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Characterization of Prostate Cancer Drug Targets

Permanent Link: http://ufdc.ufl.edu/UFE0042358/00001

Material Information

Title: Characterization of Prostate Cancer Drug Targets
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Sippel, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: carbonic, cypl, drug, isozyme, mg289, mycoplasma, p37, prostate
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Prostate cancer is the second most prevalent form of cancer in men. Over 32,000 men are expected to die of prostate cancer this year and one in six men will have prostate cancer in their lifetime. Most treatments for prostate cancer come with the risk of unpleasant side effects that reduce quality of life. The identification and characterization of novel drug targets are the first step to improving the treatment of this disease. A few of these targets include carbonic anhydrase (CA) IX that is overexpressed in solid tumors, and the Mycoplasmal proteins Cypl and MG289 which may contribute to tumorigenesis by chronic inflammation. To develop more effective drugs against CA IX, a soluble, easily expressed mimic was developed from the soluble isozyme CA II. This mimic proved effective in identifying potential mechanisms for isozyme specificity among classic sulfonamide drugs. The mimic protein was also used to evaluate isozyme specificity of several 2-Ethylestra sulfamate compounds. 2-Ethylestradiol-3-O-sulfamate demonstrated a 12-fold decrease in affinity for CA IX mimic due to a water-mediated hydrogen bond to a CA IX specific residue. Chlorzolamide was also assessed in vivo using nude mice with xenograft tumors for its effectiveness as a prostate cancer therapy. Little difference was seen in chlorzolamide treated mice as compared to controls. Further pharmacokinetic tests indicated that chlorzolamide lacks the oral bioavailability to be an effective cancer treatment. To fight Mycoplasma induced tumorigenic transformation, the immunogenic proteins had to first be characterized. The M. hyorhinis protein Cypl had been associated with cancer, but its function remained unclear. Structure solution of Cypl using a novel heavy atom for phasing revealed insights into its function. Cypl was recognized as an extracytoplasmic thiamine binding lipoprotein. Another Cypl-like protein is MG289 from M. genitalium, a sexually transmitted infection. Structurally it is very similar to Cypl, however it binds thiamine. Inferences were made addressing several metabolic questions and providing a target for fighting mycoplasmal infection. These studies represent the initial phases to the development of novel prostate cancer treatments and could be the inroads to improving the quality of life for those suffering from this pervasive disease.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Katherine Sippel.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: McKenna, Robert.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042358:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042358/00001

Material Information

Title: Characterization of Prostate Cancer Drug Targets
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Sippel, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: carbonic, cypl, drug, isozyme, mg289, mycoplasma, p37, prostate
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Prostate cancer is the second most prevalent form of cancer in men. Over 32,000 men are expected to die of prostate cancer this year and one in six men will have prostate cancer in their lifetime. Most treatments for prostate cancer come with the risk of unpleasant side effects that reduce quality of life. The identification and characterization of novel drug targets are the first step to improving the treatment of this disease. A few of these targets include carbonic anhydrase (CA) IX that is overexpressed in solid tumors, and the Mycoplasmal proteins Cypl and MG289 which may contribute to tumorigenesis by chronic inflammation. To develop more effective drugs against CA IX, a soluble, easily expressed mimic was developed from the soluble isozyme CA II. This mimic proved effective in identifying potential mechanisms for isozyme specificity among classic sulfonamide drugs. The mimic protein was also used to evaluate isozyme specificity of several 2-Ethylestra sulfamate compounds. 2-Ethylestradiol-3-O-sulfamate demonstrated a 12-fold decrease in affinity for CA IX mimic due to a water-mediated hydrogen bond to a CA IX specific residue. Chlorzolamide was also assessed in vivo using nude mice with xenograft tumors for its effectiveness as a prostate cancer therapy. Little difference was seen in chlorzolamide treated mice as compared to controls. Further pharmacokinetic tests indicated that chlorzolamide lacks the oral bioavailability to be an effective cancer treatment. To fight Mycoplasma induced tumorigenic transformation, the immunogenic proteins had to first be characterized. The M. hyorhinis protein Cypl had been associated with cancer, but its function remained unclear. Structure solution of Cypl using a novel heavy atom for phasing revealed insights into its function. Cypl was recognized as an extracytoplasmic thiamine binding lipoprotein. Another Cypl-like protein is MG289 from M. genitalium, a sexually transmitted infection. Structurally it is very similar to Cypl, however it binds thiamine. Inferences were made addressing several metabolic questions and providing a target for fighting mycoplasmal infection. These studies represent the initial phases to the development of novel prostate cancer treatments and could be the inroads to improving the quality of life for those suffering from this pervasive disease.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Katherine Sippel.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: McKenna, Robert.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042358:00001


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1 CHARACTERIZATION OF PROSTATE CANCER DRUG TARGETS By KATHERINE H. SIPPEL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Katherine H. Sippel

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3 To those who believed in me, those who put up with me, and those who decided not to strangle me in my sleep this dissertation is for you. Thank you.

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4 ACKNOWLEDGMENTS First, I would like to thank my family, my mom and dad, my brother and Bob for all their hard work, supporting me in everything until I could achieve my goals. I would like to thank the family of my heart, Krysten, Nick, Charles, and Bryan, who stood by me in the worst of times even when I couldnt stand myself. Your friendship has made me a stronger person. I could not have come this far without you. To my friend, Jeanne, who learned how to be a research scientist with me and always backed me up when I had more on my plate than I could deal with. To my friends, Courtney and Dave, who sat through bad movies, walked my dog, and generally reminded me that there was a world outside of the lab. I would like to thank my dog, Raisin for giving me a reason to take care of myself by taking care of him. My committee also deserves many thanks. They were both mentors and collaborators helping me delve into a wide variety of techniques that helped me answer a large number of questions. I want to thank the McKenna Lab, both protein and virus sides, who have made these years a pleasure. Even in the worst of times I never woke up dreading to go into lab. It has never failed to be interesting. To Dr. Art Robbins who let me pick his brain and in doing so taught me how to think like a crystallographer. And finally to Dr. Rob McKenna who has been a better mentor than I could have imagined. He has fielded questions, derailed emotional rollercoasters, and never failed to be there exactly when I needed him. He has pushed me farther than I ever thought I could go and taught me the measure of my abilities. Thank you all.

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. 4 page LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INT RODUCTION .................................................................................................... 14 Prostate Cancer ...................................................................................................... 14 Carbonic Anhydrase IX ........................................................................................... 15 Carboni c Anhydrase (CA) ................................................................................. 15 CA IX ................................................................................................................ 15 CA IXs Role in Cancer Progression ................................................................. 15 Mycoplasma p37 ..................................................................................................... 17 Chronic Inflammation and Prostate Cancer ...................................................... 17 Mycoplasmas ................................................................................................... 17 Mycoplasma Hyorhinis ..................................................................................... 18 Mycoplasma Genitalium ................................................................................... 19 Mycoplasma Hyorhinis p37 (Cypl) .................................................................... 20 Mycoplasma Genitalium MG289 ...................................................................... 21 Aims of this Study ................................................................................................... 21 2 DESIGN OF A CARBONIC ANHYDRASE IX ACT IVESITE MIMIC TO SCREEN INHIBITORS FOR POSSIBLE ANTI CANCER PROPERTIES ............... 22 Background ............................................................................................................. 22 Experimental Methods ............................................................................................ 23 In Silico Design of the CA IX Mimic .................................................................. 23 Enzyme Expression and Purification ................................................................ 23 Cr ystallization ................................................................................................... 24 Drug Soaks ....................................................................................................... 24 Diffraction Data Collection ................................................................................ 24 St ructure Determination .................................................................................... 25 18O Isotope Exchange Kinetic and Inhibition Studies ....................................... 25 Cell Lines and Culture ...................................................................................... 26 Western Blot Analysis ...................................................................................... 26 In Vitro Cytotoxicity Assay ................................................................................ 26 Results .................................................................................................................... 27 Sequence and Structural Alignment Comparison ............................................. 27 Kinetic Comparison .......................................................................................... 28

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6 Comparison Between Wild Type CA IX, CA IX Mimic, and CA II ..................... 29 Drug Inhibition Studies ..................................................................................... 30 Crystallographic Study ..................................................................................... 31 PC3 Bcl2 Western Blot and Cytotoxicity Assay ............................................. 33 Discussion .............................................................................................................. 34 3 CHARACTERIZATION OF CARBONIC ANHYDRASE ISOZYME SPECIFIC INHIBITION BY 2 ETHYLESTRA COMPOUNDS ................................................... 46 Background ............................................................................................................. 46 Experimental Methods ............................................................................................ 47 Compound Synthesis ....................................................................................... 47 Protein Expression and Purification .................................................................. 47 Oxygen18 Isotope Exc hange Inhibition Studies .............................................. 47 Crystallization and Data Collection ................................................................... 48 Structure Determination .................................................................................... 48 Results .................................................................................................................... 49 Compound Inhibition Kinetics ........................................................................... 49 Compound 14 Structural Comparisons ............................................................ 49 Compound 15 Structural Comparisons ............................................................ 50 Compound 16 Structural Comparisons ............................................................ 51 Discu ssion .............................................................................................................. 52 4 CARBONIC ANHYDRASE IX INHIBITORS APPLICATIONS IN VIVO ................ 60 Background ............................................................................................................. 60 Experimental Methods ............................................................................................ 61 Mouse Xenograft Tumors ................................................................................. 61 Tissue and Blood Samples ............................................................................... 61 Immunohistochemistry ...................................................................................... 62 TUNEL Assay ................................................................................................... 63 Mouse Pharmacokinetic Studies ...................................................................... 64 Results .................................................................................................................... 64 Xenograft Mouse Tumor Study ......................................................................... 64 Tumor Immunohistochemistry .......................................................................... 65 Hematoxylin and Eosin .............................................................................. 65 CA IX ......................................................................................................... 65 PCNA ......................................................................................................... 65 TUNEL ....................................................................................................... 66 Mouse Pharmacokinetic Studies ...................................................................... 66 Discussion .............................................................................................................. 67 5 THE STRUCTURE SOLUTION AND INSIGHTS INTO THE MYCOPLASMA HYORHINIS PROTEIN CYPL ................................................................................. 73 Background ............................................................................................................. 73 Experiment al Methods ............................................................................................ 74

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7 Expression and Purification of Cypl .................................................................. 74 Crystallization and Diffraction Data Collection .................................................. 75 Structure Solution ............................................................................................. 76 Refinement of Cypl ........................................................................................... 77 Sequence Alignments and Phylogenic Tree Anal ysis. ..................................... 78 Structural Alignment. ........................................................................................ 78 Results .................................................................................................................... 78 Overall Fold of Cypl .......................................................................................... 78 Binding of Thiamine Pyrophosphate ................................................................. 79 Comparative Structural Analysis. ..................................................................... 80 Discussion .............................................................................................................. 80 Cypl and Other Cypl like Proteins. ................................................................... 80 The Presence of TPP in Cypl. .......................................................................... 81 Why Cypl Is Probably Not an Enzyme. ............................................................. 82 Why Cypl Is Probably a Transport/Binding Protein. .......................................... 83 6 STRUCTURE SOLUTION AND INSIGHTS INTO MYCOPLASMA GENITALIUM MG289 .................................................................................................................... 94 Background ............................................................................................................. 94 Experimental Methods ............................................................................................ 95 Plasmid Cloning ............................................................................................... 95 Expression and Purification .............................................................................. 96 Crystallization, Xray Analysis, Str ucture Solution and Refinement .................. 96 Size Exclusion Chromatography ...................................................................... 97 Small Angle Xray Scattering ............................................................................ 98 Results .................................................................................................................... 98 Overall Structure of MG289 .............................................................................. 98 Thiamine Binding Site .................................................................................... 100 Dimer Interface ............................................................................................... 100 Size Exclusion Chromatography .................................................................... 101 Small Angle Xray Scattering (SAXS) ............................................................. 101 Comparison of MG289 to Homolog Cypl ........................................................ 102 Discussion ............................................................................................................ 103 7 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 112 APPENDIX A DESIGN OF A CARBONIC ANHYDRASE IX ACTIVESITE MIMIC TO SCREEN INHIBITORS FOR POSSIBLE ANTI CANCER PROPERTIES CONTINUED ........................................................................................................ 116 B CHARACTERIZATION OF ISOZYME SPECIFIC CARBONIC ANHYDRASE INHIBITION BY 2 ETHYLESTRA COMPOUNDS CONTINUED .......................... 119

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8 C STRUCTURE SOLUTION AND INSIGHTS INTO MYCOPLASMA HYORHINIS PROTEIN CYPL CONTINUED ............................................................................. 130 D STRUCTURE SOLUTION AND INSIGHTS INTO MYCOPLASMA GENITALIUM MG289 CONTINUED ............................................................................................ 137 LI ST OF REFERENCES ............................................................................................. 139 BIOGRAPHICAL SKETCH .......................................................................................... 157

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9 LIST OF TABLES Table page 2 1 Ki Values o f inhibitors for CA II, CA IX mimic and CA IX. ................................... 40 2 2 In hibitor protein interactions ............................................................................... 42 3 1 Interaction Table ................................................................................................. 55 3 2 Compound Inhibition Kinetics ............................................................................. 56 5 1 TPP interactions of Cypl ..................................................................................... 88 5 3 Pair wise sequence identity of Cypl like proteins ................................................ 91 6 1 List of Putative M. genitalium Thiamine Kinases .............................................. 111 A 1 Data and refinement st atistics for CA IX mimic alone and in complex. ............. 117 B 1 Data and refinement statistics. ......................................................................... 126 C 1 Data and Refinement Statistics of Cypl ............................................................ 134 C 2 Mycoplasma Cypllike proteins used for sequence alignment .......................... 135 D 1 MG 289 Data and refi nement statistics ............................................................. 137

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10 LIST OF FIGURES Figure page 2 1 Carbonic anhydrase inhibitors. ........................................................................... 37 2 2 Sequence and struct ure of unbound CA IX mimic. ............................................ 38 3 2 Superposition of wild type CA IX and CA IX mimic in complex with Acetazolamide. ................................................................................................... 39 2 4 Compar ison of sulfonamides in CA II and CA IX mimic. ..................................... 41 2 5 Superposition of all five drug complexes in crystal structures. ........................... 44 2 6 Ce ll proliferation analysis in PC 3 Bcl2 prostate cancer cell lines. ...................... 45 3 1 2 Ethylestra compound formulas and names. .................................................... 54 3 3 Comp ound 14. .................................................................................................... 57 3 4 Compound 15. .................................................................................................... 58 3 5 Compound 16. .................................................................................................... 59 4 1 Xenograf t tumor volume measurements. ............................................................ 69 4 2 Tumor cell morphology. ...................................................................................... 70 4 3 Immunohistochemistry of day 28 tumors. ........................................................... 71 4 4 Urine pH time course post sulfonamide drug administration. ............................. 72 5 1 Overall structure of Cypl. .................................................................................... 86 5 2 Thiamine Pyrophosphate binding site. ................................................................ 87 5 3 Structural similarity Cypl to E. coli TbpA. ........................................................... 89 5 4 Sequence ali gnment of Cypl like proteins. ......................................................... 90 5 5 An unrooted phylogenetic tree of mycoplasma Cypl like proteins. ..................... 92 5 6 Conformation of TPP. ......................................................................................... 93 6 1 The structure of MG289. ................................................................................... 108 6 2 The quaternary structure of MG289. ................................................................. 109

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11 6 3 MG 289 versus Cypl. ........................................................................................ 110 A 1 Crystal structures of the active site of CA II and CA IX mimic in complex with inhibitors. .......................................................................................................... 116 B 1 Chemical formulas of intermediate synthetic compounds. ................................ 127 B 2 Electron density maps of compounds 14, 15, and 16. ...................................... 128 B 3 CA IX mimic with 15 molecule 2. ...................................................................... 129 C 1 5 amino2,4,6triiodoisophthalic acid binding sites. ......................................... 136 D 1 Docking of MG289 dimer into SAX ab initio reconstruction. ............................. 138

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF PROSTATE CANCER DRUG TARGETS By Katherine H. Sippel December 2010 Chair: Robert McKenna Major: Medical Sciences Biochemistry and Molecular Biology Prostate cancer is the second most prevalent form of cancer in men. Over 32,000 men are expected to die of prostate cancer this year and one in six men will have prostate cancer in their lifetime. Most treatments for prostate cancer come with the risk of unpleasant side effects that reduce quality of life. The identification and characterization of novel drug targets are the first step to improving the treatment of this disease. A few of these targets include carbonic anhydrase (CA) IX that is overexpressed in solid tumors, and the Mycoplasmal proteins Cypl and MG289 which may contribute to tumorigenesis by chronic inflammation. To develop more effective drugs against CA IX, a soluble, easily expressed mimic was developed from the soluble isozyme CA II. This mimic proved effective in identifying potential mechanisms for isozyme specificity among classic sulfonamide drugs. The mimic protein was also used to evaluate isozyme specificity of several 2Ethylestra sulfamate compounds. 2Ethylestradiol 3 O sulfamate demonstrated a 12fold decrease in affinity for CA IX mimic due to a w ater mediated hydrogen bond to a CA IX specific residue. Chlorzolamide was also assessed in vivo using nude mice with xenograft tumors for its effectiveness as a prostate cancer therapy. Little difference was

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13 seen in chlorzolamide treated mice as compared to controls. Further pharmacokinetic tests indicated that chlorzolamide lacks the oral bioavailability to be an effective cancer treatment. To fight Mycoplasma induced tumorigenic transformation, the immunogenic proteins had to first be characterized. The M. hyorhinis protein Cypl had been associated with cancer, but its function remained unclear. Structure solution of Cypl using a novel heavy atom for phasing revealed insights into its function. Cypl was recognized as an extracytoplasmic thiamine binding l ipoprotein. Another Cypl like protein is MG289 from M. genitalium a sexually transmitted infection. Structurally it is very similar to Cypl, however it binds thiamine. Inferences were made addressing several metabolic questions and providing a target for fighting mycoplasmal infection. These studies represent the initial phases to the development of novel prostate cancer treatments and could be the inroads to improving the quality of life for those suffering from this pervasive disease.

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14 CHAPTER 1 INTRODUCTION Prostate Cancer Prostate cancer is the secondleading cause of cancer death in men. The American Cancer Society reports an estimated 217,730 new cases of prostate cancer will be diagnosed in 2010. This accounts for 25% of all new cases of cancer in men. Additionally 32,050 men are estimated to die from prostate cancer this year. Current approximations indicate that one in six men will have prostate cancer in their lifetime, of those one in 36 will prove fatal. Early detection methods such as digital rectal exams and screenings for prostate specific antigen (PSA) in the blood are very effective in increasing the overall survival rate of men diagnosed with prostate cancer. If diagnosed early surveillance, radical prostatectomy, radiation, and in some cases androgen deprivation therapy are effective in eradicating the disease. For advanced or aggressive cancers, however, the options become more limited. Radiation, chemotherapy and hormone therapies are common treatments but they have significant side effect s and are not always effective. This is why the prognosis of late stage diagnosis is so poor ( 1 ) Once established, prostate cancer can be devastating. Studies have shown that 6575% of patients with advanced stage prostate cancer develop bone metastasis. At this point there is only a 25% survival rate over five years ( 2 ) In 2004 the Food and Drug Administration convened the Prostate Cancer Clinical Trials Working Group to reassess how treatment effectiveness was measured in cases of advanced stage prostate cancer (i.e. metastatic or castrationresistant). The result of this meeting was a redefinition of drug success based on progressionfree survival rather than overall survival ( 3 ) There is a need to identify and characterize new prostate cancer drug

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15 targets to combat the disease, both in the formati ve stages and in the treatment of advanced disease. It is hoped that through the characterization of novel drug targets new compounds and therapies can be developed. Carbonic Anhydrase IX Carbonic A nhydrase (CA) Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible inter conversion of CO2 and HCO3 ( 4 ) class CAs are present predominantly in vertebrates, though have also been shown to present in other organisms. There are 14 CAs (CA I XIV) in humans, and the active CAs play roles in respiration, pH homeostasis, fluid production, and other functions as yet to be determined ( 5 8 ) CA IX CA IX is a membrane associated glycoprotein, composed of several domains including a short intracellular region, a single transmembrane helix, and an extracellular proteoglycan domain that encodes a catalytic CA domain ( 9 ) It appears to function as a back to back dimer, with the active sites exposed to the extracytoplasmic space. It is also one of the most active of the CA isozymes ( 10) Under normal conditions CA IX is only expressed in cells that are thought to need to maintain low extracellular pH, such as gastric mucosal cells. However, in many cancers it is over expressed as a result of hypoxia ( 6 ) CA IXs Role in Cancer Progression The regulation of the CA IX gene has been shown to be controlled by the hypoxia inducible factor 1 (HIF 1) ( 9 ) It has been hypothesized that as tumor growth progresses and becomes insufficient to maintain a supply of oxygen, the cancer cell remodels metabolically, which is partially achieved by the upregulation of CA IX. Therefore CA IX

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16 is considered to be a marker of tumor hypoxia ( 11) In hypoxic tumors, it is believed that CA IX plays a critical role in cell survival. Within these tumors there is an observed increase in CO2 concentration ( 12) It is believed that this is not a result of oxidative metabolism, but a by product of an increase in the pentose phosphate pathway. This serves to replenish the supply of NADPH and generate ribose5 phosphate, necessary for nucleotide and coenzyme production ( 13 ) The surplus of CO2 diffuses out of the cell and is converted to HCO3 and a proton by CA IX, creating a significant inc rease in extracellular proton concentration causing the acidification of the tumor microenvironment. The alteration in proton flux is also believed to affect the activity of ion transporters and channels ( 8 ) The acidity may also cause the exclusion of weakly basic chemotherapeutic agents such as mitoxantrone, paclitaxel, topotecan, and doxorubicin rendering traditional therapies less effective ( 9 ) In addition to tumor microenvironment acidification, the proteoglycan domain of CA IX has been implicated in the disruption of cell cell adhesion by breaking the connection of E cadherin to the cytoskeleton, which may lead to tumor invasion ( 14) In prostate cancer specifically, there seems to be a relations hip between CA IX, Bcl 2, an anti apoptotic oncogene, and radiation resistance ( 15) All of these factors suggest that CA IX is an attractive drug target for the treatment of m any cancers, including prostate cancer. In fact, several studies have already shown that inhibition of CA IX can lead to decreased invasiveness as well as inducing cell death under hypoxic conditions ( 6, 11, 1619)

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17 Mycoplasma p37 Chronic Inflammation and Prostate Cancer The etiology of prostate cancer is not fully understood. Like most cancers there is likely a genetic source, seen both through mutation and heredity ( 20, 21) However, it has been observed that the genetic component alone is inconsistent with the incidence of prostate cancer. Recent studies have looked to inflammation of the prostate by chronic bacterial and viral infections as one possible cause of this disease ( 22) Meta analys is of epidemiological studies has shown a link between prostatitis and prostate cancer, reporting an odds ratio of 1.4 in men who reported having a sexually transmitted infection (STI) and an odds ratio of 2.1 in men whose partners reported having an STI ( 23) There are several means by which chronic infection can induce tumorigenic transformation, including damage by reactive oxygen species ejected by inflammatory cells, mutations during damageresponse hyperproliferation, the upregulation of immune response elements and over stimulation of the immune system reducing its anti tumor surveillance ( 24, 25) Mycoplasmas Mycoplasmas (class Moll icutes ) are the smallest organisms capable of replicating unassisted. These tiny, pleomorphic, wall free bacteria survive in tandem with eukaryotes either attached to the cell membrane or intracellularly. Only 0.20.3 microns in diameter, they also have one of the smallest functional genomes, 500800 kilobases ( 26) To date there are sixteen varieties of Mycoplasma known to infect humans ( 27) However many more species go undetected in the human body, largely due to their lack of overt cytopathic effect s ( 28) The first human pathogenic strain of Mycoplasma, M. pneumoniae, was characterized in the 1960s, but this was soon followed by the

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18 discovery of several more virulent species including M. hominis and Ureaplasma urealyticum (2931) The first associations between mycoplasmas and cancer occurred in the mid1960s when mycoplasmas were detected in leukemia patients ( 32) The results of these studies are treated skeptically due to the ease of contamination and poorly developed detection methods; however more controlled in vit ro studies have begun to create a clearer picture. Coculture of various cell lines with a large assortment of different mycoplasmas lead to chromosomal aberrations ( 2931) Several more studies have shown that mycoplasmal infections induce the upregulation of immune factors such as Tumor Necrosis Factor (NF 1, Cyclooxygenase2 (COX 2), inducible Nitrous oxide synthase (iNOS), and Matrix metalloproteinase (MMP) 9 ( 3338) More recent studies have indicated that it is in a situation of chronic infection that oncogenic transformation occurs ( 39, 40) In fact, eukaryotic cells chronically exposed to mycoplasmas, specifically the species fermentans penetrans hyorhinis and genitalium have consistently formed tumors when implant ed in nude mice ( 4143) As researchers have begun to look for infectious sources of cancer etiologies such as Helicobacter pylori in the case of gastric cancers and Human Papilloma Virus in cervical cancers, the possibility is raised that chronic mycoplasmal infections may be another source of oncogenecity ( 44, 45) Mycoplasma Hyorhinis Mycoplasma hyorhinis is a swine pathogen, causing respiratory infections, chest and abdominal lining inflammation, and arthritis ( 26) Though this bacterium is not considered to infect humans it is frequently a human cell culture contaminant ( 35, 36, 38) Further connections have emerged, including the discovery of a gene from M.

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19 hyopneumoniae, another swine pathogen, that has an 85% sequence homology between PTI 1 (prostate tumor inducing gene1), a prostate cancer metastasis marker ( 46) Further research has shown that the same PTI 1 sequences shares 97% homology to a gene from M. hyorhinis (data not shown). Several studies have indicated a correlation between the bacteria M. hyorhinis and various cancers including, but not limited to, gastric carcinoma, colon carcinoma, esophageal cancer, and lung cancer ( 47, 48) Persistent exposure to M. hyorhinis has been shown to induce tumorigenic transformation in benign prostate cancer cell lines ( 43) Mycoplasma Genitalium Mycoplasma genitalium is one of the smallest organisms capable of self replication with a genome of 580 kb which codes for approxi mately 480 proteins and is considered the starting point for identifying which genes are essential for life ( 49, 50) M. genitalium is also a human sexually transmitted infection that preferentially adheres to cili ated epithelial cells in the genitourinary and respiratory regions ( 5153) It is a cause of Chlamydia trachomatis negative nongonococcal urethritis in men and urethritis, cervicitis, and pelvic inflammatory disease in women ( 54) It has also been associated with infe rtility, increased HIV 1 transmission, and malignant transformation of prostate cells ( 43, 55, 56) Testing for M. genitalium infections is not trivial and can be expensive, leaving most cases unor misdiagnosed, leading to improper treatment and an increased instance of antibiotic resistance ( 57) Like M. hyorhinis benign prostate epithelial cell lines chronically exposed to this and other mycoplasmas have shown persistent altered gene expression ( 43, 58)

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20 Mycoplasma Hyorhinis p37 (Cypl) Mycoplasma hyorhinis p37 was originally identified as a 37 kDa protein that induced invasiveness in mouse sarcoma cells. This invasivity could be reversed if the cells were pretreated with the antibody to this protein ( 59, 60) Several years following the discovery of this carcinogenic substance, a study was conducted on patients immunized intralymphatically with their own surgically debulked tumors looking for antibodies that induced remission and their associated antigens. The study revealed the same 37 kDa protein, which was identified as a membraneassoci ated protein found in M. hyorhinis and was named p37 ( 61) The presence of this antigen as an oncogenic marker was confirmed by Ilantzis et al. ( 62) P37 is a misnomer, the actual protein is 43.5 kDa, c omposed of 403 residues. It bears some sequence similarity to a periplasmic bindingproteindependant transport system found in Gram negative bacteria and some mycoplasmas ( 63, 64) It is believed to function analogously as an extracytoplasmic binding lipoprotein, given that mycoplasma have only one membrane ( 63, 64) As a result of the studies described here, p37 w as given a more descriptive, identifiable, and accurate name. The protein is now designated the extracytoplasmic thiamine binding lipoprotein (Cypl). Since Cypl was initially identified, it has been shown to increase the invasivity of cancer cells ( 59, 60, 65) It has also been revealed that Cypl may be associated with cancer malignancy and metastasis ( 66, 67) Given its contribution to tumorigenesis and the fact that antibodies to this protein have been identified in human patients, this protein might provide a unique drug target.

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21 Mycoplasma Genitalium MG289 Like M. hyorhinis M. genitalium has a Cypl like protein, MG289, with which it shares 32% identity with Cypl. Also similar to Cypl, MG289 is located in an operon tentatively coding for a binding protein dependant substrate transporter. It is annotated as a putative phosphonate binding lipoprotein, followed by MG290, a nucleotidebinding membrane permease, and MG291, an integral membrane protein ( 50, 63, 64) It is very likely that MG289 and Cypl share similar structures and functions. In an effort to understand the role of MG289 in the minimal genome, it was discovered that the gene could be disrupted nonlethally. This is likely due to the inherent redundancy of action between this and several theoretical orphaned substrate binding lipoproteins found in the M. genitalium genome. Because the function of this protein is critical enough for a minimal bacterium to have evolved multiple copies, it is still considered to be an essential gene and could provide a starting point for antibiotic design that could prophylactically prevent tumorigenic transformation of the prostate ( 6870) Aims of this Study The starting point of any drug discovery lies in understanding the target and the mechanisms by which it can be inhibited. These studies focus on the characterization of the three drug targets described above. CA IX had been characterized previously, both biochemically and structurally, which allowed energy to be focused towards identifying isozyme specific inhibition mechanisms that could be applied in vivo ( 10, 71) However Cypl and MG289 lacked any functional or structural description. Pr esented here is the compilation of efforts towards the understanding of these three targets and the mechanisms by which an effective drug design strategy might be developed.

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22 CHAPTER 2 DESIGN OF A CARBONIC ANHYDRASE IX ACTIVESITE MIMIC TO SCREEN INHIBITORS FOR POSSIBLE ANTI CANCER PROPERTIES Background To develop an effective carbonic anhydrase inhibitor (CAI) based cancer therapy one significant barrier needs to be overcome, isoform specificity.1The development of a high affinity CA IX inhibitor was hampered due to the lack of an available crystal structure of CA IX. Recently this problem has been addressed, however, CA IX is a membrane protein that has proven itself difficult to express in sufficient soluble and properly folded quantities for effective drug studies ( 10, 71, 75) CAs all share roximate dimensions of 50 x 40 x 40 3. The active site is characterized by a conical cavity that is approximately 15 deep. The zinc ion is located at the bottom of the cavity and is tetrahedrally coordinated by three histidine ligands and a bound hydrox ide/water ( 4 ) The active sites between isoforms are highly conser ved ( 73) Of the15 isozymes of CA in the human body several are essential for respiration, pH homeostasis, fluid production, and renal function ( 5, 7, 8, 74) To overcome the expression issue, a CA IX active site mim ic was generated based on a structural alignment with CA II. This engineered CA IX mimic is a double mutant of CA II (A65S N67Q CA II) that imitates the active site of wild type CA IX. This CA IX mimic was expressed and characterized both kinetically and c rystallographically, alone and in complex with several2 1 Reprinted in part with permission from Genis, C., Sippel, K. H., Case, N., Cao, W., Avvaru, B. S., Tartaglia, L. J., Govindasamy, L., Tu, C., Agbandje McKenna, M., Silverman, D. N., Rosser, C. J., and McKenna, R. (2009) Desig n of a carbonic anhydrase IX activesite mimic to screen inhibitors for possible anticancer properties, Biochemistry 48 1322 1331. Copyright 20 09 American Chemical Society. common sulfonamide inhibitors, acetazolamide

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23 (AZM), benzolamide (BZM), chlorzolamide (CHL), ethoxzolamide (EZM), and methazolamide (MZM). The crystal structure comparison of mimic to wild type CA IX i ndicates that additional mutations are needed to optimize the mimics effectiveness. Further this structural information was evaluated in relationship to inhibition studies and i n vitro cytotoxicity assays and showed a correlated structureactivity relatio nship. Experimental Methods In S ilico Design of the CA IX Mimic All 14 human CA sequences were obtained from the NCBI database and a multiple sequence alignment was performed using ClustalW ( 76) A CA IX model was built with SWISSMODEL, using the high resolution CA II crystal structure (PDB accession code: 2ILI) as a template ( 77, 78) The molecular graphics programs COOT was used to visualize and analyze the CA IX model ( 79) Enzyme Expression and Purification The CA IX mimic was made by sitedirected mutagenesis using an expression vector containing the CA II coding region ( 80, 81) Residue Ala 65 and Asn 67 were mutated to serine and glutamine, respectively. Sitedirected mutagenesis was accomplished using the Quick Change Mutagenesis kit from Stratagene using CA II as a template, and the primers (mutations in bold): 5 CCTCAACAATGGTCAT T C G TTC C A G GTGGAGTTTGATGAC 3 and 5 GTCATCAAACT CCAC C T G GAA C G A ATGACCATTGTTGAGG. The DNA sequence was confirmed by the University of Florida Sequencing Core (Interdisciplinary Center for Biotechnology Research, Gainesville FL). CA IX mimic and CA II was transformed and expressed in Escherichia coli BL21(D CA ( 82, 83) The purification of CA IX mimic and CA II was performed on an affinity

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24 column of paminomethyl benzenesulfonamide (pAMBS) as described previously ( 84) The protein was collected then buffer exchanged into 50 mM Tris Cl pH 7.8, concentrated and then analyzed for expression and purity using Coomassie stained SDSPAGE. The final protein concentration was ~15 mg ml1 as calculated by measuring the optical density at 280 nm using a molar absorptivity of 5.5 x 104 M1 cm1. Crystallization Crystals of CA II and CA IX mimic were grown at room temperature using the h angingdrop vapor diffusion method ( 85) Crys tallization drops were prepared by mixing 7 1 in 50 m M Tris (100 m M Tris HCl pH 9.0, 1.3 M n dodecyl Ether (Hampton Research, Aliso Viejo, CA). The hangingdrops were equilibrated against 1 ml precipitant solution. Crystals appeared within 5 days. Drug Soaks The CAIs AZM, BZM, CHL, EZM, and MZM were solubilized in water, with AZM, CHL, and MZM requiring the addition of 50%, 10%, and 5% of DMSO, respectively (Figure 21). CA IX mimic an d CA II crystals were incubated with the inhibitor solutions at 4mM final concentration for ~24 h prior to x ray diffraction data collection. Diffraction Data Collection All the diffraction data for both the CA IX mimic and CA II crystals were collected in house on the RAXIS IV++ ). The crystal to detector distance was set at a range of 80100 mm (dependent on the quality of diffraction) and images were collected in 1 oscillation steps with a 300 s exposure time. The data was merged and scaled using HKL 2000 ( 86) All inhibitor soaked crystals were isomorphous. Data statistics are listed in Appendix Table A1 and A2.

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25 Structure Determination All the crystal structures were determined by molecular replacement methods using the previously solved wildtype CA II structure with solvents rem oved (PDB code 2ILI) ( 77) Initially, the structures were refined in CNS using rigidbody, B factor and energy minimization refinement ( 87 ) FoFc maps provided clear unbiased electron density for the inhibitors. The PRODRG server was used to generate topology files for modeling the inhibitors ( 88) The inhibitor coordinates were placed into the density using COOT and subsequent refinement continued using conjugategradient least squares (CGLS) refinement in SHELXL followed by manual model building and the addition of waters in COOT ( 79, 89) Refinement of the structures continued until the Rcryst and Rfree converged. The inhibitor enzyme interactions were determined by observing environmental distances in COOT and the model geometries were analyzed using PROCHECK ( 79, 90) All the structures had good refinement statistics for the resolution and rmsd bond and angle v alues similar to CA II. The coordinates and structure factors for all nine structures have been submitted to the Protein Data Bank. PDB files, datarefinement, and final model statistics are given in Appendix Tables A1 and A2. The 2FoFc density for the inhibitors is shown in Appendix Figure A1. High resolution structure refinement of CA II in complex with AZM (PDB ID 3hs4) was published elsewhere ( 91) 18O Isotope Exchange Kinetic and Inhibition Studies Inhibition constants (Ki values) of all compounds were determined by measurement of the inhibition of the 18O exchange activity between CO2 and water v ia mass spectrometry, as reviewed elsewhere ( 92, 93) Experiments were carri ed out at 25 C in solutions buffered at pH 7.4 with 0.1 M HEPES. The concentration of all species of carbonate was 10 mM, 93% HCO3 -, 7% CO2, and 0.1% CO3 2-. The enzyme

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26 concentration was 7.3 nM. Inhibitor concentrations ranged up to 800 M and were analyze d by the method of Henderson for tight binding inhibitors ( 94) Cell Lines and Culture Human prostate cancer cell line PC 3 Bcl2 (characterized by marked Bcl2 overexpression, PTEN deletion, and p53 mutation) was a generous gift from Dr. Timothy McDonnell (The University of Texas M. D. Anderson Cancer Center, Houston, TX). These cells were maintained in Dulbeccos modified Eagles medium (Mediatech, Inc. Herndon, VA) with 4.5 g l1 glucose, 4 mM Lglutamine, 10% fetal bovine serum, 100 units ml1 penicillin, 100 g ml1 streptomycin, and 500 g ml1 G418. All cells were incubated at 37 oC in a humidified atmosphere of 5% CO2 in air. Western Blot Analysis For protein extraction, PC 3 Bcl2 cells were lysed in lysis buffer [250 mM Tris Cl (pH 6.8), 2% SDS, and 10% glycerol] and protein inhibitor cocktail (Sigma, St. Louis, MO). A standard protein assay was performed using the DC Protein Assay kit (Bio Rad, Hercules, CA). Western blot analysis was completed as described previously ( 95) Immunoblotting was performed by first incubating the proteins with primary antibodies against CA IX (NB100417, tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) and then with horseradish peroxidase (HRP) conjugated secondary antibody (BioRad). Protein antibody complexes were detected by means of chemiluminescence (Amersham, Arlington Heights, IL). In Vitro Cytotoxicity Assay PC3 Bcl2 cells were seeded in 96well plates at a density of 2.5 x 103 cells per well and treated with drug, control (DMSO only ), or mock. The CAIs were dissolved in DMSO and administered at concentrations ranging from 0 nM to 100,000 nM After 1 4

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27 days, of 1 mg ml1 MTT (SigmaAldrich, St. Louis, MO) solution was added to appropriate plates and allowed to incubate at 37oC for 2.5 hours ( 95) Each reaction was stopped with lysis buffer (200 mg ml1 SDS, 50 % N,N dimethylformamide, pH 4) at ro om temperature for 1 hour and the optical density was read on a microplate autoreader (BioTek Instruments, Winooski, VT) at 560 nM. Absorbance values were normalized to the values obtained for the mock treated cells to determine survival percentage. Each assay was performed in triplicate. Cellular viability was confirmed by the crystal violet exclusion test ( 95 ) Western blot, cell culture and cytotoxicity assays were performed by Wengang Cao in the lab of Charles J. Rosser, MD. Results Sequence and Structural Alignment Comparison The multiple sequence alignment showed the extracellular CA domain of CA IX (residues 141390), shared a 39% sequence identity with CA II (residues 1261). This variation in sequence is approximately the average value of sequence conservation betweeen the human CAs. Superimposition of the SWISS MODEL 3D homolgy model of the catalytic domain of CA IX onto CA II gave a C (rmsd) of 0.6 Not surprising, most of the amino acid differences between the two isoforms were on the surface. The three centrally located histidines (residues 94, 96, and 119 CA II numbering) that coordinate the zinc ion the second sphere histidine coordinating residues (Gln 92, Ser 117, and the carbonyl of residue 244), and the amino acids that form the hydrophobic CO2 binding pocket (residues Val 121, Val 143, Leu 198, Thr 199CH3, Val 207 and Trp 209 CA II numbering) were completely conserved (Figure 22A). The active site cavity of CA II is lined with hydrophilic amino acids (residues Tyr 7, Asn 62, His 64, Ala 65, Gln 67, Thr 199 and Thr 200, CA II numbering),

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28 and these residues exhibit variation between different isoforms. They are believed to create a solvent network that influence the rate limiting proton transfer step in catalysis. On close inspection of the active site cavity model of CA IX, it differed from CA II with respect to two of these amino acids, namel y, A65S and N67Q (based on CA II residue numbering) (Figure 2A). It was hypothesized that these two amino acid differences could cause differences in binding affinity of small molecule inhibitors. Therefore these point mutations were made to CA II to creat e the CA IX mimic. It is of note that there is a S65 in many CAs; in CA II from mouse, rat, and chicken; in CA VII, and in some CA CAs from Neisseria and Chlamydomonas Kinetic Comparison Duda e t al., have previously reported the pH profile of kcat/Km for CO2 hydration and the proton transfer dependent rate constant (RH2O) derived from 18O exchange for CA II, with a kcat/Km for CO2 hydration of (9.8 5) x 107 M1s1 and a maximal RH20 from the donor to zinc bound hydroxide of (8.0 1) x 105 ( 96) Similarly, the catalytic domain (residues 141390) of CA IX has been previously cloned and kinetically characterized ( 75) The pH profile of kcat/Km for CO2 hydration was derived from 18O exchange data and was described by a single ionization with an apparent pKa of 6.3, with a maximal kcat/Km of (5.5 0.1) x 107 M1s1. In addition the pH profile for RH2O of CA IX was shown to be bell shaped at pH 58, similar to CA II with RH2O corresponding to a rate limiting proton transfer from a donor of pKa of 6.4 to an acceptor (zinc bound hydroxide) with a pKa of 6.4. This was consistent with the pKa obtained from the pH profile of kcat/Km for CO2 hydration. The maximal rate constant for proton transfer from the donor to zinc bound hydroxide was (1.4 0.3) x 106 s1.

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29 For comparative purposes the same 18O protocols were employed to characterized the CA IX mimic. The pH profile (data not shown) determining the kcat/Km (M s 1) for hydration of CO2 catalyzed by CA IX mimic was (5.1 0.2) x 107 M1s1 with a (pKa)ZnH2O of 6.7 0.1, and the pH profile for the proton transfer dependent rate constant was fitted and determined to be (8.2 1) x 105s1, (pKa)donor = 6.8 0.1, and (pKa)ZnH2O = 5.7 0.1. These data were obtained at 25 C in the absence of buffer using a total concentration of all CO2 species of 25 mM, with the ionic strength maintained at 0.2 M by the addition of sodium sulfate. Comparison Between Wild Type CA IX, CA IX Mimic, and CA II In 2009 the structure of wildtype CA IX in complex with AZM was solved ( 71) This provides the opportunity to assess the feasibility of the CA II A65S N67Q mutant as a pseudo CA IX mimic. S tructural superposition of the wildtype CA IX (PDB ID, 3iai) and the CA IX mimic containing AZM (PDB ID 3dc3) reveals that the location and environment of the AZM is in fact very similar (Figure 23). The Zn2+ to N1 and two Thr 199 hydrogen bonds are cons erved between the two structures and the distances are within 0.13 of one another. Additionally the bonds to His 94, and the water mediated bond to Pro 201 are within 0.25 of each other, and the hydrophobic contacts with Val 121 and Leu 198 are nearly superimposable. However, there is a slight rotation of approximately 10 in the AZM molecule between the wild type and mimic structures. The difference between the two structures can be attributed to two distinctive features. The first is the presence of glycerol (GOL) in the wild type structure. The result of the GOL binding is apparent in the shifts of Gln 92 and Gln 67 away from the AZM molecule and the orientation of the dual conformations of His 64 and Ser 65 into an outward conformation relative to the active site. The second issue is amino acid at position 131.

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30 In wild type CA IX this is a leucine, whereas in the mimic it is still CA IIs phenylalanine. The bulkier phenylalanine provides an additional hydrophobic contact to AZM that is not present in the native protein. This may account for the some of the discrepancy between mimic and wildtype inhibition values. Therefore interactions involving hydrophobic contacts with the Phe 131 in mimic are expected to have similar Kis to the wild type CA IX, tho ugh they are likely an overestimation of the affinity. compared to CA II (PDB code 2ILI) ( 77) There were no significant conformational changes between the unbound CA II and CA IX mimic, though the Ser 65 of CA IX mimic exhibited a dual conformation (Figure 22B and 2C). The conformation of Ser 65 facing His 64 had a 1 angle of 176.3 whil e the other conformation facing away from His 64 was 70.8. Of additional interest was the proton shuttle residue His 64 in the CA IX mimic showed a dual conformation similar to that previously describe for CA II ( 93) This implies that the proton shuttling differences were most likely due to residues outside of the cavity. Drug Inhibition Studies 18O exchange methods were also used to measure the inhibition of CA II and the CA IX mimic with the inhibitors AZM, BZM, CHL, EZM, and MZM (Figu re 21) and compared to available values for CA IX (Table 21) ( 75) Assessment of the isozyme specificity reveals that AZM, the benchmark CAI, exhibited a two to three fold better binding affinity for CA IX to CA II, with a Ki of 3.0 nm for wild type CA IX, 4.9 nm for the CA IX mimic, and 10.1 nm for CA II. This result further demonstrated that the CA IX mimic was more like CA IX than CA II. Of additional interest was CHL exhibited the highest measurable Ki (1.1 nm for the CA IX mimic) approximately 2fold better than the

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31 CA II Ki of 2.0 nm. The inhibitors BZM and MZM showed no significant preferential binding to the CA IX mimic compared to CA II, though BZM was the more potent inhibitor overall, and EZM was a subnanomolar inhibitor, below the sensitivity of the 18O ex change method instrumentation. This made it impossible to accurately measure the Ki of EZM in these studies. Crystallographic Study A complete structural comparison of the binding profiles of the five CAIs was performed with both CA II and the CA IX mimi c. The sulfonamide binding site has been CAs isoforms ( 4 ) All the inhibitor complex structures for both CA II and the CA IX mimic share the same general well characterized sulfonamide binding profile. A summary for each of the inhibitors interactions with CA II and CA IX are list ed in Table 22. Hydrogen bonds and hydrophobic contact distances that change 0.2 or less fall within refinement error and are considered to be the same. The N1 group coordinates to the zinc with a hydrogen bond distance of approximately 2.0 and inter acts with the nitrogens of His 94, His 96, and His 119 at 3.33.6 as well as Thr 199 between 2.83.0 One oxygen of the sulfonamide group binds to Thr 199 at a distance of between 3.03.2 The other oxygen binds His 94 at 3.5 H119 at 3.6 and Zn2+ at 3.03.1 The sulfonamide sulfur binds Zn2+ with a distance of 3.0 There is a Van der Waals interaction with H94 at a distance of 3.23.4 as well as numerous weak hydrophobic contacts with residues Trp 209, His 119, His 96, His 94, Val 121, and Leu 198 (Table 22). In AZM the two nitrogens of the thiadiazole ring form hydrogen bonds with Thr 200 at distances of 2.93.0 and 3.1 respectively. It also forms weak hydrophobic

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32 contacts with residues His 119, His 96, Val 143 and Val 121 in both C A II and CA IX mimic (Figure 24A). The CA II AZM structure contains an additional hydrogen bond with Gln 92 with changes the conformation of the residue and lead to more hydrophobic interactions with Val 121, Phe 131, and weakly with Leu 198. For the CA I X mimic AZM complex there was an additional hydrogen bond with Thr 199 that created several weak hydrophobic contacts with Trp 209, Gln 92, and Phe 131. In both structures with BZM the O2 of the sulfonamide hydrogen bonds with the nitrogen of Gln 92 at a distance of 3.33.4 N3 hydrogen bonds with Thr 200 at 2.93.1 and more hydrophobic contacts are made with Leu 198 and Phe 131 as well as several weak hydrophobic interactions (Figure 24B). The CA II BZM structure has two additional hydrogen bonds, one to Thr 200 at 3.2 and the other to Gln 92 at 3.6 In the CA IX mimic complexed with BZM, there is a 2.9 hydrogen bond of the N2 to Thr 200. There are also weak hydrophobic contacts seen with residues His 94, Thr 200, Phe 131 and Val 135. EZM has nearly identical interactions with both CA II and CA IX mimic, which consist of the N2 hydrogen bonding to Thr 200 at 3.23.4 and of hydrophobic contacts including Thr 200, Leu 198, Trp 209, His 96, His 119, Thr 199, Pro 201, Pro 202, and Val 143 (Figure 2 4D). There are only a few weak Van der Waals interactions different between the two. MZM forms an additional hydrogen bond between O1 and His 119 at a distance of 3.43.5 and makes hydrophobic contacts with Thr 200, Pro 201, and Phe 131 in both CA II an d CA IX mimic. Most of the difference between CA II and CA IX mimic bound to MZM are in weak hydrophobic interactions. (Figure 24E). In the case of the CHL complex, both structures show O2 hydrogen bonding to His 119 at 3.6 N2 binding Thr 199 at 3.53 .6 and Thr 200 at 3.43.5 and N1

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33 hydrogen bonding to Thr 200 at a distance of 3.03.1 There are also several Van der Waals contacts with residues Leu 198, Trp 209, His 96, His 119, Thr 199, and Phe 131. The structure of CA II with CHL show several new weak hydrophobic interactions (Figure 24C). However, in the crystal structure of CA IX mimic in complex with CHL, the chlorine hydrogen bonds with Gln 92, this pulls residue Gln 67 into the active site and fixes Ser 65 into one conformation oriented t owards the His 64 with a X1 angle of 111.4 (Figure 24C and 25A and 5D). When the drugs are superimposed on one another, the similarities and differences of binding become apparent (Figure 25). The sulfonamide groups are in nearly superimposable orient ations. For the most part the ring and hydrophobic tail are locked into a planar geometry with two exceptions. In EZM the ring is rotated out slightly, probably due to the additional conjugated benzene, and the ethoxy group lies nearly perpendicular to the regular plane. In BZM the thiadiazole ring is rotated significantly out of plane and the phenylsulfonyl is rotated nearly 90 as compared to the hydrophobic tails of the AZM, CHL, and MZM and aligning with the ethoxy of EZM. PC3 Bcl 2 Western Blot and Cytotoxicity Assay A western blot was performed on untreated PC 3 Bcl2 cells to verify the expression of CA IX in this cancer cell line (Figure 26A). To determine the effects of CAIs on cell proliferation and viability, PC 3 Bcl2 prostate cancer cell lines were treated for 96 hours (at concentrations ranging from 0 nM to 0.1 mM), control or mock (DMSO only). Cellular proliferation is depicted at 0.1 mM for all agents (Figure 26B). AZM, BZM, and EZM even at higher doses had no cytotoxic effects. However, MZM at 0.01 mM and 0.1 mM demonstrated significant inhibition of proliferation and viability compared to control (29% and 32% inhibition respectively, p < 0.01). For the inhibitor

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34 CHL, significant reduction in cellular proliferation and viability was ev ident only at 0.1 mM (57% inhibition, p < 0.01). Discussion The kinetic characterization of the CA IX mimic, demonstrates that the two amino acid mutations in CA II towards CA IX do imitate CA IX in terms of chemistry at the active site zinc ion. The values of kcat/Km for the CA IX mimic are similar to those reported for the CA domain of CA IX in Wingo et. al. and in Hilvo et. al. (10, 75) However these values are lower than those reported for the CA domain with the N terminal proteoglycan domain in Hilvo et. al. (10) The kcat/Km of bot h the CA IX mimic and CA IX exhibit a two fold decrease as compared to CA II which is probably due to a change in hydrophobic properties in the active site caused by the change from the hydrophobic alanine at position 65 to the polar serine residue. In ter ms of chemistry at His 64, however, the kinetic properties of the CA IX mimic are still much like those of CA II. This is likely because there is no significant change in the residues that coordinate the waters in the active site cavity. This means there i s possibly another, nonactive site residue that causes the difference in proton transfer between CA II and CA IX. Identification of these residues and the F131L mutation might improve the mimic for future studies. To assess the feasibility of the CA IX mi mic as an analog of wildtype CA IX for drug studies, the Ki values have been compared to CA II with the classic sulfonamides. The CA IX mimic values correspond preferentially to wildtype CA IX, lending credence to use this as a model for CA IX isozyme specificity. However the CA IX mimic is not a replica of CA IX as demonstrated by the slight disparity in Ki values between CA IX

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35 mimic and wild type. The differences fall within statistical error indicating that CA IX mimic is a sufficient model for isozyme specificity. The structures of inhibitor bound CA IX mimic reveal valuable information regarding the isozyme specificity of classical sulfonamides. In the case of AZM, the Ki value is lowered by a factor of two in the CA IX mimic, making it a better inhi bitor of the mutant than CA II. This could be due to the conformational change of Gln 92 in the structure of CA IX mimic as compared to CA II; however the in vitro studies show AZM has no cytotoxic effect on the PC 3 Bcl2 cells. BZM and MZM show few diff erences kinetically or structurally, making them less likely to be preferentially effective against the tumor associated CA IX. This being stated, MZM did show cytotoxic effects against PC 3 Bcl2 cells. EZM cannot be adequately assessed kinetically due to its sub nanomolar Ki and there are few clues structurally as to its potential isozyme specificity. The results of this study suggest that CHL has the most potential for therapeutics. Comparing the structures of CA II versus CA IX mimic, the chlorine in C HL hydrogen binds to Gln 92 in the mimic and not in CA II. This may be the reason for the conformational change of Gln 67 and the locking of Ser 65 into one position oriented towards His 64. Kinetically, the Ki value is the lowest in CA IX mimic, excluding EZM. The increased inhibition is possibly due to Gln 67, which is a mutated active site residue, being pulled into a locked down conformation by Gln 92 then hydrogen bonding to the other mutant residue Ser 65. This binding mode is different from the sulfonamides in CA II that do not induce conformational changes. Also, CHL is also the only structure with a bulky chloride present and halides such as chlorine are known to affect the

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36 hydrostatics of the surrounding environment, which may improve the inhibitio n and selectivity of CHL. This evidence is supported by the cytotoxicity assay that shows CHL not only impedes propagation but also induces cell death in PC 3 Bcl2 cell. With some adjustment the CA IX mimic may prove to be an effective surrogate of CA IX for use in highthroughput kinetic and structural screenings. The fact that the crystallographic analysis and kinetic studies of CA IX mimic correlated to the cytotoxicity assay only serves to reinforce its potential. Of the five drugs studied, AZM, MZM, and CHL were identified as potential anti cancer drugs, alone or in conjunction with traditional chemotherapy. Further in vitro and in vivo studies are needed to confirm this assertion. To our knowledge this is the first time a protein mimic has been engineered from a related isozyme for the purpose of drug design.

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37 Figure 21. Carbonic anhydrase inhibitors. Acetazolamide AZM, N (5 (aminosulfonyl) 1,3,4thiadiazol 2 yl) acetamide, Benzolamide BZM 1,3,4Thiadiazole2 sulfonamide, 5((phenylsulfonyl)ami no), Chlorzolamide CHL, 5 [o chlorophenyl] 1,3,4thiadiazole2 sulfonamide, Ethoxzolamide EZM, 6 ethoxybenzothiazole2 sulfonamide, Methazolamide MZM, N (3 methyl 5 sulfamoyl 3H 1,3,4thiadiazol 2 ylidene.

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38 Figure 22. Sequence and structure of unbound CA IX mimic. (A) Amino acid sequence alignment of CA II and CA IX from residue 60 to 120 (CA II numbering). Conserved residues H64, H94, H96, H119, and all active site residues, are colored blue. Active site variants, residues 65 and 67, are highlighted in yellow. Alignment performed using Clustal W ( 76) (B) Crystal structure of CA IX mimic. Backbone (blue cartoon) active site residues (yellow sticks as labeled), Zn2+ (black sphere). (C ) Close up view of CA IX mimic. Figure B was generated and rendered in PyMOL ( 97) .

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39 Figure 32. Superposition of wild type CA IX (PDB ID 3iai) and CA IX mimic (PDB ID 3dc3) in complex with Acetazolamide. AZM is represented by lines; side chains by sticks. Coloring scheme is as follows: wild type CA IX carbons, light blue; CA IX mimic carbons, orange; oxygen, red; nitrogen, blue; sulfur, gold. The large gray spheres represent zinc. Waters are small spheres colored the same as their respective carbons.

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40 Table 21. Ki Values (nM) of inhibitors for CA II, CA IX mimic and CA IX. Inhibitor CA II CA IX mimic wild type CA IX AZM 10.10.7 4.91 3.0 a BZM 7.00.9 8.81.2 9.01.0 EZM < 1 .0 < 1 .0 CHL 2.00.4 1.10.2 1.00.3 MZM 11.81.8 11.42 9.0 a adata from ( 75)

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41 Figure 24. Comparison of sulfonamides in CA II and CA IX mimic. The backbone is shown as a cartoon. The side chains and ligand are shown as sticks and colored as follows: CA II carbons, light blue; CA IX mimic carbons, orange; oxygens, red; nitrogens, dark blue; sulfur, gold; chlorine, green. Zinc is represented by a large gray sphere. (A) AZM. (B) BZM. (C) CHL. (D) EZM. (E) MZM. Figure generated in Pymol ( 97)

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42 Table 2 2. Inhibitor protein interactions () Interactions found in all structures Interactions found in individual drugs Interactions in individual s tructures N1 a HB: H96 (3.3 3.5), T199 (2.8 3.0), Zn2+ (1.9 2.3), H94 (3.33.5), H119 (3.3 3.6) O2/O4 HB: H94 (3.5), Zn2+ (3.0 3.1), H119 (3.6) O1/O3 HB: T199 (3.03.2) S1/S3 HB: Zn2+ (3.0 3.1) bHC: H94 (3.2 3.4) cwHC: W209 (3.63.9), H11 9 (4.0), H119 (3.9) [except CA II MZM], H96 (3.7 4.0) [except CA II MZM], V121 (3.7 3.9, 3.84.0), H94 (3.84.0, 3.84.0, 3.84.0), L198 (3.63.7, 3.73.8, 3.83.9, 3.9, 3.74.0, 3.74.0) AZM: N3 HB: T200 (2.93.0) N2 HB: T200 (3.1) wHC: H119 (3.8 3.9, 3.9), H96 (3.8), V143 (4.0), V121 (3.73.8, 3.8 3.9) CA II AZM d : O3 HB: Q92 (3.2) HC: Q92 (3.2), V121 (3.5), F131 (3.5, 3.4) wHC: Q92 (3.6, 4.0, 3.8), F131 (3.6,3.7), L198 (4.0) CA IX mimic AZM: O1 HB: T199 (3.6) w HC: W209 (4.0), Q92 (3.8, 4.0), F131 (3.7, 3.8, 3.8, 3.8) BZM: N3 HB: T200 (2.93.1) O2 HB: Q92 (3.3 3.4) HC: F131 (3.3), L198 (3.3) wHC: H96 (3.7 3.8), H119 (3.9), V143 (4.0), V121 (3.9), F131 (3.53.6, 3.73 .8, 3.73.9,3.83.9, 4.0) CA II BZM: N2 HB: T200 (3.2) N1 HB: Q92 (3.6) wHC: Q92 (3.9), F131(4.0), L198 (4.0) CA IX mimic BZM: N2 HB: T200 (2.9) wHC: H94 (3.9), T200 (4.0, 4.0), F131 (3.9), V135 (3.9) CHL: O2 HB: H119 (3.6) N2 HB: T199 (3.53.6), T200 (3.43.5) N1 HB: T200 (3.03.1) HC: L198 (3.43.5), W209 (3.5) wHC: H96 (3.8), H119 (3.8, 3.9), T199 (4.0), F131 (3.8, 3.8, 3.8 3.9), L198 (3.5 3.6) CA II CHL: wHC: L198 (3.6, 3.6, 3.7, 3.7, 3.9) CA IX CHL: CL1 HB: Q92 (3.5) wHC: V121 (4.0), L198 (3.8, 3.9, 3.9, 4.0) aHB = hydrogen bond (2.0 3.6) bHC = hydrophobic contact (3.0 3.6) cwHC = weak hydrophobic contacts (3.6 4.0) dCA II complexed with AZM coordinates, PDB ID 3hs4 ( 91)

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43 Table 2 2 Continued Interactions found in all structures Interactions found in individual drugs Inter actions in individual structures EZM: N2 aHB: T200 (3.23.4) bHC: L198 (3.4, 3.53.6), T200 (2.93.0, 3.43.5) cwHC: W209 (4.0), V143 (4.0), H119 (3.8, 3.9), H96 (3.8), T199 (3.9), T200 (3.7), P201 (3.7, 3.7 3.8), L198 (3. 9 4.0), P202 (3.9) CA II EZM : wHC: T200 (4.0), F131 (4.0), L198 (4.0) CA IX mimic EZM: wHC: P202 (3.8) MZM: O1 HB: H119 (3.43.6) HC: T200 (3.03.1), P201 (3.53.6) wHC: T200 (3.8), F131 (3.6, 3.7, 3.8) CA II MZM: HC: Q92 (3.5) wHC: V121 (4.0), Q92 (3.8, 4.0, 4.0) CA IX MZM : HC: W209 (3.5), V121 (3.5), Q92 (3.4, 3.4) wHC: H96 (4.0), H1 19 (3.9, 4.0), T199 (3.9), V121 (3.9, 4.0), L198 (3.9, 3.9), Q92 (3.7, 3.8, 3.9) aHB = hydrogen bond (2.0 3.6) bHC = hydrophobic contact (3.0 3.6) cwHC = weak hydrophobic contacts (3.6 4.0)

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44 Figure 25. Superposition of all five drug complexes in crystal structures. (A) Active sites of CA II complexes. (B) Active sites of CA IX mimic complexes. AZM (red), BZM (magenta), CHL (green), EZM (orange), MZM (blue), Zn2+ (black sphere). (C) and (D) Superposition of all five drugs in CA II and CA IX mimic respectively. (E) and (F) Superposition of all five drugs in CA II and CA IX mimic respectively rotated 90 from previous view. Figure generated in PyMOL ( 97 )

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45 Figure 26. Cell proliferation analysis in PC 3 Bcl2 prostate cancer cell lines. (A) Western Blot Analysis verifying the expression of CA IX by the PC 3 Bcl2 cells. (B) Surviving fraction of PC3 Bcl2 cells treated ov er time with one of the following inhibitors; AZM (red), BZM (magenta), CHL (green), EZM (orange), MZM (blue). Percent surviving fraction normalized to mock (DMSO only).

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46 CHAPTER 3 CHARACTERIZATION OF CARBONIC ANHYDRASE I SOZYME SPECIFIC INHIBITION BY 2 ET HYLESTRA COMPOUNDS Background In the early 1990s it was discovered that the naturally occurring estrogen metabolite, 2methoxyestradiol, inhibits cell proliferation and angiogenesis independently from the estrogen receptor ( 98 ) It is believed that the mechanism of action for this compound is tubulin impairing the microtubule remodeling that occurs during cell division inducing metaphase arrest and apoptosis ( 98104) Clinical trials of 2 methoxyestra diol as a cancer treatment were halted due to an extremely low plasma concentration in patients despite high administered doses indicating a lack of bioavailability and high susceptibility to clearance by liver enzymes ( 105) Given the drugs effectiveness in vitro derivatives have been developed to help circumvent these issues. Independently it was discovered that 2substituted estrone sulfamates, a class of steroid sulfatase inhibitors were antiproliferative towards cancer cells ( 106, 107) These sulfamates, in addition to being highly bioavailable with oral administration, were capable of circumventing liver metabolism by reversibly attaching to CA II in red blood cells ( 108, 109) Numerous studies have been employed to identify the structure activity relationships associated with various derivatives of the 2substituted estrone sulfamates. Among the most promising was 2Ethylestradiol 3,17O O bis sulfamate (compound 14 in this study), 2Ethylestrone 3O sulfamate (compound 15), and 2Ethylestradiol 3O sulfamate (compound 16 ) ( 110112) 14 was shown to be highly effective anti proliferative agent in vitro with a mean 50% growth inhibition concentration (GI50

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47 and in vivo in both breast and prostate cancer xenografted mice ( 110, 112) 15 also proved to be an effective anti proliferative w ith a mean GI50 panel. 16 was shown to have antiangiogenic properties, was characterized in vitro with a mean GI50 growth inhibition in vivo with estrogen recept or negative breast cancer xenografted mice ( 111) 14 was investigated in relation to CA II, both to characterize its inhibition of the enzyme and to develop a more effective understanding of the structureactivity r elationship, the results of which will be discussed more fully later in this chapter ( 113) This study proposes to assess the effectiveness of these compounds in the context of CA II as a mechanism of liver metabolism bypass and CA IX as a means of targeting the anti proliferative action of the compounds to cancer cells. Through X ray crystallographic structure solution and 18O exchange kinetics 14, 15, and 16 were characterized in this context. This study utilizes the CA IX mimic discussed in Chapter 2 as a CA IX analog. Experimental Methods Compound Synthesis All compounds were synthesized by iThemba Pharmaceuticals (Gauteng, South Africa). Chemical formulas and full names are included in Figure 31. A full accounting of the synthetic steps is provided in Appendix B Protein Expression and Purification CA II and CA IX mimic were prepared as described in Chapter 2. Oxygen 18 Isotope Exchange Inhibition Studies Inhibition constants (Ki) of all compounds were determined were grown by measurement of the inhibition of the 18O exchange activit y as described in Chapter 2.

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48 Crystallization and Data Collection Crystals of CA II and CA IX mimic complexed with the compounds were obtained by co crystallization using the hanging drop vapor diffusion method ( 85) Crystallization 1 100 mM Tris Cl, and 7.5 mM of compound were suspended over 1 ml of 1.4 M sodium citrate and 100 mM Tris Cl. The crystallization pH for com pounds 15 and 16 was pH 8 and for 14 was pH 9. Crystals were soaked for 10 seconds in a cryoprotectant solution of 0.9 M sodium citrate, 100 mM Tris Cl, pH 8, 20% glycerol. Initial structure solutions of CA II with 15 and 16 did not give sufficient densit y for compound modeling. Additional crystals were soaked in a solution of 1 M sodium citrate, 50 mM Tris Cl, pH 8, 20% glycerol and 10 mM of the compound. The crystals were frozen to 100 K in a gas nitrogen cryostream. Data was collected as described in Chapter 2, using an 80 mm crystal to detector distance and 300 second exposure times. The data was merged using HKL 2000 ( 86) Data statistics are listed in (Appendix Table B 1). Structure Determination All the crystal structures were determined by molecular replacement methods using the previously solved wildtype CA II structu re with solvents removed (PDB code 2ILI) ( 77) The initial refinement was performed using PHENIX with 5 macrocy cles including rigid body, individual coordinate and atomic displacement parameter (ADP) refinement ( 114) FoFc maps provided phaseunbiased electron density for the respective bound inhibitors (Appendix Figure B 2). The PRODRG server was used to generate restraints for modeling the inhibitor structures during the subsequent refinement process ( 88) Refinement continued with alternating rounds in PHENIX and manual model building in COOT ( 79, 114) Ordered solvent was added, as well TLS

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49 refinement and riding hydrogens. Refinement of the structures continued until the Rcryst and Rfree converged. The compoundenzyme interactions were determined using LIGPLOT and are listed in Table 31 ( 115) The coordinates and structure factors for the five structures have been submitted to the Protein Data Bank. PDB IDs, data refinement, and final model s tatistics are given in (Appendix Table B 1). Results Compound Inhibition Kinetics 18O isotope exchange inhibition assays of CA II and CA IX mimic were conducted on compounds 14, 15, and 16 (Table 3 2) It is of note that esterase inhibition assays of compound 14 was performed with CA II previously with an inhibition constant of 230 60 nM ( 113) However this test measures the inhibition of the secondary promiscuous esterase activity of CA rather than its primary carbon dioxide hydration and has historically yielded different values. The test was repeated using 18O exchange to provide comparable numbers for analysis. Compound 14 produced very similar inhibition constants between CA II, 875 65 nM, and CA IX mimic, 680 40 nM. Compound 15 had Kis of 440 50 nM for CA II and 585 30 for CA IX mimic, revealing a small 34% decrease in affinity. In contrast compound 16 showed the most distinctive isozyme specificity with a Ki of 180 10 nM for CA II and 2090 215 nM for the CA IX mimic, a nearly 12fold difference. Compound 14 Structural Com parisons The structure of 14 in complex with CA II has been solved previously (PDB ID 2x7t) ( 113) Of note, the previously solved structure failed to adhere to traditional CA II naming convention that skips residue 126. For the purposes of comparison the numbering has been adjusted to conf orm to this standard (n+1 for all residues 126 and

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50 above). As observed previously the 17O sulfamate (D ring) preferentially binds the Zn2+ over the 3O sulfamate. This may relate to an increase in conformational flexibility of the cyclopentane over the pl anar benzene ring attached to 3O sulfamate or as suggested in Cozier et. al. the 2ethyl substitution may disfavor the 3O sulfamate coordinating to the Zn2+ ( 113 ) Between CA II and CA IX mimic the orientation of the 14 in the active site is nearly superimposable (Table 31, Figure 32A and 2B). Both are coordinated to the Zn2+ and have two hydrogen bonds to Thr 199, with values within 0.2 of one another (Table 31, Figure 32C). They share hydrophobic contacts with Pro 202, Leu 198, Val 135, and Phe 131. There is a slight difference in the distance from His 94 shifting from a 3.3 hydrogen bond in CA II to a hydrophobic contact in CA IX mimic. There is also an alternate conformation of His 64 in CA IX mimic with the inward conformation forming a hydrophobic contact. Compound 15 Struct ural Comparisons There is a significant difference between the Zn2+bound 15 between CA II and CA IX mimic (Figure 3 3A and 3B). The structure in CA IX mimic showed a clearly defined, zinc bound ligand that refined to an occupancy of 85% (Appendix Figure B 2C). Additionally another low occupancy (58%) molecule of 15 was identified to the exterior of the active site in hydrophobic contact with the D ring of the zinc bound ligand ( Appendix Figure B 3A). This secondary binding site most likely has no biologica l significance. The density of 15 in CA II was less ordered. Experiments were repeated with compound in the cryoprotectant improving the density enough for modeling at an occupancy of 60% with alternate water positions coordinating with the ordered solvent seen in unbound CA II. Though the density is contoured at a low sigma, most of the

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51 Figure B 2 B) ( 116) There was also a GOL molecule located in the active site. In the two structures the orientation of the sulfamate is similar, leading to similar distances in the Zn2+ coordination and the hydrogen bonds to Thr 199, His 96, and His 119 (Figure 4C, Table 1). The main difference between binding in CA II and CA IX mimic is in the torsion angle orienting the sulfamate moiety to the A ring. The nitrogensulfur oxygenC3 torsion angle the CA II is 157, whereas the CA IX mimic is 62. In the CA II 15 structure the inhibitor orients towards the hydrophobic region of the active site, forming van der Waals contacts with Val 121, Phe 131, Leu 141, Leu 198, Pro 202, and the GOL. His 64 is also seen in the dual conf ormation with the inward coordinating to the GOL. In the CA IX mimic structure 15 orients towards the hydrophilic side, though the bulk of the interactions are hydrophobic, including Asn 62, His 64, His 94, Phe 131, Leu 198, and Thr 200. The orientation change is most likely caused by a water mediated hydrogen bond between the carbonyl O17 and Gln 67. In CA II residue 67 is an Asn, which is too short to coordinate to this O17. This bond is fairly strong in the mimic with a distance of 2.9 to the water and 2.7 from the water to the Gln 67. The interactions of the second 15 molecule in CA IX mimic are discussed in Appendix B Compound 16 Structural Comparisons Like 15, 16 has a rather distinctive difference between the CA II and CA IX mimic structures (Figure 34A and 4B). Once again the orientation of the sulfamate is similar between the two structures, with the Zn2+ coordination, the hydrogen bond with His 96 and the two hydrogen bonds to Thr 199 (Table 31, Figure 34C). Similar to 14 there is a slight s hift in the distance from His 94 though a hydrogen bond in CA IX mimic and the hydrophobic contact in CA II. The difference in sulfamate/A ring torsion angle resembles 15 in the rotation. The nitrogensulfur oxygen C2 torsion angle is 178 in CA II and 66

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52 in CA IX mimic. CA II binds in the hydrophobic region engaged in van der Waals interactions with Val 121, Phe 131, Leu 141, Leu 198, Pro 202 and a GOL. Within the CA IX mimic 16 binds in the hydrophilic site, engaging in hydrophobic contacts with Asn 62, Val 121, Phe 131, Leu 198, and Thr 200. It also contains a water mediated hydrogen bond between O17 hydroxyl and Gln 67. These hydrogen bond distances are weaker than those seen in 15 with an O17 to water distance of 3.0 and a water to Gln 67 distance of 3.3 Discussion With the kinetic data correlated to the structures, a pattern emerges. The identical orientation of 14 in both the CA II and CA IX mimic relates to the free 3O sulfamate that lies out of the active site into the bulk solvent forming no meaningful interactions (Figure 3 2C). The ligand is oriented away from residues 65 and 67 and too long to confer any meaningful isozyme specificity. This is reflected by the negligible difference between 14s Ki in the CA II and CA IX mimic. However the differences seen between CA II and CA IX mimic structures with compounds 15 and 16 reveal a promising possibility for isozyme specificity design. The orientations of both compounds seem to relate to a water mediated hydrogen bond between O17 and the Gln 67 in CA IX mimic (Figure 3 3C and 34C). By reorienting the compound from the hydrophobic region of the active site into the hydrophilic region this reduces the number of contacts along the estrone backbone, allowing it to make van der Waals interactions w ith polar residues. In 15 the shift seen in CA IX mimic is counterbalanced by an additional hydrophobic contact and the tight water mediated hydrogen bond. This results in a relatively insignificant affinity preference for CA II over CA IX mimic. In 16, however, the hydrophobic to hydrophilic transition in CA IX mimic

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53 induces the loss of one van der Waals interaction and of the five remaining, two are with polar residues (Thr 200 and Asn 62). This drastic shift in environment also fails to be compensated by the much longer water mediated hydrogen bond. These observations taken together explain the 12fold reduction of affinity for CA IX mimic as compared to CA II. This discernable difference in affinity reinforces the viability of isozymespecific CAIs. That a single water mediated hydrogen bond can confer a pronounced change in Ki indicates that water should be a consideration in structurebased drug design. Perhaps a molecule of similar length with more hydrophilic properties along the backbone could be the key to achieving a highly desired, CA IX specific inhibitor needed to combat solid tumor growth in numerous cancers.

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54 Figure 31. 2Ethylestra compound formulas and names. Red letters indicate ring designations.

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55 Table 31. Interaction Table Compound C A II CA IX mimic 14 (WZB) a,b HB: T199 (2.6 + 3.0), H94 (3.3) Zn2+ (2.1) cHC: P202, L198, V135, F131 HB: T199 (3.0 + 2.8) Zn 2+ (1.9) H C: H94, P202, L198, V135, F131, H64 15 (VZ4) HB: T199 (2.9), H96 (3.3), H119 (3.3), Zn2+ (2 .0) HC: P202, L198, F131, L141, V121, dGOL Molecule 1 HB: T199 (2.74), H96 (3.3), H119 (3.3), Q67 e(wm) Zn2+ (2.0) HC: T200, L198, H64, N62, F131, H94, VZ4 molecule 2 Molecule 2 HB: Q92 (3.3), G132 (wm), Q136 (w m) HC: P202, L204, I91, F131, VZ4 molecule 1 16 (VZ5) HB: H96 (3.3), T199 (2.8 + 3.0), Zn2+ (2.0) HC: L198, H94, V121, L141, F131, P202, GOL HB: T199 (3.0 + 2.8), H96 (3.3), H94 (3.3), Q67 (wm), Zn2+ (2.0) HC: T200 L198, F131, V121, N62 aNumbering changed for consistency to CA II convention. For residues greater than 126 subtract 1 for the residue number in the PDB bHB hydrogen bond cHC hydrophobic contact dGOL glycerol present in active site e( wm) water m ediated hydrogen bond

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56 Table 32. Compound Inhibition Kinetics Ki (nM) Compound CA II CA IX mimic 14 875 65 680 40 15 440 50 585 30 16 180 10 2090 215

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57 Figure 33. Compound 14. (A) Orientation of 14 in the active site. CA protein shown as a gray surface rendering. 14 shown as sticks and is colored as follows: carbons from the CA II 14 complex, light blue; carbons from the CA IX mimic 14 complex, orange; oxygens, red; nitrogens, dark blue; sulfur, gold. (B) Close up view of the act ive site. (C) Stereo image of active site superposition with interacting residues shown as sticks. Residues are colored as described in (A). Zn2+ is represented by a large gray sphere. Small spheres colored the same as their respective carbons represent waters. Figure generated in Pymol ( 97)

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58 Figure 34. Compound 15. (A) Orientation of 15 in the active site. CA protein shown as a gray surfac e rendering. 15 shown as sticks and is colored as follows: carbons from the CA II 15 complex, light blue; carbons from the CA IX mimic 15 complex, orange; oxygens, red; nitrogens, dark blue; sulfur, gold. (B) Close up view of the active site. (C) Stereo im age of active site superposition with interacting residues shown as sticks. Residues are colored as described in (A). Zn2+ is represented by a large gray sphere. Small spheres colored the same as their respective carbons represent waters. B conformation waters have been removed for clarity. Figure generated in Pymol ( 97)

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59 Figure 35. Compound 16. (A) Orientation of 16 in the active site. CA protein shown as a gray surface rendering. 16 shown as sticks and is colored as follows: carbons from the CA II 16 complex, light blue; carbons from the CA IX mimic 16 complex, orange; oxygens, red; nitrogens, dark blue; sulfur, gold. (B) Close up view of the active site. (C) Stereo image of an active site superposition with interacting residues shown as sticks. Residues are colored as described in (A). Zn2+ is represented by a large gray sphere. Small spheres colored the same as their respective carbons r epresent waters. Figure generated in Pymol ( 97)

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60 CHAPTER 4 CARBONIC ANHYDRASE I X INHIBITORS APPLICATIONS IN VIVO Background When the results from Chapter 2 were analyzed, it seemed clear that Chlorzolamide (CHL) might have potential as a cancer therapy. The natural extension of this conclusion was to take a step towards the clinical aspect and test CHL in vivo A mouse model of prostate cancer had already been developed by implanting athymic nude mice with the CA IX overexpressing prostate carcinoma cell line PC 3 ( 117) In a mouse model the effectiveness of a treatment can be assessed using global measurements. These indicators include differences in tumor volume, body weight, and overall survival. In addition to those mentioned above, a mechanism for obtaining useful measurements of absorbed dose must be devised. One simple method is through the monitoring of urine pH. CA IV, located in the proximal tubule of the kidney, is strongly affected by CA inhibitors in the blood stream, which disrupts the resorption of bicarbonate leading to an increase in urine pH ( 16, 118120) By monitoring the urine pH, an estimation of bioavailability can be obtained. The effects of the drug can also be assessed on the cellular level using s ections of resected tumor tissue. H ematoxylin and eosin (H&E) staining assess es overall morphology and pathology ( 121) T erminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is used to assess apoptosis and proliferating cell nuclear antigen (PCNA) is a measure of cell proliferation ( 122, 123) All of these processes play a role in tumor progr ession and dysregulation would slow or eliminate tumor growth and can be appraised using immunohistochemistry (IHC). Since CA IX is the drug target, expression of this protein will also be monitored using IHC.

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61 Experimental Methods Mouse Xenograft Tumors M ale athymic nu/nu mice (Harlan, Indianapolis, IN) are maintained under specific pathogenfree conditions in facilities approved by the AAALAC and in accordance with current standards of the U.S. Department of Agriculture, U.S. Department of Health and Huma n Services, and the National Institutes of Health. As described previously, mice (N=19) were injected subcutaneously in the both flanks with 3x106 PC3 cells suspended in 0.1 ml buffered Matrigel (1 mg ml1) by Dr. Wengang Cao ( 15) After the tumors reached 57 mm diameter (day 13 post injection), half the mice (N=10) received a daily oral gavage of drug (CHL, 5 mg kg1 day1) or vehicle (5% DMSO) followed by an additional gavage of sterile saline (performed by Stacy Porvasnick). CHL was prepared as a stock solution of 72.5 mM (6.25 mg ml1) in 50% DMSO and diluted tenfold in sterile saline immediately prior to administration. Urine pH was measured using urinalysis strips (Multistix 9 SG, Bayer). Tumor dimensions, body weight and urine pH were recorded twice weekly for approximately 6 weeks after cell injection. Tumor volumes were calculated according to the following formula: length (L) x width (W)2 x 0.523. Tissue and Blood Samples On days 28, 36, 42, 49, and 55 post injection a representative sample of mice from each of these treatment groups were euthanized, and their tumors resected. Tumors were fixed for 24 hours in formalin and then paraffinembedded. Tumors collected on day 28 were selected for further IHC. This was the tumor harvest closest to day 23, which was the time point at which the tumor growth difference was the most significant (p= 0.12). Slides were cut from one control and one CHLtreated tumor and

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62 one slide each stained with H&E by the University of Florida Molecular Pathology Core (Gainesville, FL) Immunohistochemistry IHC staining for PCNA (Dako; Carpinteria CA; 1:500) and CA IX(M75 mouse monoclonal antibody),was performed on paraffinembedded samples collected 28 days post cell injection. The ti ssue sections were deparaffinized in xylene, rehydrated in increasing concentrations of ethanol, and peroxidases were blocked in 3% hydrogen peroxide in methanol as described above. Antigen retrieval was performed by incubating slides in 10 mM citric acid buffer, pH 6.0 for 15 min at 95C. For immunostaining with PCNA, tissue sections were blocked for 5 hours in 100 mM citric acid buffer, pH 6.0 containing 1% BSA, 1% goat serum and 1.5% horse serum. Then the sections were incubated at room temperature over night with primary antibodies diluted in the blocking buffer. Following primary antibody incubation slides were incubated at room temperature for 3 hours in the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame CA, 1:400). This w as followed by avidinbiotin complex (Vectastain Elite ABC Kit; Vector) and immersion in DAB substrate (Vector). Slides were counterstained with Gills hematoxylin, dehydrated in increasing concentrations of ethanol followed by xylene, and coverslips were mounted with Permount followed by sealing with nail polish. For CA IX staining with the M75 mouse monoclonal antibody, the M.O.M.TM Kit (Vector Labs) was used to reduce background staining ( 124) Briefly, sections were blocked with M.O.M. blocking reagent for an hour, then washed with PBS and incubated in M.O.M. Protein Concentrate for 5 minutes. Sections were incubated at room

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63 temperature for 30 minutes in primary antibody (1:5000 in Protein Concentrate). Following a wash with 1x PBS, the slides were incubated for 10 minutes in M.O.M. Biotinylated AntiMouse IgG Reagent, followed by avidinbiotin complexation (Vectastain Elite ABC Kit; Vector Labs) and immersion DAB substrate (Vector Labs). As described above the sl ides were counterstained with Gills hematoxylin and mounted. Stained slides were photographed using a Zeiss Axioplan 2 imaging microscope and Openlab 5.0.3 Beta Improvision software. Ten random frames at 200x magnification were selected for quantitation and counted manually. PCNA slides were counted for positive (brown stained nuclei) versus total number of cells. Statistical significance was calculated using a twosample unequal variance (heteroscedastic) Students T test in Microsoft Excel. CA IX was as sessed visually for the presence of positive staining in the frame. No further quantitation was performed. TUNEL Assay Paraffin embedded samples were assayed for apoptosis using the TACS XL In Situ apoptosis Detection Kit (R&D Systems, Minneapolis MN), f ollowing the manufacturers protocol. Briefly, the slides were deparaffinized followed by rehydration. The sections were then treated with Cytonin solution for 30 min, and peroxidase was blocked as described above for 5 min. The samples were incubated for 1 hour, 15 minutes at 37C with terminal deoxynuclotidyl trasferase, for 1 hour at 37C with anti BrdU, and then with peroxidaseconjugated streptavidin. The slides were soaked for 10 minutes in diaminobenzidine followed by counterstaining with methyl green, then mounted and photographed as described above. For quanatitation, 10 frames at 200x magnification were selected and the number of TUNELpositive cells per frame was counted. Statistical analysis was performed as described above.

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64 Mouse Pharmacokinetic Studies Acetazolamide (AZM, Sigma Aldrich) and chlorzolamide (CHL, a gift from Dr. Thomas Maren to Dr. David Silverman) were prepared as stock solutions in 50% DMSO at a concentration of 6.25 mg/ml for the 5 mg/kg body weight dose and at 12.5 mg/ml for 10 mg/kg body weight dose. The stock solutions were diluted tenfold in sterile saline immediately prior to administration. Control mice received a vehicle control of 5% DMSO in sterile saline. All mice received a drug or control gavage followed by a second saline gavage as described in the Xenograft Mouse Study. Tests were performed on Black 6 mice (a gift from Dr. Barry Byrne). All mice received a 1 ml subcutaneous injection of Lactated Ringers solution (B. Braun Medical Inc.) the evening prior to drug a dministration to prevent dehydration. At 0 hours the inhibitors were administered to the mice orally by gavage, in the case of AZM and CHL. The pH of the urine was measured at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, and 48 hours using urinalysis strips (Multis tix 9 SG, Bayer). Data are expressed as the change in urine pH compared to control and shown as SEM; N=8 mice per group. Results Xenograft Mouse Tumor Study The in vitro studies discussed in Chapter 2 indicated that CHL was more isozyme specific than AZM This experiment was translated into an in vivo mouse model for clinical assessment ( 72 ) Nineteen nude mice were implanted with PC 3 prostate cancer cells. Treatments with CHL (5 mg kg1 body weight daily) or vehi cle began 13 days post implantation. Initially the tumor growth was equal between the control and CHLtreated tumors (Figure 41). Following day 34 the tumor volumes of the CHLtreated mice trended towards larger volumes though they lack statistical signif icance. The overall

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65 survival rate shows no treatment difference (Data not shown). In fact, most of the mice survived until the end of the study without reaching IACUC termination criteria (tumor > 1.5 cm diameter or ulcerated). Like the survival curve body weight was measured and showed little variation over the course of the experiment (data no shown). Urine pH was also documented during the course of the study, however the values varied erratically depending on the time of measurement in relation to the drug administration. Tumor Immunohistochemistry Hematoxylin and E osin Tumors collected 2 weeks post treatment (day 27) were selected for extended testing via IHC. Twp slides were initially stained with H&E to assess morphology (Figure 4 2). Both control and CHL treated groups showed regions of low density cells as well as densely packed areas. Both tumors had eosinophilic clumps corresponding to vascular spaces. CA IX Tumors were assessed by IHC for CA IX expression (Figure 43). In both control and CHLtre ated tumors the staining was patchy with heavier staining around the periphery of the tumor. Regions of positive staining were present in all 10 fields taken at 200x magnification in both the control and CHLtreated tumor (Figure 43A). This assessment has not been quantified. PCNA Ten fields from each tumor (200x magnification), one control and one CHLtreated, were assessed for the presence of PCNA (Figure 43A). Like CA IX, PCNA staining was patchy across the entirety of the tumor, with heavier staining on the periphery. This is consistent with tumor growth given the crowded nature of the interior with fewer

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66 nutrients and less room to proliferate. Quantification of the control tumor showed that 46 3% (SEM) of the cells stained positive as compared to 51 6% of the CHLtreated tumor (Figure 43B). TUNEL Apoptosis within the tumor was appraised via TUNEL staining (Figure 43A). There were few apoptotic cells present in either control or CHLtreated cells, likely due to the early stage of tumor development. In the control cells there was a marginally larger number of apoptotic cells (5 5 cells per field SE or SD?) located mostly near the border of the tumor (Figure 43C). CHL treated tumors exhibited fewer TUNEL positive cells (2 1 cells per field) w hich were dispersed randomly throughout the tumor. Mouse Pharmacokinetic Studies As a result of the erratic urine pH measurements observed in the xenografted mice, a pharmacokinetic analysis of CHL in mice was warranted. To evaluate the effect of sulfonami de administration in vivo mice were given a single dose (5 or 10 mg kg1) of AZM, a classic sulfonamide inhibitor, or CHL with urine pH measured at multiple time points over 48 hours (Figure 44). The urine pH of AZM treated mice increased 2.2 units within 1 to 2 hours. The urine pH of the 5 mg kg1 mice returned to normal by 6 hours whereas the 10 mg/kg dose was still 0.8 units above the control mouse average at 8 hours Therefore the higher dose of AZM did not result in a higher maximum pH change, but did appear to have a slightly longer duration of activity beyond 8 hours CHL produced only a very small increase in urine pH within the first 30 min to 1 hour, with all effect dissipating by 4 hours. There was no discernable difference between the 5 mg kg1 and 10 mg kg1 doses of CHL.

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67 Discussion The purpose of this study was to assess the application of the isozyme specific CHL in vivo The treatment of PC 3 xenograft mice with CHL seemed to have no significant consequence on the tumor growth over the cour se of the experiment. CHL treatment produced no decrease in tumor size. By day 37 post injection the average tumor size was larger than control mice (Figure 41A). There was also no effect on the longevity or body weight of the mice. Further immunohistochemistry of the day 28 tumors support these conclusions. H&E staining of the tumors revealed that CHLtreated mice exhibited negligible differences from the control with patchy cell density and some distinctive vasculature in each. CA IX qualitatively shows very little difference between the two, though the drug acts as an inhibitor of CA IX enzyme activity and would probably have little effect on its expression at the protein level. There is no significant change in proliferation or apoptosis in CHLtreated tumors as determined by PCNA and TUNEL staining, respectively. Neither result indicates a positive response to CHL treatment. However it is important to note that these IHC results are derived from a single tumor (i.e. N=1), with the error bars indicating tumor heterogeneity. It is difficult to appropriately interpret meaningful results from such a small number of observations. Given the lack of treatment effect over the course of the xenograft study, a question arose as to the bioavailability of CHL. CHL is know to have lipophilic properties and quickly fell out of solution upon dilution to the final administered concentration, which prompted the additional saline gavage. The pharmacokinetic study was employed to address this concern. The issue proved to be relevant, with the CHLtreated mice showing little urine pH variability even in the initial stages as compared to the AZM -

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68 treated ones. This could be due to an isozymespecificity issue, indicating that CHL is a less effective inhibitor of the renal CA I V, but it is more likely an adsorption problem. This is supported by the lack of dosedependence, with the 10 mg kg1 mice showing a nearly identical urine pH profile. When considered together, these data collectively indicate that CHL treatment does not translate in vivo Both global indicators (tumor growth and overall survival) and cellular ones (H&E and IHC) agree. There is no discernable effect of CHL on PC 3 derived xenograft tumors. This seems due largely to the lack of bioavailability. While CHLs potency and moderate isozyme specificity is appealing, it does not appear to have potential as an oral cancer therapy. CHL may still be effective if an alternative administration such as liposomes or nanoparticles could be employed, however it would requir e extensive development.

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69 Figure 41. Xenograft tumor volume measurements. Mice were injected subcutaneously with 3x106 PC3 tumor cells on day 0. CHL administrat ion by gavage was begun on Day 13. Control mice values (N=9) are colored blue. CHL treated mice values (N=10) are colored pink. Error bars represent the standard error of the mean (SEM). No values are statistically significant. Tumor volumes were calculated according to the following formula: length (L) x width (W)2 x 0.523.

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70 Figure 42. Tum or cell morphology. Mice were injected subcutaneously with 3x106 PC3 tumor cells on day 0. CHL administrat ion by gavage was begun on Day 13. Tumors were resected on Days 28. A slide from both a control (A C) and a CHL treated (D F) tumor was stained in hematoxylin and eosin. Views are shown at 200x (A and D), 400x (B and E) and 630x (C and F) magnification arrows indicated microvessels.

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71 Figure 43. Immunohistochemistry of day 28 tumors. (A) A slide from both a control and a CHL treated tumor was stained for CA IX, PCNA, or TUNEL. Views are d as a percent of total number of cells. (C) Quantitation of TUNEL positive nuclei expressed as total positive cells per frame. Error bars represent standard error.

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72 Figure 44. Urine pH time course post sulfonamide drug administration. Data was normalized to a vehicle time course. Lines are colored as follows: AZM 5 mg kg1 body weight (blue), AZM 10 mg kg1 body weight (yellow), CHL 5 mg kg1 body weight (pink), CHL 10 mg kg1 body weight (cyan). Error bars represent standard error of the mean.

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73 CHAPTER 5 THE STRUCTURE SOLUTION AND INSIGHTS INTO THE MYCOPLASMA HYORHINIS PROTEIN CYPL Background Cypl is a protein present on the outer membrane of the bacteria Mycoplasma hyorhinis .1 Reported here is the structure determination of Cypl using I3C as a heavy atom derivative for single isomorphous replacement with anomalous scattering (SIRAS) phasing. Utilizing this structure, as well as sequence alignments to other mycoplasmal Cypllike proteins, and structural alignments to other proteins, the putative role of Cypl is addressed. Evidence will be offered which supports the current hypothesis that Cypl is Crystallization and preliminary X ray data of Cypl were previously reported ( 127) However low (~15%) sequence identity to previously solved structures made molecular replacement phasing methods impossible ( 128) Additionally, the low pH of the crystallization conditions (pH 3) interfered with the binding of heavy atoms necessary for phasing through direct methods. The report of a heavy atom derivative, 5amino2,4,6triiodoisophthalic acid (I3C) presented a straight forward and inexpensive opportunity to phase the structure of Cypl ( 129) I3C proved to be uniquely suited to solve the Cypl structure as it allowed for the binding of the heavy atom compound at low pH. An in house room temperature (RT) X ray diffraction data collection from a single Cypl crystal, quick soaked in I3C, was sufficient to identify the positions of five iodines and generate phases for structure solution. 1 Reprinted in part with permission from Sippel, K. H., Robbins, A. H., Reutzel, R., Domsic, J., Boehlein, S. K., Govindasamy, L., AgbandjeMcKenna, M., Rosser, C. J., and McKenna, R. (2008) Structure determination of the cancer associated Mycoplasma hyorhinis protein Mhp37, Acta Crystallogr D Biol Crystallogr 64 11721178. Copyright 2008 International Union of Crystallography ( http://journals.iucr.org/ ) and Sippel, K. H., Robbins, A. H., Reutzel, R. Boehlein, S. K., Namiki, K., Goodison, S., Agbandje McKenna, M., Rosser, C. J., and McKenna, R. (2009) Structural insights into the extracytoplasmic thiamine binding lipoprotein p37 of Mycoplasma hyorhinis, J Bacteriol 191, 2585 2592. Copyright 2009 Jou rnal of Bacteriology.

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74 an extracytoplasmic binding lipoprotein, specifically, a thiamine pyrophosphate (TPP) bindin g protein rather than an enzyme utilizing TPP as a cofactor ( 63, 64) Furthermore, sequence data suggests that the other mycoplasmal Cypl like proteins will also bind thiamine or simila r molecules. Experimental Methods Expression and Purification of Cypl An Nterminal truncated form of Cypl (residues 24403 ) was expressed and purified using methods previously described in Ketcham et al. 2005 ( 65) Plasmid pMH38113 contained the entire coding sequence for Cypl. The first 23 residues of Cypl contain the signal sequence and the lipoprotein moiety that was removed to enhance purification. Additionally, all of the TGA codons (mycoplasmal codon for T rp) were changed to TGG to optimize its expression in E. coli. This product was ligated into the expression vector pET31f1m1 (gift of Dr. P. Laipis, Department of Biochemistry, University of Florida, Gainesville, FL). E. coli strain BL21(DE3)pLysS was subs equently transformed with the plasmid. Cloning was performed by Dr. Susan Boehlein. The protein was expressed by inoculating 1 liter of minimal media (37C) supplemented with tryptone (10 g l11 ml1), and glucose (0.75% w v1). The culture was induced at an OD600nm of 0.7 to 1.0 with a final concentration of 1 mM IPTG. Cells were grown for an additional 2.5 hours at 37C and harvested. The cells were lysed by vortexing the pellet in 1/10 the original volum e of 20 mM phosphate buffer (pH 7.95) followed by three 15second sonication cycles. The resulting crude cell lysate was centrifuged at 40,000 g for 20 minutes at 4C. The supernatant underwent ion exchange chromatography using a 5 ml BioRad EconoPac S cation

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75 exchange column attached to the bottom of a 50 ml BioRad anion exchange Q column, and equilibrated with 20 mM sodium phosphate buffer (pH 7.95) at a flow rate of 2.5 ml min1. Approximately 125 mg of total protein was loaded on the column. The flow through containing Cypl was adjusted to pH 6.1 with 2 M acetic acid, and loaded on a 5ml cation exchanger, BioRad EconoPac S cartridge, equilibrated with 20 mM sodium acetate, pH 6.1 (buffer A). The column was washed with 5% buffer B (20 mM sodium acet ate, pH 6.1 and 1 M NaCl) and the Cypl protein was eluted with 15% buffer B. The eluted sample was then buffer exchanged into 50 mM Tris Cl, ph 7.5 and concentrated to ~8 mg ml1 using a Centriprep 10 spin column (Millipore, Bedford MA). Crystallization a nd Diffraction Data Collection The crystallization conditions for Cypl were slightly modified from those previously reported by Reut zel et al., 2002 ( 127) The crystals were grown using the batch method in micro bridges under paraffin oil immersion at RT. Crystallization drops were prepared 1 in 50 mM Tris of the precipitant solution, (100 mM citric acid at pH 3.0 containing 40% PEG 4000, 100 mM NH4 decyl D maltoside detergent (Hampton). Useful crystals appeared within 7 days. The heavy atom reagent I3C (Sigma) was soaked into native C solution directly to the crystallization drop. The crystals acquired a pale yellow color within 5 minutes and were immediately mounted in a quartz capillary for RT data collection. The native and I3C X ray diffraction data was collected in house as descr ibed in Chapter 2. The data was obtained using a crystal to detector distance of 100 mm, with 1 oscillation steps and 8 minute exposures per image. Additionally a native dataset was collected at Cornell High Energy Synchrotron Source (CHESS), beamline A1 and

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76 solved. These data were collected using 1 oscillation steps with a crystal to detector distance of 120 mm and an exposure time of 3 seconds The data frames were indexed, integrated and scaled with HKL2000 ( 86) Diffraction d ata statistics are presented in Appendix Table C 1. Structure Solution The crystal structure of Cypl was solved using SIRAS methods, based upon the location of five iodine positions and their associated anomalous differences. The iodine positions were det ermined using the SHELXC and SHELXD software ( 89, 130 ) Protein phases for both the monoclinic space groups, P2 and P21, were cal culated since the diffraction data did not include sufficient 0k0 reflections for an unambiguous space group assignment, and both enantiomorph solutions were checked. Iodine positions from SHELXD were displayed in COOT ( 79) and appeared in two clusters, one an equilateral triangle, and the other an isolated pair of sites. All iodineiodine intracluster distances were in the range 5.6 5.9 as expected from the structure of the I3C reagent ( 129) SHELXE was then used to compute the protein phases and the resulting electron density maps were examined for reasonable protein density. The best phases resulted from the P21 space group and the inverse enantiomer of the original heavy atom constellation. This electron density map was used to fit the sequence using PHENIX AutoBuild ( 131) Within six cycles of fitting, over 120 amino acids were fit to the electron density. The final Aut oBuild model contained 311 placed residues, of which 250 were assigned amino acid sequences. Thus ~66% of the 379 amino acids in the truncated sequence were assigned, yielding an initial Rcryst of 29% and Rfree of 32%.

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77 Refinement of Cypl The Cypl struct ure determination was completed by use of iterative rounds of model building in COOT ( 79) followed by cycles of refinement in SHELXL from the SHELX97 software package ( 89 ) The side chain electron density and local environment of residue 256 was inconsistent with a phenylalanine at that position, and it was replaced by a serine. This assignment was confirmed by resequencing of the plasmid (University o f Florida Sequencing Core, Interdisciplinary Center for Biotechnology Research, Gainesville FL) Waters were added using the automated water divining option in SHELXL ( 89) The coordinates of this model were then used to calculate phases for the 1.9 resolution native data and later the 1.15 resolution data, followed by standard refinement cycling methods. No significant differences were observed between the native and I3C soaked structures. In a cleft between two distinct domains of the Cypl structure was well defined electron density that suggested a pyrophosphate linked to an undefined density that terminated in a planar entity. After several attempts were made, the ligand was eventually identified as TPP. The coordinates for TPP were retrieved from the HIC UP server ( 132 ) and was fit into the electron density. Molecular bond and angle restraints were generated for TPP in the SHELXPRO utility in the SHELX97 software. The final high resolution model contains residues 39 to 403, refined to 1.15 resolution with an Rcryst of 12.9% and an Rfree of 16.6%, with good stereochemistry, as shown by PROCHECK ( 90) Full refinement statistics are presented in Appendix Table C 1. Full refinement details, a structural post mortem, and a description of I3C binding sites are described in Appendix C.

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78 Sequence Alignments and Phylogenic Tree Analysis. Sequence alignment and phylogenetic analysis was performed on twelve mycoplasma Cypl like proteins. Species, description, host organism, and Entrez Protein accession number are listed in Appendix Table C 2. Sequence alignments were performed using ClustalW in the SDSC Biology Workbench ( 7 6, 133) Pairwise and Multiple sequence alignments were performed using the Gonnet Series weight matrix, with an open gap penalty of 10.0 and a gap extension penalty of 1.00. All other parameters were set at default. An unrooted tree was generated using P HYLIP, also within the Biology Workbench ( 133, 134) Structural Alignment. A structural alignment of Cypl was performed using the DALI server ( 135) Results Overall Fold of Cypl approximately equal sized compact domains (Domain I and II), separated by a deep cleft at the interface (Figure 51A). The overall shape is an irregular prolate ellipsoid, with approximate dimensions of 70 x 50 x 40 Domain I consists of a six stranded 5 1B). A short, loosely folded loop borders the Domain I entrance to the central cleft Amino acid residues 39140 lie within the Domain I, Domain II contains residues 141342, and residues 342 403 cross back into Domain I adding two strands and two helices helix which spans the interface between domains 1A and 1B). The first 15 residues of the N terminus were not observed in the crystal structure, indicating high flexibility in

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79 this region. A hydrophilic loop extends partially above the cleft entrance loop (residues 55 to 63, Figure 51C). A map of the electrostatic surface of Cypl shows a typical protein electrostatic profile, with no significant exposed hydrophobic patches (Figure 51D). Of note is the patch of basic residues along the inside of the clef t corresponding to the TPP binding site characterized below. Binding of Thiamine Pyrophosphate TPP is bound between the two domains and makes numerous interactions with both main chain and side chains of the adjacent amino acids (Table 51 and Figure 52). The methyl amino pyrimidine ring is sandwiched between the Trp 314 indole plane and the peptide bond plane between residues Leu 379 and Gly 380. No interaction is apparent between the pyrimidine and amino nitrogen atoms with water molecules, although they are exposed to solvent in the cleft. The C2 of the thiazole ring interacts with the protein, via the carbonyl oxygen of Leu 379 (3.0 ) and the phenolic oxygen atom of Tyr 343 (3.2 ). A close interaction of OD2 of Asp 344 to the thiazole sulfur atom of 3 .2 completes the interactions between the ring atoms and the protein. Side chain atoms of Tyr 215 and the OD1 atom of Asp 344 are in Van der Waals contact with the ethyl carbon atoms of the TPP (Figure 52B). Many of the interactions with the protein are with the pyrophosphate end of the helix. Two lysine side chains, Lys 258 and Lys 129, neutralize charges on the pyrophosphate. Hydrogen bonds from two waters to the PA phosphate oxygen atoms complete the interactions of this phosphate. PB, the terminal phosphate is involved in many more hydrogen bonding interactions, including Lys 129 (3.1 ), the phenolic hydroxyl of Tyr 215 (2.6 ), the OG atom of Ser 255 (2.6 ), and the amide nitrogen and hydroxyl oxygen atoms of bot h Ser 256 (2.8 ) and Ser 257 (2.7

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80 ). The OG of Ser 256 bridges to the phosphate oxygen via a water molecule. It should be noted that Ser 256 was originally phenylalanine in the published amino acid sequence. In a final note, the pyrophosphate lies adjacent to the positive end of the 2B). Comparative Structural Analysis. The Cypl structure was submitted to the DALI server to identify several varieties of proteins with similar structural homology ( 135) All of the proteins listed were some form of binding protein. There were 504 structures with Z scores of greater than two, and none had greater than 16% identity to the sequence of Cypl. Of the structures obtained Interestingly the first 76 matches were to some variant of transferrin (sero, ovo, lacto ). T he top match was to the H249Q mutant N lobe of human serum transferrin, with an rmsd of 4.1 for 250 C ( 136) Several other proteins including a Type II ionotropic glutamate binding domain (APMA receptor), a periplasmic thiamine binding protein (TbpA), and an ATP phosphoribosyl transferase (PRT) were selected for comparison and were structurally superpositioned onto Cypl using COOT ( 79, 137139 ) A sample superposition of Cypl and TbpA are shown in Figure 53. Discussion Cypl and Other Cypl like Proteins. The sequence of Cypl was compared to the sequences of Cypl like proteins in eleven other species of mycoplasmas (Appendix Table C 2). Five of the aligned sequences are shown in Figure 54. There is no significant clustering of sequence identity between the various species, with most pair wise agreements ranging between 20% to 40% identity. However there are a few exceptions. M. mycoides M. capricolum

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81 and M. agalactiae, all of which infect ruminants, s how significant identity, up to 70% in the case of M. mycoides and M. capricolum as well as 58% identity between M. genitalium and M. pneumoniae, two human pathogens (Table 53). This is reflected in the phylogenetic tree (Figure 55). Each species deviat es in a similarly diverse manner from the others. The tree is almost completely centrally rooted with little grouping. Given that two sequences only need 25% identity to be structurally homologous it is likely that all of these proteins will share very sim ilar structures ( 140 ) It is important to note that several residues associated with TPP binding in Cypl are identical or conserved between species (Table 51 and Figure 54). All residues described use Cypl numbering. Within the TPP binding site residues Ser 255, Trp 343, Tyr 215, Trp 314, Asp 344, Glu 308, and Lys 258 were identical or showed significant conservation for all twelve species. Of interest are two of t he residues necessary to pyrophosphate stabilization in the Cypl structure, Ser 256 and Ser 257. Ser 256 (confirmed by sequencing) is a conserved alanine in eight of the other sequences. The Ser 257 is only conserved in four sequences, the remaining eight being glycine. Given the role of Ser 257 in stabilizing the second phosphate, it is likely that the other Cypl like proteins bind thiamine, thiamine monophosphate or another similar molecule. The Presence of TPP in Cypl. The presence of TPP was unanticipated. The most likely source of the TPP would be the E. coli expression system. This leads to the conclusion that TPP must be tightly bound, given that it was not introduced during either purification or crystallization, and is likely the endogenous ligand. In an attempt to elucidate the function of TPP binding in Cypl, the crystal structures of several enzymes that require TPP for catalysis were examined. It was found that in all TPP dependent enzymes, the TPP cofactors

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82 assume a similar V shape, relating t he thiazolium ring to the pyrimidine ring, in an energetically unfavorable conformation ( 141) In this conformation, the 4 amine, most probably in the imino form, is the catalytic base that extracts the proton from C2 of the thiazolium ring in the catalytic mechanism ( 141, 142) When the TPP conformation from yeast transketolase (PDB code 2TRK) is least squares superposed onto the thiophene ring of TPP from Cypl t he 4 amine is too distant and sterically mis oriented in regards to the C2 carbon atom to be catalytically active (Figure 56A) ( 143) The TPP of Cypl more closely resembles the thiamine monophosphate observed in the crystal structure of periplasmic thiamine binding pr otein (TbpA) from Esherichia coli (PDB code 2QRY; Figure 56B) ( 139) In E. coli and in other Gram negative bacteria, the periplasmic binding protein is part of an ATP binding cassette (ABC) transporter that also includes a membrane permease, and an ATPase. Why Cypl Is Probably Not an Enzyme. With these observations it became apparent that TPP is probably not a cofactor for an active enzyme. First, the Cypl bound TPP is not in the catalytically active conformation (Fi gure 56). Second, while the possibility exists that Cypl in the crystal is in a noncatalytic conformation, to our knowledge, no crystal structure of a TPP dependent enzyme has been found in a catalytically inactive conformation. Third, it is believed that a Mg2+ ion, which bridges oxygen atoms from both TPP phosphates, is bound to the apoform of the enzyme prior to TPP binding. The conserved amino acid sequence necessary to bind magnesium in active enzymes is G(D,E)G(~27Xaa)(N,D) ( 141) No such conserved sequence appears in the Cypl sequence (Figure 54). Fourth, the active sites of TPP dependent enzymes are near C2, and are solvent accessible in most crystal structures. There are often side chains of basic am ino acids nearby to

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83 stabilize the intermediates that are predicted by the catalytic mechanism. These features are not seen in the TPP bound Cypl structure. The carbonyl oxygen of Leu 379 lies only 3.0 from C2, and necessary basic amino acid are not present. Fifth, in TPP dependent enzymes, a conserved glutamic acid side chain forms a hydrogen bond to N1 of the TPP pyrimidine, and is believed to be critical to stabilizing the imine form of the 4amino group. According to the mechanism, the 4amino nitrogen is the nucleophile that attacks the proton on C2, creating the reactive carbanion. The closest glutamic acid to N1 in Cypl is Glu 309 at a distance of 11.2 Why Cypl Is Probably a Transport/Binding Protein. Genetic analysis performed by Dudler et. al in 1988 shows that Cypl is located on a M. hyorhinis 5.2kb operon including p29 and p69 which putatively codes for parts of an ABC transporter similar to those found in Gram negative bacteria ( 63, 64) The first open reading frame (ORF) codes for Cypl and contains a 23 amino acid export signal peptide that appears to be cleaved as the protein matures. The C S N sequence on the N terminal of the mature protein is consistent with a lipoprotein. The other two ORFs code for p29 and p69, respectively. The overall operon is flanked by inverted repeats indicating Rhoindependent transcription termination. P29 appears to have sequence similarity to bacterial proteins that interact with both substrate binding proteins and integral membrane proteins. P69 bears a hydropathy plot similar to integral membrane proteins of ABC transport systems, though it does lack the EAA sequence typically seen in these proteins ( 63) The overall structural topology of Cypl is fairly consistent with the Type II periplasmic binding proteins, also described as Type D substrate binding proteins (Figure 53) ( 144146) This is supported by the overwhelming majority of DALI matches

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84 being some sort of binding protein. It has strong structural similarity to the N lobe of transferrin, but not the entire protein ( 125) However there ar e some critical differences. Transferrin is a secreted protein and not membraneassociated like Cypl ( 147 ) The other structurally similar proteins are not ideal functional models either, given their propensity for complex formation. The AMPA receptor and PRT are octamers ( 137, 138) Even the TbpA crystallizes as a dimer, which may or may not be physiologically relevant ( 139) Cypl has shown no indication of functioning as a dimer. In native gels (data not shown) the protein migrates predominantly as a single monomeric band and the electrostatic surface of the protein shows no dimer like hydrophobic patches along the opening of the binding cleft (Figure 51B) ( 138, 139 ) While it may behave as part of an ABC binding cassette in mycoplasma, its role both in the virulence of mycoplasma and in tumorigenic transformation remains unclear. Cypls role in mycoplasma infection has never been addressed. Perhaps in vivo Cyp l is involved in the transport of thiamine into the mycoplasma, which may sequester thiamine away from surrounding host cells, weakening them and creating an opportunity for infection. A precedent for this scenario exists has been described for Bacillus th iaminolyticus which is pathogenic because it degrades thiamine, thus depleting the supply for its host ( 139) Research has also shown that TPP or thiamine are necessary components of lactate oxidation in M. gallisepti cum ( 148) These data indicated that a thiamine binding protein would be an essential component for mycoplasma survival. Additional studies are required to adequately identify a function for Cypl. Further biological and crystallographic studies are necessary to fully characterize this protein and its role in infection as well as tumorigenic transformation of human cells. It is

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85 apparent that Cypl like proteins may offer an important therapeutic target in future drug studies, either for inhibition of Cypl induced tumor progression, or for ameliorating persistent pathogenic infections.

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86 Figure 5 dary helices cyan, strands magenta). B) Structural colored from low to high (blue to red) temperature factors. Also shown are the binding sites for TPP (open red circles) and I3C (open purple circles). (D) Surface charge distribution. Blue indicates regions of positive and red negative charge. Figures A and C generated using Pymol ( 97) Figure B generated using TopDraw ( 149) Figure D generated using Grasp ( 150)

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87 Figure 5 2. Thiamine Pyrophosphate binding site. A) 2FoFc electron density of TPP. Contoured at is colored as follows: TPP carbons, yellow; protein carbons, gray; sulfur, gold; oxygen, red; nitrogen, blue; phosphate, orange. Waters are red spheres. Figure generated using Pymol ( 97)

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88 Table 5 1. TPP interactions of Cypl Atom and bond type Residue (distance in ) O1B HB a Y215 (2.6), S257 (2.9 and 2.7) O2B HB K129 (3.0), S256 (2.9) O3B HB S255 (2.5), K258 (3.0), K129 (3.5) O1A HB Y215 (3.5) O2A HB K258 (2.7), HOH 1062 (2.85) O3A HB HOH 1073 (3.5) O4A HB HOH 1073 (2.9), HOH 1048 (2.8) S1 HB D344 (3.5) N3 HB Y343 (3.3) N4 HB HOH 1126 (2.9) N3 HB HOH 1270 (3.1) N1 HB HOH 1027 (2.7) HC b N200, V201, E308, S257, S255, K258, W314, L379, D344, Y215 aHB = hydrogen bond bHC = hydrophobic contact

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89 Figure 5 3. Structural similarity Cypl to E. coli TbpA. Coil C alpha tracing of Cypl (blue), with TbpA (PDB code 2QRY chain C, color orange). Figure generated using Pymol ( 97)

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90 Figure 5 4. Sequence alignment of Cypl like proteins. M. hyorhinis (hyor), M. genitalium (geni), M. hyopneumoniae (hyop), M. pneumoniae (pneu), M. penetrans (pene). Sequences are highlighted by percentage identity and consensus. Symbols above columns denote residues involved in ligand interactions: TPP binding residues are denoted by red arrowheads. Figure generated using Jalview ( 151)

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91 Table 5 3. Pair wise sequenc e identity (%) of Cypl like proteins. Abbreviations are as follows, M. hyorhinis (hyor), M. genitalium (geni), M. hyopneumoniae (hyop), M. pneumoniae (pneu), M. penetrans (pene), M. pulmonis (pulm), M. gallisepticum (gall), M. agalactiae (agal), M. synoviae (syno), M. arthritidis (arth), M. mycoides (myco), M. capricolum (capr) hyor geni hyop pneu pene pulm gall agal syno arth myco capr hyor 31.2 39.1 29.5 32.1 39.2 33.4 28.8 30.6 31.6 24.3 24.2 geni 31.2 33.3 58.1 31.3 35.0 41.6 22.7 26.9 29.7 22.6 23.6 hyop 39.1 33.3 33.0 31.6 43.2 33.8 27.7 30.7 33.8 24.6 25.9 pneu 29.5 58.1 33.0 33.0 31.5 40.4 25.5 24.7 29.0 23.5 25.8 pene 32.1 31.3 31.6 33.0 33.0 33.2 25.0 28.9 30.6 24.4 26.5 pulm 39.2 35.0 43.2 31.5 33.0 33.2 26.9 29.1 32.7 22.8 26. 0 gall 33.4 41.6 33.8 40.4 33.2 33.2 23.1 27.8 30.5 23.7 24.1 agal 28.8 22.7 27.7 25.5 25.0 26.9 23.1 37.1 26.9 44.3 46.0 syno 30.6 26.9 30.7 24.7 28.9 29.1 27.8 37.1 25.8 31.2 31.7 arth 31.6 29.7 33.8 29.0 30.6 32.7 30.5 26.9 25.8 25.1 25.8 m yco 24.3 22.6 24.6 23.5 24.4 22.8 23.7 44.3 31.2 25.1 70.3 capr 24.2 23.6 25.9 25.8 26.5 26.0 24.1 46.0 31.7 25.8 70.3

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92 Figure 5 5. An unrooted phylogenetic tree of mycoplasma Cypl like proteins. M. hyorhinis (hyor), M. genitalium (geni), M. hyop neumoniae (hyop), M. pneumoniae (pneu), M. penetrans (pene), M. pulmonis (pulm), M. gallisepticum (gall), M. agalactiae (agal), M. synoviae (syno), M. arthritidis (arth), M. mycoides (myco), M. capricolum (capr). Figure generated in PHYLIP ( 134) 20% difference

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93 Figure 56. Conformation of TPP. (A) TPP from yeast transketolase, PDB code 1TRK superposed onto the thiazole ring of TPP of Cypl. (B) Thiamine monophosphate from periplasmic thiamine binding protein, P DB code 2QRY superposed onto TPP of Cypl. The carbons of Cypl TPP are shown in yellow. The carbons of transketolase TPP and thiamine monophosphate are colored cyan. All other atoms are colored as follows: oxygen, red; nitrogen, blue; phosphorus orange; s ulfur, gold. Figure made using PYMOL ( 97)

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94 CHAPTER 6 STRUCTURE SOLUTION AND INSIGHTS INTO MYCOPLASMA GENITALIUM MG289 Background Mycoplasmas (class Mollicutes ) are tiny, pleomorphic bacteria, approximately 200300 nm in diameter, that liv e in association with eukaryotes either by attachment to the cell membrane of its host or intracellularly.3 Mycoplasma genitalium is one of the smallest organisms capable of self replication with a genome of 580 kb which codes for approximately 480 proteins and is considered the starting point for identifying which genes are essential for life ( 49, 50) Experimental and bioinformatic methods have identified a subset of as few as 206 to as many as 387 genes that are considered minimal for an organism to survive and self replicate ( 50, 6870, 154) As described in chapter 1, M. genitalium is also a human sexually transmitted infection that preferentially adheres to ciliated epi thelial cells in the genitourinary and respiratory regions ( 5153) These organisms are highly pervasive, infecting plants and animals including humans, and highly specialized to thrive within specific tissues in its host species. Mycoplasmas are believed to have evolved from low G+C content Grampositive bacteria undergoing significant genome reduction, adapting to the resources available in its host and eliminating unnecessary genes including those responsible for oxidative phosphorylation, ATP generation via pentose phosphate pathway, and the biosynthesis of amino acids, nucleotides, lipids, and cofactors ( 49, 50) Mycoplasmas also adapted reduced substrate specificity allowi ng fewer proteins to accomplish the tasks of many ( 153, 154) 3 Reprinted with permission from Sippel, K. H., Venkatakrishnan, B., Boehlein, S. K., Sankaran, B., Quirit, J. G., Govindasamy, L., AgbandjeMcKenna, M., Goodison, S., Rosser, C. J., and McKenna, R. (2010) Insights into Mycoplasma genitalium metabolism revealed by the structure of MG289, an extracytoplasmic thiamine binding lipoprotein, Proteins: Structure, Func tion, and Bioinformatics Accepted article. Copyright 20 10 John Wiley and Sons.

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95 MG289 is a protein from M. genitalium is annotated as a phosphonate binding protein and is located in an operon tentatively coding for a binding protein dependant substrate transporter. The gene can be disrupted nonlethally; however, since the function of this protein is critical enough for a minimal bacterium to have evolved multiple copies, it is still considered to be an essential gene ( 6870) It is homologous to the M. hyorhinis extracytoplasmic thiamine binding protein Cypl, also known as p37, though they are only 32% identical ( 50, 155) Based off of the s tructure solution of Cypl and sequence alignments, it was theorized that MG289 would bind to thiamine (VIB) or thiamine monophosphate due to a lack of phosphate stabilizing residues ( 125, 126) The cloning, express ion, purification and preliminary X ray analysis of MG289 were reported previously ( 155) Presented here is the structure solution and refinement to 1.95 resolution. The MG289 model is mixed ains with a binding cleft, similar to Cypl and the Type II periplasmic binding proteins of Gram negative bacteria ( 126, 144, 145) As predicted, the ligand found in the binding cleft was VIB. MG289 crystallizes wit h two molecules in the asymmetric unit. Experimental and computational analysis was undertaken to identify whether this dimerization was crystallographic or biological in nature. The discrepancy between the binding partners of Cypl and MG289 are addressed and a hypothesis regarding the phosphorylation of nucleotides and cofactors in M. genitalium discussed. Experimental Methods Plasmid Cloning A plasmid containing the N terminal truncated form of the coding sequence (residues 26386) was amplified using M genitalium (strain G37) genomic DNA (The strain was a gift from Dr. Joel Basemen, University of Texas, and the genomic DNA was

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96 provided by Dr. Leticia Reyes, Department of Infectious Disease and Pathology, College of Veterinary Medicine, University of Fl orida). The N terminal truncation was made to improve the solubility of the protein by removing a putative signal sequence and lipoprotein moiety. The PCR product was purified by agarose gel electrophoresis, digested with NdeI and XhoI, and then ligated into a pET31f1m1 expression vector. Silent mutations were added for codon optimization in E. coli. The plasmid was sequenced to verify it identical to published sequences (Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesvill e, FL). The cloning was performed by Dr. Susan Boehlein. Expression and Purification Protein was expressed and purified as described previously ( 65) Briefly, the plasmid was transformed into BL21(DE3)pLysS cells. One liter of LB supplemented with 100 mg l1 ampicillin was inoculated and cultured until reaching an OD600nm of 0.71.0. IPTG was added to a final concentration of 1 mM, then the cells were allowed to grow for an additional 3 hours before harvesting. Cells were lysed using a french press in 1/100 volume of 20 mM phosphate buffer (pH 7.95). The protein was purified as described in Chapter 5. Purity was confirmed by 10% SDSPAGE stained with Coomassie Blue. Concentrations were calculated by absorbance at 280 nm using a calculated extinction coefficient of 54620 M1 cm1. Crystallization, X ray Analysis, Structure Solution and Refinement The purified MG289 was buffer exchanged into 50 mM Tris Cl pH 7.5 and concentrated to ~8 mg ml1 using a Centriprep 10 spin column (Millipore, Bedford, Massachusetts, USA). Crystallization was performed using hanging drop vapor diffusion ( 85)

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97 M ammonium sulfate, 0.1 M sodium acetate trihydrate, pH 4.6, and 15% PEG 4000) 1 thiamine monophosphate chloride di hydrate, acetylsalicylic acid, cholic acid, 1,2,3heptanetriol, vanillin, N acetyl D mannosamine in 20 mM HEPES sodium, pH 6.8; Hampton Research, Aliso Viejo, CA, USA) equilibrated against 1 ml of precipitant solution. Useful crystals appeared in 3 weeks. A single crystal was directly flash cooled in a gaseous nitrogen stream without any additional cryoprotection beyond the crystallization condition. X ray diffraction data was collected on beamline 5.0.2 at the Advance ). Two hundred useable images were collected with data to a maximum resolution of 1.95 The data was indexed and scaled using HKL2000 ( 86) Data statistics are listed in Appendix Table D 1. Molecular r eplacement was performed in MOLREP implemented in the CCP4i suite using the structure of M. hyorhinis Cypl (PDB 3eki) as a search model ( 126, 156, 157) Refinement was carried out using alternating rounds of comput ation using PHENIX and manual model refinement in COOT ( 79, 114) Positive residual density in the binding cleft and the monomer interface was modeled as thiamine (VIB) and acetate (ACT) respectively. For a more detailed description of the structure solution refer to Supplemental Methods. Final refinement statistics are listed in Appendix Table D 1. Size Exclusion Chromatography Purified MG289 and Cypl in 50 mM Tris Cl pH 7.5 were each diluted to a concentration of 2.5 mg ml1 Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated with 150 mM sodium chloride, 50 mM sodium phosphate, pH 7.2. The flow rate was 0.4 ml min1 and

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98 peaks were detected by absorbance at 280 nm. Molecular weight estimates were calibrated using BioRad Gel Filtration Standard. Small Angle X ray Scattering Purified MG289 was buffer exchanged into 50 mM sodium phosphate buffer pH 7.5 and concentrated to 0.5 mg ml1. The SA XS data was collected at the G1 beamline at the Cornell HighEnergy Synchrotron Source (CHESS). The wavelength used was 1.296 with a detector distance of 1210 mm. The data was collected between angular displacement values (s) of 0.001 and 0.275 1. The images were processed for intensity and s values using DATASQUEEZE ( 158) GNOM was used to compute the radii of gyration and the pairwise distribution function. DAMMIN was used to generate the 3D ab initio model. Ten DAMMIN simulations were averaged using the DAMAVER program to generate a final model. This model was converted to a SITUS volume map for docking purposes and the MG289 X ray dimer was docked manually into the map using the CHIMERA program ( 159, 160) Theoretical SAXS curves from the crystal structures of the monomer and the dimer were computed using the CRYSOL. GNOM, DAMMIN, DAMAVER, and CRYSOL are all located within the ATSAS suite ( 161) Results Overall Structure of MG289 The structure of MG289 was solved to a resolution of 1.95 with an Rcryst and Rfree of 22.1% and 25.5% respectively. Data and refi nement statistics are listed in Appendix Table D (Domains I and II) connected by a hinge region formed by two nonsequential strands and the C terminal helix (Figure 61A). The two domains form a deep cleft with thiamine (VIB) tightly bound between them. There are two MG289 monomers in the

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99 crystallographic asymmetric unit (ASU) (Figure 61B). The overall shape of the protein is an irregular prolate ellipsoid with the approximated dimensions of 70 40 30 3 (Figure 61C). Domain I is slightly smaller than Domain II. Domain I includes residues 32115, and 308hinge region into Domain II. Domain II includes residues 116307. Residues 281283 were not modeled due to a lack of density. Domain II contains six strands flanked by (residues 353367), lies parallel to the hinge region sitting nearly equally between Do mains I and II. The distribution of temperature factors (B factors) in the structure is consistent with sheets having the lowest B factors that increase as the residues become more solvent exposed (Figure 61B) Domain I has ~20% lower temperature factors than Domain II partly owing to a densely packed hydrophobic core that imposes high order. This is also the result of an approximately 7% loss of helical structure with Domain II being composed of long semi ordered loops forming 42% of the total secondary structure. An additional consideration is that there is a difference in the average temperature factor between the two chains in the ASU. Due to the high resolution of the data the refinement was carried out wit hout noncrystallographic symmetry restraints resulting in an average B factor of 44.5 2 for Chain A and 33.6 2 for Chain B. This 24.5% increase in B factor may be correlated to a 34.4% loss of crystal contact surface area in Chain A as compared to Chain B ( 162, 163)

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100 Thiamine Binding Site VIB was located at the base of the hinge region at the junction between the two domains in both MG289 monomers in the ASU. In both chains the bulk of the residue interactions ar e hydrophobic (Figure 6with Trp 275, and has Van der Waals interactions with Gly 344, Ile 343, His 42, and Tyr 167. The VIB also has a direct hydrogen bond of ~3.0 to the OD2 of Asp 41 and there is a water mediated hydrogen bond to the OH of Tyr 167 in chain B. Also an additional water is bound to N4A at a distance of 3.2 in chain B. The thiazole ring engages in exclusively hydrophobic interactions binding Tyr 307, Asp 269, and Ile 343. The ethanol moiety of VIB exhibits two conformations between the two monomers of the ASU. In chain A the ethyl portion of the tail lies parallel to the phenol ring of Tyr 176 and the hydroxyl hydrogen bonds through water to Asp 269, and in chain B, C7 interacts hydrophobically with Tyr 176 and Asp 308, and has a direct hydrogen bond to the N of Asp 308 at a distance of 3.0 Therefore, it appears structurally that either conformation is equally favored. Dimer Interface The overall accessible surface area of the MG289 monom er, as calculated by the PISA server, is 15940.0 2, of which 1041 2 (9.6%) is buried in the interface between the two molecules in the ASU ( 163) There is an estimated gain of 4.2 kcal/mol upon complexation. The composition of the interface residues is as follows: 12 hydrophobic residues, 18 polar uncharged residues and 2 polar charged residues for chain A and 12, 17 and 3 residues respectively for chain B (Figure 62A). There are ten hydrogen bonds in the interface, mostly between asparagine, glutamine, and threonine residues, and no disulfides, covalent interactions, salt bridges or large continuous hydrophobic patches

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101 were detected. The overall complexation significance score (CSS) of the interface was 0.113. CSS ranges from 0 to 1 as the biological relevance of the interaction increases. This indicates that the interface may be a very weak interaction that would only play an auxiliary role in complexation. Size Exclusion Chromatography In an effort to confirm the computational results, experimental methods were employed to identify the quaternary structure of MG289 in solution. S ize exclusion chromatography (SEC) was used to derive an approximate molecular weight of the soluble species. As the shape of a protein is a factor in SEC, in addition to the globular molecular weight standards, the MG289 homolog Cypl was used for comparat ive purposes. Cypl is highly similar in shape to MG289 and known to be monomeric (Figure 6 2B) ( 126) The MG289 has a computed molecular weight of 39.5 kDa and has an elution peak at 15.9 ml, which corresponds to approximately 45 kDa. This is compared to Cypl with a computed molecular weight of 43.4 kDa. Cypl elutes at 15.7 ml corresponding to a molecular weight of 49 kDa. There was a small peak at ~14 ml, however SDS PAGE of the fraction showed that it was contaminating protein (data not shown). Small Angle X ray Scattering (SAXS) Given the deviation of the SEC estimated molecular weight of MG289 from computed values, a second method of experimental verification was employed. The theoretical radius of gyration (Rg) o f the monomer and the dimer were 22.4 and 31.8 respectively, indicating that SAXS would be amenable to addressing the question of monomer versus dimer. The experimental Rg of MG289 was measured to be 33.7 (Figure 62C). The overall shape of the dimer is a slightly twisted disk, whereas the

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102 monomer is dumbbell shaped. The shape of the pairwise distribution curve can describe this information. The curve of the experimental data strongly resembles the dimer (Figure 62D). The experimental data was used t o derive a low resolution ab initio model of MG289 in solution. The overall shape and size of this model is consistent with a dimer (Appendix Figure D 1). Comparison of MG289 to H omolog Cypl When the overall structure of MG289 is compared to the M. hyorhi nis homolog Cypl, there are strong sim ilarities between to two proteins (Figure 63A). The rmsd of for the 365 comparable atoms. As mentioned previously the two share 32% sequence identity with high conservation in the ligand binding site residues. The largest structural variation is in the truncation of two surface loops in MG289 as compared to Cypl, one along the exterior of the binding cleft and one located proximal to the dimer interface (Figure 63A). The binding cleft loop is shortened in MG289 bringing it closer to the ligand binding site, which leads to several additional residues stabilizing the methyl pyrimidine ring in VIB that do not have equivalent amino acids in Cypl (Figure 4B). Comparison of the Cypl and MG289 ligand binding sites show that, as predicted, several of the ligand binding residues are highly c onserved, at least for those interacting with the methyl pyrimidine and thiazole ring (Figure 63) ( 126) The tryptophan, glycine, and three tyrosine residues are nearly superimposable. The conservative substitution of isoleucine for leucine also causes very little change in the pyrimidine ring orientation. Residues 41 and 42 of MG289, which interact with the methyl pyrimidine ring, have no equivalent amino acids in Cypl because they are located on the shortened binding cleft loop. The greatest structural differences are located in the tail region. TPP has an

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103 ethylene bridged pyrophosphate tail, whereas VIB merely has the ethanol group. In Cypl three serines and two lysine residues stabilize the TPP, whereas in MG289, one of the serines is replaced by an alanine, one lysine by a glutamine, and the other lysine by an arginine. Discussion The overall structure of MG289 resembles a periplasmic substrate binding protein (SBP). More specifically, it resembles the Type II periplasmic binding proteins derived from domain displacement as described by Fukami Kobayashi ( 144) Recently a more extensive classification system for SBPs was developed ( 146) Based on this new classification system MG289 is a class D SBP, subcl ass II based off of the VIB ligand. This assertion was confirmed by the secondary structural matching results obtained from the DALI server, as the first 200+ structure matches are Class D ( 164) Since mycoplasmas lack a periplasmic space having evolved from Gram positive bacteria and combined with the fact that they have a lipoprotein moiety, this supports the idea that MG289 is probably extracytoplasmic, being presented on the exterior of the cell membrane. The quaternary structure of MG289 has not been fully answered. The computational and SEC data indicates a monomeric protein whereas the SAXS data indicates a dimer. Computational ly the dimer lacks many of the critical features of a biological interface, including a large percentage of buried surface area, an adequate number of hydrophobic residues, and any interactions stronger than a hydrogen bond ( 165) The SEC experiments support the computational results. Though there was a 5 kDa difference between the calculated molecular weight and the experimental one, this can be accounted for by the distinctive shape of MG289 which would affect its mobility

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104 in a size exclusion column. Its match to the elution curve of Cypl, a monomer, confirms its quaternary structure in solution ( 126) However, the SAXS data contradicts the computation and SEC data fitting to a dimeric structure. It is likely that the interaction is concentration dependant. The SAXS data was collected at a concentration of 0.5 mg ml1, whereas the SEC is 0.05 mg ml1 at its most concentrated. Prokar yotic SBPs are usually monomeric when bound to an ABC transporter ( 146) Occasionally they can purify as dimers, however in those cases the SBPs would shift towards the monomeric form upon ligand binding ( 166, 167) In the context of the native lipidassociated form the protein must interact with its transporter to transfer its ligand. The observed interface may be the result of a sticky integral membrane binding region of the protein surface trying to bury itself while in concentrated solution. When compared to the M. hyorhinis homolo g Cypl, there are strong sim ilarities between the two proteins. The largest distinction is in the absence of several large loops in the binding cleft and at the binding interface (Figure 63A). This interface loop might play a role i n the dimeric nature of the protein in concentrated solution. Cypl crystallizes in the P21 space group with only one molecule in the ASU. Cypl has an extended loop in what would be the dimer interface region, which may account for both the monomeric struct ure of Cypl in solution and the lack of a necessary crystal contact that would lead to the two molecules in the ASU and the P212121 space group of MG289 (Figure 63A) ( 125, 126) The ligand binding residues show a high degree of conservation between MG289 and Cypl, though there are differences (Figure 3B). The residues of the tail region seem to show the greatest sequence variation. The three serine and two lysine residues

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105 strongly stabilize the pyrophosphate in Cypl. In MG289, the residues are two serines, an alanine, a glutamine and an arginine that do not interact with the VIB. These differences would not preclude the possibility for low affinity binding of a phosphate or even pyrophosphate moiety, but they are obviously not the preferred ligand. There is also the addition of residues 41 and 42 from the shortened binding cleft loop in MG289. This does not seem to make a difference in the context of thiamine selection, however it may contribute to the affinity for o ther ligands. Further studies of the kinetics of MG289 and Cypl would be needed to fully characterize the significance. Both ligand binding sites lack strong hydrogen bonds in favor of less specific hydrophobic interactions that may allow for some of the predicted substrate promiscuity including the transport of unsynthesizable guanylate or phosphonates ( 69, 70, 168) When comparing MG289 and Cypl, the most obvious question that arises is why does M. hyorhinis have a preference towards binding TPP while M. genitalium binds VIB? Thiamine is a critical component of Mycoplasma minimal media ( 148, 169) This is because most Mycoplasmas are solely dependant on glycolysis, fermentation, and substrate phosphorylation to meet energetic requirements ( 50, 169) Two enzymes critical to survival are pyruvate dehydrogenase and transketolase, both of which need TPP as a cofactor ( 154, 169) Generation of TPP from thiamine requires its phosphorylation, usually by some form of nucleoside diphosphokinase (ndk). In most known genomes, including prokaryotes, there is some form of highly conserved ndk, however no such gene exists in Mollicutes. It is theorized that to compensate nonorthologous gene displacement occurred allowing other sugar kinases to perform the duties of ndk ( 68, 153, 154, 168, 169) From the crystallogr aphically observed

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106 preferential binding of Cypl for TPP rather than VIB it seems likely that M. hyorhinis lacks the kinase necessary for TPP production inside the cell; a kinase produced by M. genitalium Several possible ndk replacements have been suggest ed including 6phosphofructokinase (MG215), phosphoglycerate kinase (MG300), pyruvate kinase (MG216) and acetate kinase (MG357) that can phosphorylate nucleotide monoand diphosphates, a dephosphoCoA kinase (MG264) and a putative deoxyribonucleoside kina se (MG268) ( 68, 153, 154) We have recently sequenced approximately 96% of the M. hyorhinis genome, with the remainder being mostly repetitive, noncoding sequences. We searched the M. hyorhinis genome for homologs of all known or putative sugar or nucleoside kinase sequences found in M. genitalium (data not shown). Seven M. genitalium sugar or nucleoside kinases had no detectable homologs in the M. hyorhinis genome. These include MG268, mentioned above, as well as a choline/ethanolamine kinase (MG356), uridine kinase (MG382), glycerol kinase (MG038), HPr kinase/phosphorylase (MG085), an inorganic phosphate/ATP NAD kinase (MG128), and a riboflavin biosynthetic protein (MG145). Full descriptions of the kinases and accession numbers are listed in Table 61. MG268, MG382, and MG128 seem to have a higher probability of being the actual thiamine kinase given the nature of their endogenous substrate. Another consideration is the mechanism of VIB transport into the cell. The remaining genes encoded on the operon include a nucleotide binding protein and a transmembrane protein, typical of prokaryotic ABC transporters. It is theorized that the transporter may function as a VIB/proton symporter using proton motive force as a p ropellant, however this does not explain the nucleotide binding protein on the operon

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107 ( 169) Nucleotide binding proteins or domains are equipped for substrate hydrolysis, usually ATP to power transport ( 170) This implies that thiamine and probably other nucleoside transport is achieved via ABC transporters rather than symporters as previously theorized.

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108 Figure 61. The structure of MG289. (A) Secondary structural topology of the MG 289 monomer. Helices are colored cyan and strands, magenta. Figure generated in TopDraw ( 149) e MG289 dimer colored from low to high (blue to red) temperature factor. Ligands are shown as sticks; carbon colored orange; oxygen, red; nitrogen, blue; and sulfur, gold. VIB binding sites (open red circles) and ACT binding sites (open purple circles). (C ) Stereo image of the MG289 monomer with VIB ligand bound. Protein is colored as described in (A), and VIB as described in (B). (D) The VIB binding site of chain A. The 2FoFc ( 116) Protein carbons are colored grey; the rest colored as described above. Waters are represented as red spheres. (E) The VIB binding site of chain B. The 2FoFc described in (D). Figures (B), (C), (D), and (E) generated in PYMOL ( 97)

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109 Figure 62. The quaternary structure of MG289. (A) The dimer interface. Chain A is shown as a molecular surface, while chain B is a cartoon. Noninterface residues are colored grey; nonpolar, green; polar uncharged, violet, and polar charged, orange. Figure generated in PYMOL ( 97) (B) Size exclusion chromatograph of MG289 (solid line) and Cypl (dotted line) with UV absorbance (mAU) at 280 nm plotted against elution volume (ml). (C) Guinier plot of the squared angular displacement values (s2) versus the intensity (I). Experimental data ( ) is charted against the s caled theoretical plots of the monomer ( ) and the dimer ( ). distance from the center of gravity (r). Experimental data (solid line) charted against the scaled theoretical plots of the monomer (dashed line) and the dimer (dotted line). ( r )=( 2) I ( q ) q sin( qr )d q wavelength in and the 2 value)/ number of observati ons].

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110 Figure 63. MG 289 versus Cypl. (A) Secondary structure matching alignment of MG289 (blue) chain A and Cypl (orange). Regions of significant difference are indicated by open red circles. (B) Superposition of ligand binding site from MG289 and Cypl MG289 chain A carbons are colored blue; chain B carbons, cyan; Cypl carbons, light orange; oxygen, red; nitrogen, blue; sulfur, gold; phosphate, orange. Blue text indicates MG289 residue numbering; Orange, Cypl numbering. indicates no equivalent residues. Figure generated in Pymol ( 97)

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111 Table 61. List of Putative M. genitalium Thiamine Kinases MG locus Gene n ame Function Uniprot KB a ccession 038 glpK glycerol kinase P47284 085 hprK HPr kinase/phosphorylase P47331 128 ppnK putative inorganic polyphosphate/ATP NAD kinase P47374 145 ribF putative riboflavin biosynthesis protein P47391 268 n/a putative purine deoxynucleoside k inase P47510 356 n/a putative choline/ethanolamine kinase Q49423 382 Udk uridine kinase P47622

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112 CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTION S The successful treatment of cancer is highly dependent on understanding the factors involved in tumorigenesis and progression. However each type of cancer, even the same type of cancer in different individuals, can be highly heterogeneous. On the clinical side of oncology, there is a need to adequately diagnose what factors are involved on a patient to patient basis. Additionally, from a research perspective, a diverse and extensive understanding of the innumerable players involved in cancer must be obtained to provide clinicians with multiple treatment options so that the patients care can be optimized. Presented here were insights into understanding a few of those factors. In the case of CA IX, though its role in cancer progression has been characterized, effective means of evaluating drug efficacy in vitro has been lacking. This issue was combated through the desi gn of a CA IX mimic, using CA II as a base and mutating the active site (A65S N67Q) to behave like CA IX. This has proven to be relatively effective, though the mimic is still in need of finetuning. Future experiments include the addition of a F131L mutat ion. The kinetics behave similarly to CA IX and its ease of crystallization allows for structural evaluation of drug binding. Utilizing this mimic to evaluate classic sulfonamides, a mechanism for the moderate in vitro isozyme specificity of CHL was identified. The 2Ethylelstra dual action CA/microtubule polymerization inhibitors have a much larger degree of isozyme specificity, with compound 16 demonstrating a 12fold greater affinity for CA II over CA IX mimic. The crystallization experiments showed str ucturally that the loss of affinity was related to a water mediated hydrogen bond

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113 coordinated to Gln 67, a residue unique to CA IX. Water mediated hydrogen bonds are rarely incorporated into structure based drug design, however these studies demonstrate that this element is critical for obtaining an effective isozyme specific inhibitor. This information provides new paths for potential compound development. In the case of compound 16 for use as a chemotherapeutic agent, the specificity for CA II is important to circumvent liver metabolism, however CA IX binding might also provide a targeting mechanism to direct the drug towards tumors. In an effort to translate the in vitro results of the initial CA IX mimic experiment to an in vivo setting, the effectiveness of CHL was evaluated in a mouse model. The global indicators of successful treatment such as reduced tumor growth and overall survival showed no difference between the CHLtreated and control mice. Immunohistochemistry of the tumors revealed no difference between those treated with CHL versus the control. Because of the concern about the bioavailability of CHL, a small pharmacokinetic study was employed to monitor drug uptake though the monitoring of urine pH. As compared with AZM, which showed a signifi cant change in urine pH over the first few hours, CHL demonstrated little pH change that did not seem to improve with increased dosage. The end result was another example among many that prove in vitro results rarely correlated to in vivo success. The poss ibility of an alternate drug administration methodology might allow CHL to be employed in vivo however there are many more promising compounds that might prove to be more effective. Efforts were also directed towards the characterization of potential onc ogenic agents from Mycoplasma, M. hyorhinis Cypl and the related M. genitalium MG289.

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114 Though extensive research has been invested into Cypls role in cancer, less has been spent identifying its function. Structure solution of this molecule through the use of a novel heavy atom for phasing introduced the first glimpse into the proteins purpose through the identification of a bound ligand, TPP. Analysis of the secondary and tertiary structure matched the overall fold to a substratebinding protein. This information coupled with the chemically inert orientation of TPP in the molecule indicated that the molecule was a binding protein. Furthermore bioinformatic analysis indicated that the Cypllike proteins found in other species of Mycoplasma would bind analogous molecules though more likely versions with fewer phosphates. This information coupled with the extracytoplasmic location of the protein make it a promising target for antibiotics thereby preventing oncogenic transformation. Using Cypl as a starting point, the structure of MG289 was solved. The structure of MG289 has revealed many interesting insights into mycoplasma metabolism as a whole. In the context of a minimal genome it discloses a possible mechanism for promiscuity in ligand binding through a dear th of specificity conferring hydrogen bonds. It behaves as a weak dimer in concentrated solution, which may reveal a biological relevant interface in the context of a plasma membrane and interactions with a transmembrane protein. The preference of the prot ein for VIB, the precursor rather than TPP the enzymatic cofactor, indicates that it possesses a kinase capable of processing VIB. The list of putative kinases was reduced to seven possibilities. Further studies will be necessary to identify the overall st ructure of the transporter, the true kinase and the extent of its binding diversity. Hopefully, this knowledge will be used to further aid our

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115 understanding about the metabolic requirements of a minimal genome and may lead to the development of better drugs for the treatment of M. genitalium infections. These discoveries are only the first steps to understanding the mechanisms by which prostate cancer develops and progresses. New CAIs are being produced rapidly. Kinetic assessment and structure solution us ing an improved CA IX mimic will reveal more insight into structure based drug design and how to utilize waters to confer isozyme specificity. In vitro and in vivo models are in place to assess the viability of these new compounds as future cancer therapies. Further characterization is needed to understand the role of Cypl and MG289 in Mycoplasma metabolism and in cancer progression. Currently a kinetics assay is being developed to identify Cypl binding constants for thiamine and its analogs. This informati on combined with crystal structures of alternate binding substrates will provide the information for structurebased design of antibiotics. Studies into the other proteins encoded in the Cypl operon would also help to develop an understanding of critical m etabolic processes to fight Mycoplasma infections. Though the development of isozyme specific CAIs or antibiotics for mycoplasmal infection may not be the magic bullet by which all prostate cancers can be eradicated, they do present options for more ef fective treatments in many cases. This information will add another tool to the clinicians toolbox, improving prognoses and offering therapeutic alternatives with fewer side effects.

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116 APPENDIX A DESIGN OF A CARBONIC ANHYDRASE IX ACTIVESITE MIMIC TO SCREEN INHIBITORS FOR POSSIBLE ANTI CANCER PROPERTIES CONTINUED Figure A 1. Crystal structures of the active site of CA II and CA IX mimic in complex with inhibitors (A) CA II with AZM, (B) CA IX mimic with AZM (C) CA II with BZM (D) CA IX mimic with BZM (E) CA II with CHL (F) CA IX mimic with CHL (G) CA II with EZM (H) CA IX mimic with EZM (I) CA II with MZM (J) CA IX mimic with MZM. The Zn2+ atom is labeled and shown as a black sphere; side chain residues are labeled and atom coloring are as follows: carbon (yellow), oxygen (red), nitrogen (blue), sulfur (orange), chlorine (green). The blue 2Fo PyMOL ( 97 )

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117 Table A 1. Data and refinement statistics for CA IX mimic alone and in complex. aValues in parenthesis are for highest resolution shell. bRsym| 100. cRcrystobs| |Fcobs|)100. dRfree is calculated the same as Rcryst, except it uses 5% of reflection data omitted from refinement. Parameter No Drug MZM CHL AZM EZM BZM Space Group P2 1 P2 1 P2 1 P2 1 P2 1 P2 1 Unit cell parameters (,) a=42.8 b=41.8 c=72.8 a=42.9 b=41.8 c=73.0 a=42.9 b=41.9 c=73.0 a=42.9 b=41.8 a=42.8 b=41.7 c=72.8 a=42.8 b=42.0 c=72.7 Resolution () 20 1.60 (1.66 1.60) a 20 1.80 (1.86 1.80) 20 1.60 (1.66 1.60) 20 1.70 (1.76 1.70) 20 1.50 (1.55 1.50) 20 1.70 (1.76 1.70) Redundancy 3.0 2.8 2.1 3.1 3.8 3.9 Completeness (%) 9 4.9 (91.4) 93.8(91.1) 79.8 (76.5) 96.0(93.4) 92.3(87.2) 93.1 (89.2) R sym b 0.074( 0.279) 0.101(0.328) 0.064 (0.297) 0.068 (0.311) 0.081 (0.430) 0.089 (0.488) R cryst c /R free d 0.151/0.200 0.145/0.214 0.146/0.206 0.141/0.193 0.155/0.190 0 .143/0.197 rmsd for bond lengths/angles() 0.008/0.026 0.006/0.022 0.008/0.024 0.007/0.024 0.009/0.026 0.007/0.023 Average B Factors ( 2 ) Main/side/ solvent/ drug 15.6/23.6/ 29 .1/ n/a 16.1/23.8/ 27.1/ 16.0 15.7/23.0/ 28.7/ 16.4 17.3/24.6/ 37.5/ 17.8 18.6/25.5/ 29.8/ 22.5 19.8/27.1/ 29.3/ 32.1 Ramachandran s tatistics (%) Most favored 88.4 88.0 87.0 90.7 88.9 86.6 Additional /Outlier 11.6/0 12.0/0 13.0/0 9.3/0 11.1/0 13.5/0 PDB ID 3dc9 3dcs 3dcc 3dc3 3dcw 3dbu

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118 Table A 2. Data and refinement statistics for CA II complexed structures. Parameter MZM CHL EZM BZM S pace Group P2 1 P2 1 P2 1 P2 1 Unit cell parameters (,) a=42.8 b=41.7 c=72.9 a=42.9 b=41.8 c=72.9 a=42.8 b=41.7 c=72.9 a=42.8 b=42.1 c=72.7 Resolution () 20 1.60 (1.66 1.60)a 20 1.65 (1.711.65) 50 1.48 (1.531.48) 20 1.70 (1.761.70) Redundancy 2.7 2.3 2.9 2.7 Completeness (%) 98.3 (96.3) 93.0 (89.2) 94.2 (89.3) 96.7 (94.1) R sym b 0.067 (0.358) 0.055 (0.393) 0.057 (0.214) 0.067 (0.323) R cryst c /R free d (%) 0.148/0.197 0.145/0.190 0.149/0.180 0.146/0.195 rmsd for bond lengths ()/angles() 0.006/0.025 0.007/0.024 0.011/0.02 7 0.007/0.023 Average B Factors ( 2 ) Main/side/solvent/drug 17.3/24.8/ 29.9/18.2 15.5/23.7/ 28.2/16.0 16.2/23.3/ 32.3/13.6 18.9/26.0/ 29.8/27.5 Ramachandran Statistics (%) Most favor ed 88.4 88.0 87.6 88.0 Additional & Generously allowed/disallowed 11.6/0/0 12.0/0/0 12.0/0.5/0 11.6/0.5/0 PDB ID 3DAZ 3D9Z 3DD0 3D8W aValues in parenthesis are for highest resolution shell. bRsym100. cRcrystobs| |Fcobs|)100. dRfree is calculated the same as Rcryst, except it uses 5% of reflection data omitted from refinement.

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119 APPENDIX B CHARACTERIZATION OF ISOZYME SPECIFIC CARBONIC ANHYDRASE INHIBITION BY 2 ETHYLESTRA COMPOUNDS CONTINUED Experimental Methods Chemical Synthesis All chemicals were purchased from Aldrich Chemical Co. Organic solvents of A.R. grade were used as supplied. Anhydrous N,Ndimethylformamide and N,Ndimethylacetamide were purchased from Aldrich and stored under a positive pressure of N2 after use. Tetrahydrofuran was distilled from sodium. Sulfamoyl chloride was prepared by an adaptation of the method of Appel and Berger and stored in a tightly sealed container in the fridge ( 171) Chromatography was performed on silica gel ( 70 230 mesh, Macherey Nagel). Thin layer chromatography was performed on Alugram SIL G/UV254 aluminium backed plates (Macherey Nagel). Products were visualized with basic potassium permanganate solution. 1H NMR spectra were recorded in deuterated chloroform solution (unless otherwise indicated) with a Varian 400 NMR spectrometer at 400 MHz. Chemical shifts are reported in parts per million (ppm, d) relative to tetramethylsilane (TMS) as an internal standard. The chemical formula for intermediate compounds 1 2 3 4 5 and 13 are described in Appendix Figure B 1 and for compounds 14, 15, and 16 are in Chapter 3, Figure 31. 17,17Ethylenedioxy Estrone 17,17Ethylenedioxy estrone was synthesized according to a literature procedure ( 111) A suspension of estrone (27.24 g, 101 mmol), toluene (300 cm3), ethylene glycol (30.00 cm3, 592 mmol) and p toluenesulfonic acid (0.250 g, 1.45 mmol) was refluxed for 16 hours under DeanStark conditions. About 2.20 ml of water was collected. The purple reaction mixture was cooled to ambient temperature and poured onto a saturated

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120 solution of sodium hydrogen carbonate (300 cm3) and diluted with ethyl acetate (500 cm3). The organic layer was separated and the aqueous layer was extract ed with additional ethyl acetate (200 cm3). The combined organic extract was washed with water (400 cm3) and brine (400 cm3). The yellow organic extract was dried over sodium sulfate, filtered and evaporated to give crude compound 2 (31.5 g, 100 mmol, 99% yield) as an off white solid which was used without further purification. An analytical sample was prepared by recrystallization from methanol: 1H NMR (400 MHz, CDCl3) d 7.14 (d, J = 8.4 Hz, 1H), 6.61 (dd, J = 2.7, 8.4 Hz, 1H), 6.55 (d, J = 2.7 Hz, 1H), 5. 10 (s, 1H), 4.003.84 (m, 4H), 2.912.70 (m, 2H), 2.401.20 (m, 13H), 0.88 (s, 1H). The NMR data matched those in the literature ( 111) Compound 3 Compound 3 was synthesized according to a literature procedure ( 111) Sodium hydride (60% dispersion in oil, 5.72 g, 143 mmol) was added in a portionwise manner to a stirred 0C solution of compound 2 (30.0 g, 95 mmol) in anhydrous N, N dimethylformamide (420 cm3). The cooling bath was removed and stirring was continued at ambient temperature until the evolution of hydrogen had ceased. This took about 4 hours. The orange reaction mixture was recooled to 0C and methyl chloromethyl ether (14.50 cm3, 191 mmol) was cautiously added dropwise. Upon complete addition, the cooling bath was removed and the milky reaction mixture was allowed to stir at ambient temperature for 16 hours. Ammonia (2 M, 180 cm3) was cautiously added to destroy exces s methyl chloromethyl ether and sodium hydride. The aqueous reaction mixture was extracted once with ethyl acetate (850 cm3) and the organic extract was washed with brine (5 x 300 cm3). The organic extract was dried over sodium sulfate, filtered and evapor ated to give an oil. Column chromatography (10%

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121 ethyl acetate/hexane) afforded compound 3 (28.12 g, 78 mmol, 82% yield) as a viscous colourless oil that solidified on standing: Rf 0.54 (9:1 hexane/ethyl acetate). 1H NMR (400 MHz, CDCl3) d 7.20 (d, J = 8.5 Hz, 1H), 6.82 (dd, J = 2.7, 8.5 Hz, 1H), 6.76 (d, J = 2.7 Hz, 1H), 5.13 (s, 1H), 4.02 3.84 (m, 4H), 3.47 (s, 3H), 2.902.77 (m, 2H), 2.402.17 (m, 2H), 2.10 1.20 (m, 11H), 0.88 (s, 3H). The NMR data matched those in the literature ( 111) Compound 4 Compound 4 was synthesized according to a modified literature procedure ( 111) A wellstirred solution of tetramethylethylenediamine (20.00 cm3, 133 mmol) in dry tetrahydrofuran (210 cm3) was cooled to 78C and then treated with n butyllithium (1.6 M, 80.00 cm3, 128 mmol) over 10 minutes. The reaction mixture was stirred at that temperature for an additional 15 minutes. Compound 3 (15.01 g, 41.9 mmol) in dr y tetrahydrofuran (210 cm3) was added by way of canula over 10 minutes. The reaction mixture was allowed to gradually warm to 0C. This took about 6 hours. The reaction mixture was maintained at this temperature for an additional 30 minutes and then allowe d to stir at ambient temperature for 15 minutes. The reaction mixture was recooled to 78C and iodoethane (10 cm3, 124 mmol) was added over 5 minutes. The reaction mixture was allowed to warm to ambient temperature over 2 hours and then carefully quenched with a saturated solution of aqueous ammonium chloride (50 cm3). The aqueous reaction mixture was diluted with ethyl acetate (1000 cm3) and water (50 cm3) and the organic phase was separated. The organic extract was washed with an aqueous solution of sodium thiosulphite (10% m/v, 200 cm3), more water (2 x 100 cm3) and finally with brine (100 cm3). The organic extract was dried over sodium sulfate, filtered and evaporated to give a pale yellow oil (17.02 g). Two column chromatographic

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122 (2.5% ethyl acetate/hexane) purifications afforded compound 4 (7.00 g, 18.1 mmol, 43% yield) as a viscous colourless oil. An analytical sample was recrystallized from methanol: Rf 0.10 (97.5:2.5 hexane/ethyl acetate). 1H NMR (400 MHz, CDCl3) d 7.08 (s, 1H), 6.78 (s, 1H), 5.16 (s, 2H), 4.02 3.82 (m, 4 H), 3.48 (s, 3H), 2.92 2.73 (m, 2H), 2.62 (q, J = 7.4 Hz, 2H), 2.412.16 (m, 2H), 2.101.95 (m, 1H), 1.94 1.70 (m, 4H), 1.701.28 (m, 6H), 1.19 (t, J = 7.5 Hz, 3H), 0.88 (s, 3H) The NMR data matched those in the literature ( 111 ) Further elution of the column afforded starting material (8.00 g, 22.3 mmol, 53% recovery). Compound 5 Compound 5 was synthesized according to a literature procedure ( 111) Methanol (68 cm3) was cooled to 0C and cautiously treated with acetyl chloride (24 cm3) and stirred for 10 minutes under nitrogen. The methanolic HCl solution was then added to a slurry of compound 4 (5.000 g, 12.94 mmol) in methanol (32 cm3) and stirred for 1 hour until all the solids had dissolved. Water (100 cm3) was added slowly, and the reaction mixture was cooled in an icebath causing precipitation of the product as a white powder which was collected by filtration and washed with water (2 x 50 cm3). The solid was air dried under suction for 2 hours before being further dried under high vacuum for 1 hour to afford compound 5 (3.750 g, 12.57 mmol, 97% yield) as a fluffy white solid. An analytical sample was recrystallized from methanol: Rf 0.55 (1:2 hexane/ethyl acetate). 1H NMR (400 MHz, CDCl3) d 7.05 (s, 1H), 6.52 (s, 1H), 4.63 (s, 1H), 2.882.79 (m, 2H), 2.60 (q, J = 7. 6 Hz, 2H), 2.56 2.37 (m, 2H), 2.291.91 (m, 5H), 1.721.34 (m, 6H), 1.22 (t, J = 7.6 H z, 3H), 0.91 (s, 3H). The NMR data matched those in the literature ( 111)

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123 Compound 13 Compound 5 (0.300 g, 1.00 mmol) was added to a solution of water/ tetrahydrofuran/ ethanol (1:1:1, 30 cm3) and stirred at 0C. S odium borohydride (0.076 g, 2.01 mmol) was added in one portion. Stirring was continued for 16 hours. Excess sodium borohydride was decomposed with acetic acid (1.00 cm3), the solvent evaporated, and the residue extracted with ethyl acetate (10 cm3). The organic extract was washed with water (5 cm3) and brine (5 cm3). The organic extract was dried over sodium sulfate and evaporated to give a white powder. The powder was recrystallized from diethyl ether/hexane to give compound 13 (0.280 g, 0.93 mmol, 93% yi eld) as a fluffy white powder: 1H NMR (400 MHz, CDCl3) ) d 7.05 (s, 1H), 6.49 (s, 1H), 4.59 (s, 1H), 3.73 (t, J = 8.5 Hz, 1H), 2.87 2.70 (m, 2H), 2.59 (q, J = 7.5 Hz, 2H), 2.402.28 (m, 1H), 2.242.05 (m, 2H), 1.991.92 (m, 1H), 1.911.81 (m, 1H), 1.761.62 (m, 1H), 1.56 1.19 (m, 11H containing t, J = 7.5 Hz, 3H ), 0.78 (s, 3H). Compound 14 Sulfamoyl chloride (0.231 g, 2.00 mmol) was added to an icecold solution of compound 13 (0.100 g, 0.33 mmol) in N,Ndimethylacetamide (5.00 cm3). The reaction was allo wed to warm to ambient temperature overnight. Cold brine (5 cm3) was added and the aqueous reaction mixture was extracted with ethyl acetate (3 x 20 cm3). The organic layer was dried over sodium sulfate, filtered and evaporated to to give a yellow solid. Column chromatography (10% acetone/chloroform) followed by trituration with chloroform/hexane mixtures afforded compound 14 (0.065 g, 0.14 mmol, 43% yield) as an off white solid. The solid was stored in the freezer when not in use: Rf 0.10 (1:9 acetone/chloroform). 1H NMR (400 MHz, d6DMSO ) d 7.71 ( br s, 2 H), 7.17 ( br s, 2H), 7.16 ( s, 1H), 7.06 (s, 1H), 4.39 (dd, J = 7.0, 9.0 Hz, 1H), 2.862.77 (m, 2H), 2.67 (q, J =

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124 7.5 Hz, 2H), 2.442.14 (m, 3H), 2.071.98 (m, 1H), 1.941.85 (m, 1H), 1.82 1.71 (m, 2H), 1.511.13 (m, 9H containing t, J = 7.5 Hz, 3H ), 0.82 (s, 3H). Compound 15 Synthesized according to a literature procedure ( 111) Sulfamoyl chloride (0.347 g, 3.00 mmol) was added to an icecold solution of compound 5 (0.300 g, 1.00 mmol) in N,Ndimethylacetamide (1.50 cm3). The reaction was allowed to warm to ambient temperature overnight. Ethyl acetate (25 cm3) and water (25 cm3) were added, and the organic layer was separated and washed with additional water (4 x 25 cm3) and brine (25 cm3). The organic extract was dried over sodium sulfate, filtered and evaporated to give a yellow oil. Column chromatography (10% acetone/chloroform) afforded compound 15 (0.300 g, 0.80 mmol, 79% yield) as a white powder. The solid was stored in the freezer when not in use: Rf 0.4 (1:9 acetone/chloroform). 1H NMR (400 MHz, CDCl3) d 7.20 (s, 1H), 7.11 (s, 1H), 4.93 (s, 2H), 2.95 2.85 (m, 2H), 2.70 (q, J = 7. 6 Hz, 2H), 2.562.38 (m, 2H), 2.342.22 (m, 1H), 2.221.91 (m, 4H), 1.701.37 (m, 6H), 1.22 (t, J = 7. 6 Hz, 3H), 0.91 (s, 3H). The NMR data matched those in the literature ( 111) Compound 16 Synthesized according to a literature procedure ( 111) To a sol ution of compound 15 (0.150 g, 0.40 mmol) in tetrahydrofuran (2.5 cm3) and 2propanol (10 cm3) was added sodium borohydride (0.030 g, 0.79 mmol) in a portionwise manner. The reaction was stirred at ambient temperature for 2 hours. A saturated solution of ammonium chloride (1.50 cm3) was carefully added to the reaction mixture and the aqueous reaction mixture was left to stand overnight. Water (30 cm3) was added and the aqueous reaction mixture was extracted with ethyl acetate (2 x 50 cm3). The organic extra ct was washed with water (20 cm3) and brine (20 cm3). The organic extract was

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125 dried over sodium sulfate, filtered and evaporated to give a yellow solid. Column chromatography (20% ethyl acetate/hexane) afforded compound 16 (0.137 g, 0.36 mmol, 91% yield) as a white solid. The solid was stored in the freezer when not in use: Rf 0.40 (3:2 hexane/ethyl acetate). 1H NMR (400 MHz, CDCl3 + 2 drops d6DMSO ) d 7.18 (s, 1H), 7.10 (s, 1H), 6.63 ( br s, 2H), 3.83 3.62 (m, 1H), 2.83 (dd, J = 4.1, 8.8 Hz, 2H), 2.71 (q, J = 7.5 Hz, 2H), 2.53 (d, J = 5. 60 Hz, 1H), 2.382.29 (m, 1H), 2.252.04 (m, 2H), 1.98 (td, J = 3. 3, 12.6 Hz, 1H), 1.93 1.84 (m, 1H), 1.751.63 (m, 1H), 1.561.16 (m, 11H containing t, J = 7.5 Hz, 3H ), 0.77 (s, 3H). The NMR data matched those in the literat ure ( 111 ) Results CA IX Mimic Compound 15 Second Molecule The second molecule of 15 was identified at 58% occupancy sitting on the exterior of the active site (Appendix Figure B 3A). It was sitting end on, the D ring engaged in hydrophobic contacts with the D ring of the Zn2+bound 15 (Appendix B Figure 2B). The O17 carbonyl was engaged in a hydrogen bond of 3.3 with the A conformation of Gln 92. Additionally there were water mediated hydrogen bonds to Gly 1 32 and Gln 136. The estrone backbone had van der Waals interactions with Pro 202, Leu 204, Ile 91 and Phe 131. Given the extremely low occupancy of the molecule and the weakness of the interactions, it is believed that it is merely an advantageous binding and does not contribute to the inhibition.

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126 Table B 1. Data and refinement statistics. Parameter 14 CA IX mimic 15 CA II 15 CA IX mimic 16 CA II 16 CA IX mimic Space Group P2 1 P2 1 P2 1 P2 1 P2 1 Unit cell parameters (,) a = 42.2 b = 41.3 c = 72.2 a = 42.2 b = 41.2 c = 71.5 a = 42.3 b = 41.3 c = 72.4 a = 42.2 b = 41.2 c = 71.3 a = 42.1 b = 41.3 c = 71.6 Resolution () 25.3 1.5 (1.55 1.5) a 23.1 1.45 (1.5 1.45) 28.7 1.45 (1.5 1.45) 23.8 1.45 (1.5 1.45) 23.1 1.5 (1.55 1.50) Redundancy 5.1 (4.8) 4.7 (3.5) 5.8 (4.5) 4.4 (3.5) 3.6 (3.4) Completeness (%) 93.5 (89.3) 95.3 (80.3) 94.0 (76.9) 94.1 (77.9) 95.9 (91.1) R sym b 0.057 (0.39) 0.055 (0.21) 0.051 (0.181) 0.048 (0.25) 0.025 (0.090) R cryst c /R free d (%) 12.9/ 16.3 14.2 / 15.9 13.2 / 14.7 13.4 / 17.0 13.7 / 17.4 rmsd for bond lengths ()/angles() 0.012/ 1.56 0.009/ 1.40 0.009/ 1.38 0.013/ 1.67 0.012 / 1.50 Average B Fa ctors ( 2 ) Main/side/solvent/drug 1 2.5/16.7/ 15.8/ 31.3 15.6/19.8/ 29.8/ 25.4 12.7/16.7/ 27.9/ 21.8 17.2/20.4/ 31.0/ 17.1 14.4/18.6/ 29.2/ 18.4 Ramachandran Statistics (% ) Most favored /a llowed/outliers 96.9/3.1/0 97.2/2.8/0 96.3/3.7/0 96.3/3.7/0 96.9/3.1/0 PDB ID 3oik 3oku 3okv 3oim 3oil aValues in parenthesis are for highest resolution shell. bRsym100. cRcrystobs| |Fcobs|)100. dRfree is calculated the same as Rcryst, except it uses 5% of reflection data omitted from refinement.

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127 Figure B 1. Chemical formulas of intermediate synthetic compounds.

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128 Figure B 2. Electron density maps of compounds 14, 15, and 16. All maps are 2FoFc averaged kick maps ( 116) Coloring is as follows: carbons from CA II complex, light blue; car bons from CA IX mimic complex, orange; oxygens, red; nitrogens, dark blue, sulfur gold. Zinc is represented by a large gray sphere. Small spheres colored the same as their respective carbons represent waters. (A) 14 complexed with CA IX mimic contoured to 15 15 c 16 c omplexed 16 complexed with CA IX mimic ( 97)

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129 Figure B 3. CA IX mimic with 15 molecule 2. Coloring is as follows: carbons from CA II 15 complex, light blue; carbons from CA IX mimic 15 complex, orange; oxygens, red; nitrogens, dark blue, sulfur gold. Zinc is represented by a large gray sphere. Small spheres colored the same as their respective carbons represent waters. (A) 2FoFc averaged kick maps of the second 15 molecule contou( 116 ) (B) Image of the active site with interacting residues shown as sticks. Figure generated in Pymol ( 97)

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130 APPENDIX C STRUCTURE SOLUTION AND INSIGHTS INTO MYCOPLASMA HYORHINIS PROTEIN CYPL CONTINUED Results Refinement of the Coordinates of the Heavy At om Reagent Using model phases from the complete Cypl structure and the iodine heavy atom derivative data, the locations of the two I3C compounds (site A and B; Figure 51C) were examined to see how they interacted with the protein. The energy minimized str ucture for the I3C was generated using the PRODRG website and the molecular bond and angle restraints for the heavy atom compound were generated in SHELXPRO ( 88) Both the I3C molecules were associated with solvent exposed surfaces on Domain II of Cypl (Figure 51C). The site A molecule was refined with full occupancy, and because of the weaker electron density the site B molecule was refine at 0. 5 occupancy (Appendix Table C 2). The full occupancy I3C molecule (Site A, Appendix Figure C 1A and 1B). Several hydrogen bond interactions were observed; between the N1 of the I3C and the carbonyl oxygen of Leu 335 (3.1 ), the O4 and NZ of Lys 232 (2.5 ), and between O3 of one carboxyl group through a water to ND2 of Asn 179. The other carboxyl group induces a rotamer shift of the side chain of Asp 167 (compared to the native structure), thus avoiding an unfavorable steric interaction. Of note was an unusually close contact from I2 to O of Ala 334 (3.0 ) (Appendix Figure C 1B). The 0.5 occupancy I3C molecule (Site B, Appendix Figure C 1C and 1D), was located in a more exposed surface region, sandwiched between two

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131 symmetry related Cypl molecules. However the electron densities of the iodines were still well defined (Appendix Figure C 1C). The O3 share a hydrogen bond interaction with the amide nitrogen atom of Trp 240 (2.9 ) and NZ of Lys 273 (3.3 ), and the O4 forms a hydrogen bond to the ami de nitrogen of Asn 241 (2.9 ). In addition the O2 of the other carboxylate group forms a hydrogen bond to NZ of the symmetry related Lys 273 (3.0 ) (Appendix Figure C 1D). Identification of Thiamine Pyrophosphate The identification of the TPP cofactor proved to be a significant challenge to solving this structure (Figure 52). Because of the pyrophosphatelike and planar electron densities, the initial guess was ADP, which was placed into the residual FoFc electron density and subjected to additional cycles of refinement. However, the fit of ADP was unsatisfactory and the resulting electron density map was inconsistent with a ribose or deoxy ribose nucleotide so the sugar was removed from the model. After subsequent refinement and refitting cycles, a l arge, electron dense portion of the central ring suggested a methyl thiazole, which was added to the model. The thiazole gave a better fit to the electron density than a 5 carbon sugar but the electron density at the adenine was still ambiguous. A search of nucleotide and cofactor structures suggested TPP which produced an excellent fit to the unknown density (Figure 5 2A). Attempts to Solve the Cypl Crystal Structure in Retrospect With the Cypl structure solved it seemed of interest to address whether it was improvements in software or the use of the I3C itself that led to the successful solution. To assess whether alternative heavy atom phasing methods would have located the I3C in the structure, several alternatives were attempted.

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132 PHENIX AutoSol was tried, but the HASSP algorithm failed to locate more than two sites ( 131 ) Similarly, the heavy atom search routine in CNS ( 87 ) failed to find the correct cluster of sites. Perhaps high temperature factors / low occupancies on the iodine positions caused these Patterson search routines to fail. This may have been because both I3C molecules were in similar orientations, i.e. orientated in the same planar direction, and therefore the Patterson vectors overlap. Once it was determined that SHELXD was ideal for identifying the coordinates of the heavy atoms, the sam e routine of SHELXC, D, and E followed by AutoBuild was applied to previously collected data from a bromide halide soaked Cypl crystal (data not shown) ( 172) Although SHELXD was able to identify seven bromines in the structure, the phases generated by SHELXE did not result in interpretable electr on density maps, AutoBuild failed after eleven cycles. High temperature factors and poor occupancy caused the routine to break down. As mentioned previously, there is very little sequence identity between Cypl and known protein structures in the Protein database which had prevented the use of molecular replacement phasing techniques ( 128, 173) Once solved, the Cypl structure was put through the DALI server to identify proteins that exhibited structural (not sequenc e) homology ( 164) This search resulted in a multitude of matches and the closest hit was selected for use in molecular replacement using routines in CNS ( 87) This was human transferrin N lobe mutant H249Q chain A (PDB code 1JQF) which had the lowest rmsd value, 4.2 for 246 C

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133 13% sequence identity (Figure 4) ( 136) Using the transferrin domain as separate chains failed to give a correct solution to phase the Cypl data. While the secondary structural topology of transferrin is similar, the overall structure of either domain probably deviated too greatly from that of the Cypl domains for molecular replacement to be successful.

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134 Table C 1. Data and Refinement Statistics of Cypl Parameter Native (1.9 ) PDB 3E78 I3C (1.9 ) PDB 3E79 Native 1.15 Space Group P2 1 P2 1 P2 1 Unit Cell a=50.4 b=69.8 c=60.0 a=50.1 b=69.2 c=60.3 a=50.4 b=67.9 c=59.8 Resolution () 25 1.9 (1.9 8 1.90) a 50 1.9 (1.97 1.90) 50 1.15 (1.19 1.15) R sym b (%) 6.7 (37.6) 9.8 (37.9) 6.4 (21.8) 16.3 (5.0) 10.8 (3.7) 19.6 (6.3) Completeness (%) 96.2 (93.2) 92.2 (87. 0) 92.6 (84.1) Mean Multiplicity 3.5 (3.5) 4.1 (4.1) 6.4 (4.4) R cryst c /R free d (%) 18.6/24 18.7/24.4 12.9/16.6 rmsd for bond lengths ()/angles() 0.006 / 0.022 0.006 / 0.021 0 .013 / 0.029 Ramachadran Plot (%) Most favoured / Allowed/outlier 89.1 / 10.9 / 0 90.6 / 9.4 / 0 89.5 / 10.5 / 0 Average Temperature Factors ( 2 ) Main chain/Side chain/ 28.3 / 36.9 / 23.1 / 31.2 / 10.9 / 15.6 / Waters/TPP/I3C/Ca/ 40 .8 / 23.4/ n/a /36.0 /43.6 / n/a / n/a 31.8 / 20.6 / 32.9/20.4 / 25.7 / 13.7 / n/a/11.6 / n/a / 12.2/ 20.1 aValues in parenthesis are for highest resolution shell. bRsym100. cRcrystobs| |Fcobs|)100. dRfree is calculated the same as Rcryst, except it uses 5% of reflection data omitted from refinement.

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135 Table C 2. Mycoplasma Cypllike proteins used for sequence alignment Species subsp. (stra in) Entrez Protein d escription Entrez Protein accession c ode Host Sequence Length a Ref. hyorhinis p37 CAA32357 swine 403 ( 63 ) genitalium G37, phosphonate ABC transporter, SBPb, putative NP_ 072955 human 368 ( 69 ) hyopneumoniae (7448) p37like ABC t ransporter SBP YP_287754 swine 421 ( 174 ) pneumoniae (M129) high affinity transport system protein p37 NP_110103 human 380 ( 175 ) penetrans (HF 2) high affinity transport system protein NP_758050 human 402 ( 176 ) pul monis p37like, ABC transporter SBP CAC13398 murine 412 ( 177 ) gallisepticum high affinity transport protein A AAF78031 avian 366 DS c agalactiae (PG2) alkylphosphonate ABC transporter SBP YP_00125641 1 ovine, caprine 438 ( 178 ) synoviae (53) putativ e p37like ABC transporter SBP YP_278219 avian 404 ( 1 74 ) arthritidis (158L3 1) p37like ABC transporter SBP ACF07046 murine 380 ( 179 ) mycoides mycoides SC (PG1) ABC transporter SBP NP_975778 bovine 477 ( 180 ) capricolum capricolum (ATCC 27343) ABC transporter, SBP YP_424691 caprine 486 ( 69 ) aValues are the number of amino acids in the coding sequence bSBP substrate binding protein cDS direct submission

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136 Fig ure C 1. 5amino2,4,6triiodoisophthalic acid binding sites. (A) and (B) I3C Site A; A) 2FoFc electron density conto interactions of I3C. (C) and (D) I3C Site B; C) 2FoFc electron density follows: I3C carbons, yellow; protein carbons, gray; oxygen, red; nitrogen, blue; iodine, purple. Waters are red spheres. Atom numbering for I3C is as labeled in panel A. Figures generated in Pymol ( 97)

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137 APPENDIX D STRUCTURE SOLUTION AND INSIGHTS INTO MYCOPLASMA GENITALIUM MG289 CONTINUED Table D 1. MG 289 Data and refinem ent statistics (PDB ID 3myu). Parameter Space Group P2 1 2 1 2 1 Cell dimensions () a= 49.3, b= 90.4, c= 175.4 Resolution () 45.18 1.95 (2.02 1.95) a R sym (%) b 7.6 (56.0) 22.0 (2.7) Completeness (%) 90.9 (71.0) Redundancy 7.2 (6.5) R cryst c /R free d /R all data (%) 22.0 / 25.5 / 22.2 rmsd for bond lengths ()/angles() 0.006/0.938 Residues in model (chain id) 32 280, 284 367 (A) 32 280, 283 367 (B) B factors ( 2 ) Protein main /side chain 37.1 / 40.8 VIB / ACT 31.2 / 46.1 Water 38.6 Ramachandran Plot (%) Favored/Allowed/Outlier 97.0/3.0/0 aValues in parenthesis are for highest resolution shell. bRsym100. cRcrystobs| |Fcobs|)100. dRfree is calculated the same as Rcryst, except it uses 5% of reflection data omitted from refinement.

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138 Figure D 1. Docking of MG289 dimer into SAX ab initio reconstruction. A cartoon rendering of the crystal structure (blue) was manually docked into the ab initio reconstruction (orange spheres) derived from the SAXS data. The reconstruction was performed in DAMMIN and averaged in DAMAVER within the ATSAS suite ( 161) Models were docked manually using CHIMERA ( 160) Figure generated in PYMOL ( 97)

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157 BI OGRAPHICAL SKETCH Katherine Sippel was born in the backwoods of Kentucky, but was quickly spirited away to the wilds of the Texan suburbs. She was an odd child who read and doodled too much. Remarkably she survived her teens graduating from The Colony High School in 2001. She received her Bachelor of Science from Texas A&M University Corpus Christi in 2006, where she majored in chemistry and minored in biology and creative writing. She has been a graduate student of the University of Florida since Fall 2006, where she has worked under the tutelage of Dr. Robert McKenna. In her short time here she has published nine papers in seven different journals. She has been the recipient of the Alumni Fellowship and the Medical Guilds mini grant. She has presented at numerous national and international conferences and received the Department of Biochemistrys Boyce Award as well as third place at the College of Medicines 2010 research competition. She hopes to have a long, profitable career in science.