Biochemical and Pharmacological Characterization of Cytochrome B5 Reductase As a Potential Novel Therapeutic Target in Candida A

University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School January 2011 Biochemical and Pharmacological Characterization of Cytochrome b5 Reductase as a Potential Novel Therapeutic Target in Candida albicans Mary Jolene Patricia Holloway University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the American Studies Commons, Biochemistry Commons, Microbiology Commons, and the Molecular Biology Commons Scholar Commons Citation Holloway, Mary Jolene Patricia, "Biochemical and Pharmacological Characterization of Cytochrome b5 Reductase as a Potential Novel Therapeutic Target in Candida albicans" (2011). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/3730 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Biochemical and Pharmacological Characterization of Cytochrome b5 Reductase as a Potential Novel Therapeutic Target in Candida albicans by Mary Jolene Holloway A dissertation written in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Andreas Seyfang, Ph.D. Co-Major Professor: Robert Deschenes, Ph.D. Michael J. Barber, D.Phil Gloria Ferreira, Ph.D. Alberto van Olphen, Ph.D. Date of Approval: November 14, 2011 Keywords: Fungal drug resistance, protein biochemistry and structure, ergosterol biosynthesis, yeast knockouts, oxidoreductases Copyright © 2011, Mary Jolene Holloway i DEDICATION This work is dedicated to my mother, Theresa Holloway, for being my coach and friend. This wouldn’t have been possible without you. ii ACKNOWLEDGEMENTS I would like to thank Dr. Andreas Seyfang, my major professor, and Dr. Robert Deschenes, my co-major professor, for helping me achieve this goal. Their guidance and perspective have allowed me to navigate through this challenging acadmic program, and their integrity as scientists and mentors has allowed me to flourish as a researcher. A special thank you to my dissertation committee members, Dr. Michael J. Barber, Dr. Gloria Ferreira, Dr. Alberto van Olphen, and Dr. Dennis Kyle for your assistance, direction, constructive criticism, input, ideas, and especially encouragement. To Dr. David Mitchell for his assistance as a brilliant and talented yeast geneticist, as well as Dr. Roman Manetsch for his collaborative efforts in drug discovery. To my parents, Edward and Theresa Holloway, for your unwavering support and love throughout all my life. To my family and friends, especially Helena Holloway and Beverly Minick, for helping me keep my remaining sanity in an overwhelming time. To Jude, my favorite conversation partner and feline companion. And to my amazing fiancé, Kyle English, for being my rock and best friend. iii TABLE OF CONTENTS List of Tables iii List of Figures iv List of Abbreviations vii Abstract ix 1) Introduction 1 a) Candida albicans and Human Disease 1 b) Antifungal Therapeutics 3 i) Classes of Antifungal Agents 6 ii) Emerging Drug Resistance and Challenges in Treatment 7 c) Cytochrome b5 Reductase 7 i) Overview 7 ii) Methemoglobinemia 10 iii) Fungal cb5r 10 d) Research Aims and Approaches 12 2) Materials and Methods 16 a) Materials 16 i) Molecular Biology Reagents 16 ii) Enzymology and Spectroscopy Reagents 16 iii) Saccharomyces cerevisiae Knockout Strains 17 b) Methods 17 i) Cloning of C. albicans CBR1 and MCR1 Cytochrome b5 Reductase Isoforms and Cytochrome b5 17 ii) Site-Directed Mutagenesis of the Non-canonic CTG Codon in C. albicans CBR1 18 iii) Recombinant Protein Optimization of Expression and Purification 20 iv) Protein Homology Modeling 21 v) UV/Vis Absorbance Spectroscopy 21 vi) Initial-Rate Enzyme Kinetics 21 vii) UV/Vis Circular Dichroism 22 viii) Thermal Stability Assays 22 (1) Fluorescence Spectroscopy 23 (2) Determination of T50 23 (3) Thermodynamic Analysis 23 ix) Kinetic Inhibition Assays 24 i x) Yeast Growth Curves 24 (1) Growth of C. albicans in the Presence of Potential Inhibitors 24 (2) S. cerevisiae Knockout Strains Growth in the Presence of Various Environmental Stressors and Selected Antifungal Agents 25 xi) Sterol Analysis 26 xii) Protein:Protein Interactions 26 (1) C. albicans Spheroplasts 27 (2) His6-tagged Protein Pull-Down Assay 27 (3) Mass Spectroscopy and Analysis 28 3) Results and Discussion 29 a) Expression and Biochemical Characterization of C. albicans Cbr1 and Mcr1 cb5r Isoforms 29 b) Pharmacological Characterization of C. albicans cb5r using Inhibition Studies and Structural Analysis 49 c) Examination of Knockout Phenotypes in the Yeast Model System Saccharomyces cerevisiae 64 d) A Preliminary Investigation into the Protein:Protein Interactions of C. albicans Cbr1 and Cytoplasmic Proteins 94 4) Conclusions and Future Directions 105 Literature Cited 108 About the Author ii LIST OF TABLES Table 1 Primers Used in the Construction of the C. albicans cb5r 20 Protein Expression System. Table 2 Purification of Recombinant C. albicans Cb5r Cbr1 Protein 35 Table 3 Comparison of Initial-Rate Enzyme Kinetics of Rat and 41 C. albicans cb5r Proteins. Table 4 DICHROWEB Analysis of Secondary Structure Composition 44 between Rat and C. albicans cb5r Proteins. Table 5 Mean Ergosterol and DHE Content of S. cerevisiae 91 Knockout Strains Table 6 Peptide-specific Antibodies against the C-terminal Region 97 Unique for the Two C. albicans cb5r Isoforms (ER-specific Cbr1 and Mitochondrial-specific Mcr1). Table 7 Proteins Indentified in Mass Spectrophotometric Analysis 101 iii LIST OF FIGURES Figure 1 Pathogenicity of Candida albicans Attachment and Invasion 3 Figure 2 Current Antifungal Drug Targets 5 Figure 3 Inhibitors of the Ergosterol Biosynthesis Pathway. 6 Figure 4 Crystal Structure of Soluble Human Cb5r in Complex with FAD 9 Figure 5 Electron Flow Between cb5r and Cytochrome P450 Proteins, 10 Utilizing both NADH and NADPH as Electron Donors Figure 6 Phylogenic Analysis and Sequence Similarity of Rat and 31 C. albicans Cb5r Figure 7 Structural Comparison of Mammalian and Fungal Cb5r 33 Figure 8 Purification of C. albicans Cbr1 and Mcr1 Recombinant 35 Proteins Figure 9 IMPACT-TWIN Vector Schematic used in Purification of 36 C. albicans cb5 Figure 10 Purification of C. albicans cb5 Recombinant Protein 37 Figure 11 Spectral Analysis of C. albicans and Rat Cb5r. 38 Figure 12 Initial-Rate Enzyme Kinetics of Recombinant C. albicans Cbr1 39 Figure 13 Initial-Rate Enzyme Kinetics of Recombinant C. albicans Mcr1 40 Figure 14 Thermal Stability and Thermodynamic Analysis of Rat Cb5r 42 and C. albicans Cbr1 Figure 15 Circular Dichroism Spectroscopy Comparing Rat Cb5r 43 and C. albicans Cbr1 Figure 16 LIGPLOT Analysis of Rat cb5r and C. albicans Cbr1 45 Interactions with the FAD Prosthetic Group iv Figure 17 Effect of Herbicidal Inhibitors on C. albicans Cell Growth 51 Figure 18 The Structure of NAD+ 53 Figure 19 The Structure of the Aminocoumarin Antibiotics, 54 Novobiocin, Clorobiocin, and Coumermycin A1 Figure 20 Inhibitory Effects of Selected Compounds against Rat Cb5r 55 and C. albicans Cbr1 Figure 21 Ki values of Selected Substrate Analogues and Novobiocin 57 against C. albicans Cbr1 Figure 22 LIGPLOT Analysis of Cbr1-NAD Interactions 58 Figure 23 Selected Compounds from Fragment-Based Drug Design 60 Figure 24 Selected Ki Values of Compounds Synthesized as 61 Potential Cbr1 Inhibitors Figure 25 Substrate Specificity of C. albicans Mcr1 and Rat cb5r 62 Figure 26 Schematic of Gene Knockout Technique 66 Figure 27 Confirmation of S. cerevisiae Single Knockout Strains 67 by kanMX4 Cassette Figure 28 Confirmation of S. cerevisiae Single Knockout Strains 68 by Motif Primer Design Figure 29 S. cerevisiae Wild-type and Knockout Control Growth Curves 69 Figure 30 S. cerevisiae Wild-type and Knockout Growth at 37°C 70 Figure 31 S. cerevisiae Wild-type and Knockout Growth in 71 Reduced Nutrient Availability and Starvation Conditions Figure 32 S. cerevisiae Wild-type and Knockout Growth under 73 Acidic Stress Figure 33 S. cerevisiae Wild-type and Knockout Growth under 74 Oxidative Stress Figure 34 S. cerevisiae Tetrad Growth Following Mating of 77 cbr1Δ and mcr1Δ Strains v Figure 35 Confirmation of S. cerevisiae Double Knockout Strains 78 by kanMX4 Cassette Figure 36 Confirmation of All S. cerevisiae Knockout Strains by 79 kanMX4 Cassette Figure 37 Confirmation of All S. cerevisiae Double Knockout Strains 80 by Motif Figure 38 S. cerevisiae Wild-type and Knockout Growth in Liquid 82 Culture in the Presence of Selected Antifungal Compounds Figure 39 S. cerevisiae Wild-type and Knockout Growth in the Presence 84 of Selected Compounds Figure 40 Diagram of the Ergosterol Biosynthetic Pathway and 86 Alternative Sterol Metabolism in C. albicans Figure 41 S. cerevisiae Wild-type and Knockout Growth in the Presence 87 of AmB and KTZ with Ergosterol Supplementation in the Growth Media Figure 42 UV Absorption Spectra of S. cerevisiae Wild-type and 89 Selected erg Mutants, erg3∆, erg5∆, and erg6∆ Figure 43 UV Absorption Spectra of S. cerevisiae Wild-type 90 and cb5r Knockouts in the Presence of 5µM Ketoconazole Figure 44 Decrease in Ergosterol Content with Increasing Ketoconazole 91 Concentrations Figure 45 Protein:Protein Interactions of C. albicans Cbr1 and Candida 98 Cell Lysate using the Pierce Pull-Down

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