Thesis Presented to the Graduate Faculty Of
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A SEARCH FOR MULTI-DRUG RESISTANCE PUMP INHIBITOR MOLECULES BY ISOLATION OF NATURAL PRODUCTS Approved by: ________________________________ Edward R. Biehl, Professor ________________________________ John A. Maguire, Professor ________________________________ David Y. Son, Professor ________________________________ Pia D. Vogel, Professor A SEARCH FOR MULTI-DRUG RESISTANCE PUMP INHIBITOR MOLECULES BY ISOLATION OF NATURAL PRODUCTS A Thesis Presented to the Graduate Faculty of Dedman College Southern Methodist University In Partial Fulfillment of the Requirements For the degree of Master of Science With a Major in Chemistry By Alan Wilfred Humason (B.S., Chemistry, University of Massachusetts at Amherst) May 14, 2005 Humason, Alan B.S., University of Massachusetts at Amherst, 1979 A Search for Multi-Drug Resistance Pump Inhibitor Molecules By Isolation of Natural Products Advisor: Professor Edward R. Biehl Master of Science conferred May 14, 2005 Thesis completed April 22, 2005 A search for multi-drug resistant pump inhibitory molecules was conducted in several plants that have clinically, or in traditional medicine, demonstrated useful activity against bacteria. The investigation was conducted by extracting the plants with organic solvents, and fractionating the resultant extracts chromatographically. These fractions were then subjected to testing against Staphylococcus aureus, in conjunction with a sub-inhibitory dose of the antibiotic berberine. Extracts exhibiting inhibitory activity were further fractionated using vacuum liquid chromatography, flash liquid chromatography and conventional and preparative thin layer chromatography, attempting to isolate a single molecule which exhibits multi- drug resistance pump inhibition. The plants that were investigated, Eriogonum brevicaule Nuttall, Hydrastis canadensis, Thuja occidentalis, Rhus trilobata, Pouteria pallida, and Gunnera macrophylla, yielded over two hundred fractions, including several active ones. The utility of each of the chromatographic techniques was demonstrated and critiqued. iii TABLE OF CONTENTS LIST OF FIGURES.............................................................................................................v LIST OF TABLES .............................................................................................................vi ACKNOWLEDGEMENTS ..............................................................................................vii Chapter I. INTRODUCTION......................................................................................................1 II. EXPERIMENTAL SECTION....................................................................................7 III. EXTRACTION PARAMETERS FOR SPECIFIC SAMPLES ...............................13 Eriogonum brevicaule Nuttall ...............................................................................13 Hydrastis canadensis.............................................................................................24 Pouteria pallida – N014795..................................................................................27 Thuja occidentalis – N102279/ N102249..............................................................30 Rhus trilobata – N102791 .....................................................................................32 Gunnera macrophylla – N014045.........................................................................35 IV. MDRP ACTIVITY SUMMARY .............................................................................37 V. CONCLUSION ........................................................................................................45 Appendices A. MOLECULAR STRUCTURE OF CERTAIN ANTIBIOTICS ..............................48 B. EXTRACTION LOG...............................................................................................49 C. MDRP ACTIVITY TESTING RESULTS...............................................................54 D. SPECTROSCOPIC ANALYSIS BY DR. RAMADAS SATHUNURU.................67 REFERENCES..................................................................................................................73 iv LIST OF FIGURES Figures 1. Molecular Structures – Reserpine ...........................................................................4 2. Molecular Structure – 5”-Methoxy hydrocarpin-D.................................................4 3. Molecular Structure – Berberine .............................................................................5 4. Molecular Structure – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid.....................43 5. Molecular Structure of Certain Antibiotics ...........................................................48 6. Molecular Structure – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid.....................67 7. Molecular Structure – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid acetate.........68 8. HRMS – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid..........................................70 9. 1H NMR – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid.......................................71 10. 1H NMR – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid acetate ..........................72 v LIST OF TABLES Table 1. Eriogonum brevicaule Nuttall Hillside Fractionation Parameters ........................18 2. Eriogonum brevicaule Nuttall FxCP Fractionation Parameters............................19 3. Eriogonum brevicaule Nuttall Leaves Fractionation Parameters..........................19 4. Eriogonum brevicaule Nuttall Roots Fractionation Parameters............................20 5. Eriogonum brevicaule Nuttall Re-extraction Parameters......................................21 6. Flash Chromatography Parameters for AH73/AH103/AH127 .............................23 7. Flash Chromatography Parameters for Pouteria pallida.......................................28 8. Flash Chromatography Parameters for Thuja occidentalis ...................................31 9. Flash Chromatography Parameters for Rhus trilobata AH250 .............................34 vi ACKNOWLEDGMENTS I would like to thank the many scientists who assisted me with this paper, both with information and with their efforts. Dr. Frank Stermitz, who provided flora samples and technical advice. Dr. Kim Lewis, whose laboratories at Tuft’s University and Northeastern University performed the biological activity testing, and who also assisted with technical advice. Dr. George Tegos, and Mr. Anthony Ball, who did the activity testing, under Dr. Lewis. Dr. Edward Biehl, who was my thesis advisor and mentor. Dr. Pia Vogel, who reviewed and corrected the biological references in this thesis, and the thesis itself, thus completing her review twice. Dr. Ramadas Sathunuru, who gave me invaluable guidance and assistance in the laboratory, as well as investing his own time in this research. It is his work that appears in Appendix D. I must also express my thanks and affection to my wife, Melissa McNamara Humason, and my children, Alana Rose Humason and Aidan Bartholomew Humason, for their support and their acceptance of my absences while completing this work. vii Chapter I INTRODUCTION To describe the search for a multi-drug resistance pump inhibitor, one must first define the terms. We can actually build this definition one term at a time. In the counter-action of bacteria, the traditional medical approach is to use antibiotics, such as penicillin1,2, tetracycline3, erythromycin4, and the “last resort”5 antibiotic vancomycin.6 These are the drugs. The need has always existed for the development of new antibiotics, because the old ones become ineffective as bacteria become resistant to them.7-10 This is the drug resistance. One method used by bacteria to counteract drug therapy is to develop a mechanism to eject the antibiotics from each cell. This mechanism takes the form of a complex protein that spans the cell wall. This protein is programmed to identify an antibiotic and systematically pump it outside the cell wall. This is the drug resistance pump. As new antibiotics are developed, the bacterial drug resistance pumps succeed in ejecting the new drug, which may be chemically very different from old ones. The structural diversity of current antibiotics is shown in Appendix A. This ability to eject many different antibiotics is what constitutes the multi-drug resistance pump (MDRP.)11 1 The anthropogenic approach to overcoming the MDRP is not useful for flora. Plants have the ability to produce their own antibiotics. For example, Hydrastis canadensis, known as goldenseal, is a source of the naturally occurring antibiotic berberine. The plant does not, however, have the ability to create new antibiotics as the bacteria reject the old ones. Therefore, flora takes a different approach. This defense mechanism is to create a molecule, dissimilar to the antibiotic, which can disrupt the pump mechanism. These molecules will enter the multi-drug resistance pump and bind, thereby blocking the bacteria’s antibiotic defense. Then, the original antibiotics become efficacious again. These compounds are the multi-drug resistance pump inhibitors.12 The goal of this study is to identify one or more compounds that demonstrate multi-drug resistance pump inhibition. Plants that have shown useful activity against bacteria may employ MDRP inhibitors, although they may use other defense mechanisms. These plants are the focus of this study. The manner of the search is outlined as follows: 1) Obtain samples of those plants that have clinically, or in traditional medicine, been used antibacterially. 2) Isolate the smaller