Western Michigan University ScholarWorks at WMU Dissertations Graduate College 5-2010 High-Throughput Screening Studies of Inhibition of Human Carbonic Anhydrase II and Bacterial Flagella Antimicrobial Activity Albert A. Barrese III Western Michigan University Follow this and additional works at: https://scholarworks.wmich.edu/dissertations Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Biology Commons Recommended Citation Barrese, Albert A. III, "High-Throughput Screening Studies of Inhibition of Human Carbonic Anhydrase II and Bacterial Flagella Antimicrobial Activity" (2010). Dissertations. 500. https://scholarworks.wmich.edu/dissertations/500 This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. HIGH-THROUGHPUT SCREENING STUDIES OF INHIBITION OF HUMAN CARBONIC ANHYDRASE II AND BACTERIAL FLAGELLA ANTIMICROBIAL ACTIVITY by Albert A. Barrese III A Dissertation Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Department of Biological Sciences Advisor: Brian C. Tripp, Ph.D. Western Michigan University Kalamazoo, Michigan May 2010 UMI Number: 3410393 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMT Dissertation Publishing UMI 3410393 Copyright 2010 by ProQuest LLC. All rights reserved. This edition of the work is protected against unauthorized copying under Title 17, United States Code. uest ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 HIGH-THROUGHPUT SCREENING STUDIES OF INHIBITION OF HUMAN CARBONIC ANHYDRASE II AND BACTERIAL FLAGELLA ANTIMICROBIAL ACTIVITY Albert A. Barrese III, Ph.D. Western Michigan University, 2010 Carbonic anhydrase (CA) enzymes catalyze the reversible hydration of carbon dioxide to form bicarbonate and release a proton. There are currently five known CA structural families, a-, p-, y-, 5-, and ^-classes, as reviewed in Chapter One. In Chapter Two, the hypothesis that a high-throughput screen (HTS) of diverse biologically active compounds could yield new structural insights into CA inhibition and identify new inhibitors for this enzyme was experimentally tested. Human CA II was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4- nitrophenyl acetate (4-NPA) in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of <2%. Chapter Three examines the functional mechanism of thioxolone. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II. When thioxolone binds in the active site of CA II it is cleaved and forms 4-mercaptobenzene-l,3-diol which then binds to the zinc active site via the thiol group, and is therefore the active CA inhibitor. The preliminary development and validation of a novel methodology for the high-throughput screening of antimicrobial compounds and inhibitors of bacterial motility is described in Chapter Four. Two basic techniques were combined to enable rapid screening for motility inhibitors; the classical bacterial swarming agar motility assay and the use of 96-well microplates common to HTS drug discovery techniques with a standard absorbance microplate reader. The feasibility of screening the Salmonella typhimurium SJW1103 strain, which is wild-type for flagellar motility and has peritrichously arranged flagella, was examined. The non-physiological substrate, 4-nitrophenyl acetate, does not react with all known isozymes of a-class CAs or the y-class CA from M. thermophila. Preliminary studies describing the subcloning and expression CA I and a truncated, soluble form of CA IV, are described in Chapter Five. This chapter also details the preliminary exploration of several alternative pH absorbance assays for measuring the catalytic activity of CAs. Finally, some possible directions for future research, based on the experimental results presented in Chapters 2-5, are discussed in Chapter Six. Copyright by Albert A. Barrese III 2010 ACKNOWLEDGMENTS I would like to thank Dr. Tripp for his guidance, support, dedication and patience with me during my graduate study at Western Michigan University. He was a great mentor and I feel that the skills that I learned from him will start my scientific career off in a successful way. I would also like to thank my committee members; Dr. John Geiser, Dr. James Kiddle, and Dr. Wendy Ransom-Hodgkins. Their suggestions and thoughtful insights have given me the opportunity to critically think about my work and at times see a new direction for my work to proceed in. Lastly I would like thank my family and friends for their support and patience without them this work would not have been possible. Albert A. Barrese III ii TABLE OF CONTENTS ACKNOWLEDGMENTS ii LIST OF TABLES vii LIST OF FIGURES ix CHAPTER I. OVERVIEW OF CARBONIC ANHYDRASE ENZYMES, INHIBITORS, AND BACTERIAL FLAGELLIN PROTEINS 1 Introduction 1 Carbonic Anhydrase Enzyme Classification, Protein Structures, and Catalytic Mechanisms 1 Inhibitors of Carbonic Anhydrase 15 Medical Applications of Carbonic Anhydrase Inhibitors 17 Bacterial Flagellin Protein 19 References 25 II. CA II INHIBITION STUDIES WITH THE GENESIS PLUS AND NCI DIVERSITY SET COMPOUND LIBRARIES 37 Introduction 37 Experimental Procedures 38 Compound Libraries 39 Esterase Activity Screening Assay 41 Inhibitor IC50 Measurement 42 iii Table of Contents-Continued CHAPTER Inhibitor Kd Measurement 45 Results 47 GenPlus Compound Library: Preliminary Identification of Hits 47 Reproducibility 55 Signal to Noise Characteristics 57 Enzyme and Substrate Stability 59 Hit Confirmation 60 I. Sulfonamide CAIs 60 II. Non-Sulfonamide CAIs 63 Hit Characterization 67 Results of IC50 Assays with 1 uM CA II 68 Results of IC50 Assay with 5 uM CA II 68 Hill Slope as an Indicator of Promiscuity 69 Results of IC50 Assay with 1 uM CA II and Triton X-100 70 Dissociation Constant Assay Results 72 Comparison of IC50 and Kd Values 77 NCI Compound Library 79 Discussion 90 iv Table of Contents-Continued CHAPTER Limitations of 4-NPA Esterase Assay 90 Esterase vs. Fluorescence Screening Assays 92 References 94 III. MECHANISTIC STUDIES OF HUMAN CARBONIC ANHYDRASE II INHIBITION BY THIOXOLONE 101 Introduction 101 Experimental Procedures 104 Enzyme Expression and Purification 104 Materials 106 Preparation of 4-mercaptobenzene-l,3-diol (Compound 3) by acid hydrolysis of thioxolone (Compound 1) 106 Carbonic Anhydrase 4-NPA Esterase Inhibition Studies 109 Michaelis-Menten Enzyme Kinetic Studies 110 Liquid Chromatography-Mass Spectroscopic Studies 113 Results 115 Structure-Activity Relationship Study 115 Enzyme Kinetic Studies 129 Liquid Chromatography-Mass Spectroscopy Analysis of Thioxolone and Hydrolysis Products 136 Discussion 138 References 143 v Table of Contents-Continued CHAPTER IV. A HIGH-THROUGHPUT SCREENING ASSAY FOR DETECTING INHIBITION OF FLAGELLAR MOTILITY 148 Introduction 148 Experimental Procedures 149 Results and Discussion 152 References 166 V. CARBONIC ANHYDRASE SUBCLONING, EXPRESSION, AND C02 HYDRATION ACTIVITY ASSAY DEVELOPMENT... 168 Carbonic Anhydrase Isozyme Subcloning and Expression 168 Expression and Purification of y-class Carbonic Anhydrase (Cam) 192 Sodium Bicarbonate pH Assay 196 References 217 VI. FUTURE WORK 219 Reference 224 APPENDIX Recombinant DNA Biosafety Approval 225 vi LIST OF TABLES 2.1. Potential carbonic anhydrase II inhibitor compounds identified in initial screen of Genesis Plus compound library, as confirmed by two statistical criteria 49 2.2. Summary of IC50 values and K& values for Genesis Plus hit compounds and model sulfonamide compounds 74 2.3 Carbonic anhydrase II inhibitor compounds identified in a screen of the NCI Diversity set compound library 83 3.1. Structures and apparent IC50 values for inhibition of human carbonic anhydrase II by thioxolone analogs 119 3.2. Michaelis-Menten kinetic parameters determined for concentration- dependence of thioxolone inhibition of 4-nitrophenyl acetate hydrolysis by carbonic anhydrase II 131 3.3. Mechanism-based enzyme inactivation parameters determined for thioxolone inhibition of 4-nitrophenyl acetate hydrolysis by carbonic anhydrase II 136 4.1. Complete list of antibacterial compounds detected in screen of Genesis Plus library with offset motility detection method 161 5.1. Mammalian a-class carbonic anhydrase clone vector and sequence information 170 5.2. Sequencing results for the pET28c-CA I and pET28c-CA IV 176 5.3. Partial blastx search results with CA I DNA sequence results 177 5.4. Partial blastx search results with CA IV DNA sequence results 178 5.5. The translated peptide sequence for the subcloned carbonic anhydrase I and IV genes in the pET 28c-CA I and pET 28c-CA IV plasmids 179 vii List of Tables-Continued 5.6. Comparison of the translated CA I pET28c-CA I plasmid DNA sequence to the wild-type protein sequence 180 5.7. Comparison of pEt 28c-CA I V plasmid DNA sequence to the wild-type CA IV sequence 181 5.8. Results of non-linear fit of standard CA II esterase assay IC50 data using Graph Pad Prism 4.0 sigmoidal dose-response model/curve fit 206 5.9. Results of non-linear fit for microplate format CA II CO2 hydration pH assay IC50 data using Graph Pad Prism 4.0 sigmoidal dose-response model/curve fit 206 5.10. Statistical results of fluorescein MultiDrop 8-channel peristaltic test for three successive dispensing operations into 96-well microplates 209 5.11. The concentration of reagents right after mixing the aqueous solution of sulphuric acid, cobalt sulphate, and potassium dihydrogen phosphate with an aqueous solution of sodium bicarbonate 210 5.12. CA II carbonic acid dehydrating to CO2 pH assay.