spec of NL-I-97 77’ Figure C1 – MIC of tamoxifen in SA 79 Figure C2 – MIC of tamoxifen in MRSA 79 Figure C3 – MIC of desmethyl tamoxifen in SA 80 Figure C4 – MIC of desmethyl tamoxifen in MRSA 80 Figure C5 – MIC of didesmethyl tamoxifen in SA 81 Figure C6 – MIC of didesmethyl tamoxifen in MRSA 81 Figure C7 – MIC of NL-I-43 in SA 82 Figure C8 - MIC of NL-I-43 in MRSA 82 Figure C9 – MIC of NL-I-44 in SA 83 Figure C10 – MIC of NL-I-44 in MRSA 83 ix Figure C11 – MIC of NL-I-45 in SA 84 Figure C12 – MIC of NL-I-45 in MRSA 84 Figure C13 – MIC of NL-I-69 in SA 85 Figure C14 – MIC of NL-I-69 in MRSA 85 Figure C15 – MIC of NL-I-70 in SA 86 Figure C16 – MIC of NL-I-70 in MRSA 86 Figure C17 – MIC of NL-I-71 in SA 87 Figure C18 – MIC of NL-I-71 in MRSA 87 Figure C19 – MIC of NL-I-72 in SA 88 Figure C20 – MIC of NL-I-72 in MRSA 88 Figure C21 – MIC of NL-I-97 in SA 89 Figure C22 – MIC of NL-I-97 in MRSA 89 Figure C23 – MIC of 4-hydroxy tamoxifen in SA 90 Figure C24 – MIC of 4-hydroxy tamoxifen in MRSA 90 x SUMMARY Antibiotic resistance is increasingly a health and financial burden on the global population. Use and misuse of antibiotics has led to increased frequencies of antibiotic-resistant infections worldwide, leading to fatalities as well as greatly increasing healthcare costs. To combat this, researchers have done much work to expand to the field of antibiotics, delving back into old compounds and testing massive libraries of compounds with rapid screening techniques. Tamoxifen is one such compound that is primarily used as an anticancer agent, but displays many useful other characteristic, including antibacterial effects. However, the mechanism of the antibacterial effects of tamoxifen are poorly documented. My research was aimed at both improving the effectivity of tamoxifen as an antibacterial and elucidating the mechanism of action of tamoxifen. xi CHAPTER 1. ANTIBIOTICS Antibiotics have a relatively short history, with development beginning in the 1930’s, slowly as the idea was adopted, exploding in the 1960’s, and decelerating significantly to the present day as many natural sources of novel antibiotics are exhausted. A brief history of antibiotics, along with a survey of the majority of relevant antibiotic classes, their histories, mechanisms, and uses are presented in this chapter. 1.1 The discovery and development of antibiotics In 1928, Alexander Fleming took a vacation from his work at St. Mary’s, leaving behind Petri dishes with bacterial cultures. Upon his return, he found the plates a mess of contamination. As he sorted through and discarded the ruined cultures, he noticed that one plate had mold growing along the edge. Furthermore, there was a clear zone between the mold and the nearest bacterial colony.