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Novel Compounds Inhibiting Hiv Infection, Breast NOVEL COMPOUNDS INHIBITING HIV INFECTION, BREAST CANCER METASTASIS, AND BACTERIAL GROWTH by Joyce Brewer Shepard A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry MONTANA STATE UNIVERSITY Bozeman, Montana May 2012 ©COPYRIGHT by Joyce Brewer Shepard 2012 All Rights Reserved ii APPROVAL of a dissertation submitted by Joyce Brewer Shepard This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citation, bibliographic style, and consistency and is ready for submission to The Graduate School. Dr. Martin Teintze Approved for the Department of Chemistry and Biochemistry Dr. Bern Kohler Approved for The Graduate School Dr. Carl A. Fox iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non-exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Joyce Brewer Shepard May, 2012 iv DEDICATION This work is dedicated first and foremost to Karen and Jeffrey Brewer. They deserve more than one dedication can give for being so supportive, listening, giving the best advice, and most of all, for loving so much. This is for you. This is also dedicated to Dr. Eric Shepard, an extraordinary husband. Without Eric, there would be no thesis. His perseverance, confidence, strength, and optimism is catching. His support is unwavering and his love is unequaled. It is finally done. And lastly, this is dedicated to the mountains of Montana. It may be abnormal to thank geography, but the mountains in Montana inspire many things such as; quilt patterns; rock collections of Amethyst, Smokey Quartz, Calcite, Agate, and others; and inspire “Big Sky” thinking more than all. Forever and Always, On Wisconsin. v ACKNOWLEDGMENTS I would like to thank Dr. Martin Teintze, my primary advisor, who taught me so much biochemistry and countless scientific techniques. I never found the limit of his knowledge, which was awe inspiring. I would also like to thank Dr. Royce Wilkinson, who trained me how to do many techniques and was always willing to discuss ideas with me. Dr. Jovanka Voyich deserves many thanks as well. She welcomed me into her lab for the MRSA experiments and shared her insight and enthusiasm. Thank you. A gigantic thank you to Dr. Jean Starkey, retired Research Associate Professor of the Microbiology Department. Jean become my co-mentor for the breast cancer project, and even hired me to run mice studies on her very own PDT project; reminding me how much fun science is. She was and will always be an inspiration and role model for me as a Scientist and for living life. May only happy memories be hers. Thank you, Jean. I would like to thank my committee members and collaborators from MSU who helped me over the years. Dr.Edward Dratz, my co-advisor for the crosslinking project; Dr. Valerie Copié; Dr. Brian Bothner, who directed me for two years in the MSU MS Facility as the Proteomics Specialist; Dr. Lilya Kirpotina for help with the calcium flux experiments; Robert Watkins, Cassidy Cooper, and Elizabeth Erikson for help with the MRSA project. Thanks to my friends and sisters for endless support, Bonnie Rizzardi, and Brenda Frank for always being there for me. Thank you all for helping me achieve this goal. vi TABLE OF CONTENTS 1. INTRODUCTION .............................................................................................. 1 Novel Guanide, Biguanide, and Phenylguanide Compounds with Applications in HIV Infections, Breast Cancer Metastasis, and Growth Inhibition of Antimicrobial Resistant Bacteria ....................................... 1 HIV Infection ..................................................................................................... 2 Breast Cancer and Metastasis ........................................................................ 16 Antimicrobial Compounds and Resistance ..................................................... 29 References ..................................................................................................... 39 2. NOVEL COMPOUNDS CONTAINING MULTIPLE GUANIDE GROUPS WHICH BIND THE HIV CO-RECEPTOR CXCR4 .......................................... 48 Contribution of Authors and Co-Authors ......................................................... 48 Manuscript Information Page .......................................................................... 49 Title Page ....................................................................................................... 50 Abstract .......................................................................................................... 51 Introduction ..................................................................................................... 52 Materials and Methods ................................................................................... 55 Starting Material Amine Compounds ....................................................... 55 (i) Trishexylaminomelamine (THAM) synthesis ............................. 55 (ii) Trisethylaminomelamine (TEAM) synthesis ............................. 56 (iii) Trisbutylaminomelamine (TBAM) synthesis ............................ 56 Synthesis of Guanide Derivatives ............................................................ 56 Synthesis of Biguanide Derivatives .......................................................... 57 Synthesis of Phenylguanide Derivatives .................................................. 57 Synthesis of a Photoactive, Fluorescent T140 peptide ............................ 58 CXCR4-T140 Cross-link Inhibition Assay ................................................ 58 CCR5-Maraviroc Analog Cross-link Inhibition Assay ............................... 59 Assay for Anti-HIV Activity ....................................................................... 59 Toxicity Assays ........................................................................................ 60 Results ........................................................................................................... 60 Guanide, Biguanide, and Phenylguanide Synthesis ................................ 60 CXCR4-T140 Cross-link Inhibition Assay ................................................ 61 Inhibition of HIV Infection ......................................................................... 63 Cytotoxicity .............................................................................................. 64 Discussion ...................................................................................................... 64 Tables and Figures ......................................................................................... 68 Acknowledgments .......................................................................................... 76 References ..................................................................................................... 77 vii TABLE OF CONTENTS - CONTINUED 3. NOVEL GUANIDE CONTAINING MULTIPLE GUANIDE GROUPS THAT INHIBIT BREAST CANCER METASTASES .................................................. 82 Contribution of Authors and Co-Authors ......................................................... 82 Manuscript Information Page .......................................................................... 83 Abstract .......................................................................................................... 84 Introduction ..................................................................................................... 85 Materials and Methods ................................................................................... 87 Cell Lines ................................................................................................. 87 CXCR4-Inhibitor-T140 Binding Assay ...................................................... 88 Cell Viability Assay................................................................................... 88 CXCR4 Expression Using Flow Cytometry .............................................. 88 Calcium Flux Assay ................................................................................. 89 Tissue Culture Wound Assay ................................................................... 89 Matrigel Assay ......................................................................................... 90 Mouse Toxicity ......................................................................................... 90 A. Alexidine Dihydrochloride ......................................................... 90 B. Spermine Bisguanide ................................................................ 91 C. Spermidine Trisphenylguanide, Spermidine Bis-2- Naphthylguanide, or Spermine Tris-2-Naphthylguanide ............ 91 Lung Colony Metastasis Assays .............................................................. 91 A. Alexidine and
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