Assessment of Aspergillus Kinases As Targets for Antifungal Drug Discovery
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ASSESSMENT OF ASPERGILLUS KINASES AS TARGETS FOR ANTIFUNGAL DRUG DISCOVERY A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 NARJES CHYAD ALFURAIJI School of Biological Sciences Infection, Immunity and Respiratory Medicine Manchester Fungal Infection Group 1 List of Contents List of Contents ...................................................................................................................... 2 List of Tables ....................................................................................................................... 10 List of Figures ...................................................................................................................... 12 List of Abbreviations ........................................................................................................... 16 Declaration ........................................................................................................................... 19 Copyright Statement ............................................................................................................ 19 Dedication ............................................................................................................................ 20 Acknowledgments ................................................................................................................ 21 Chapter 1 : Fungi and Fungal Infections .............................................................................. 23 1.1 Introduction to fungi .................................................................................................. 23 1.1.1Polyploidy and fitness .......................................................................................... 24 1.1.2 Fungal biofilms ................................................................................................... 26 1.1.3.Importance of fungi ............................................................................................. 27 1.2 Fungal infections (Mycoses) ...................................................................................... 28 1.2.1 Superficial mycoses ............................................................................................ 30 1.2.2 Subcutaneous mycoses ........................................................................................ 31 1.2.3 Systemic mycoses ............................................................................................... 32 1.2.3.1 Systemic mycoses caused by primary pathogens......................................... 32 1.2.3.2 Systemic mycoses caused by opportunistic pathogens ................................ 33 1.2.3.2.1 Candidiasis ............................................................................................ 33 1.2.3.2.2 Aspergillosis .......................................................................................... 34 1.2.3.2.3 Mucormycosis (zygomycosis) .............................................................. 35 1.2.3.2.4 Pneumocystosis ..................................................................................... 36 1.2.3.2.5 Fusariosis .............................................................................................. 36 1.3 Antifungal pharmacology .......................................................................................... 37 1.3.1 Polyene antifungal drugs ..................................................................................... 37 1.3.2 Azole antifungals ................................................................................................ 39 1.3.2.1 Topical azoles .............................................................................................. 40 1.3.2.2 Oral Azoles .................................................................................................. 42 1.3.3. Echinocandins .................................................................................................... 44 1.3.4 Pyrimidine analogues .......................................................................................... 45 2 1.3.5 Allylamines, thiocarbamate and morpholines ..................................................... 46 1.3.6 Griseofulvin ........................................................................................................ 47 1.3.7 Developmental antifungal drug ........................................................................... 47 1.3.8 Combination therapy ........................................................................................... 48 1.4 Resistance to antifungal drugs ................................................................................... 50 1.4.1 Resistance to Polyenes ........................................................................................ 51 1.4.2 Resistance to echinocandins ................................................................................ 51 1.4.3 Resistance to flucytosine ..................................................................................... 52 1.4.4 Resistance to Azoles ........................................................................................... 53 1.4.4.1 Mechanisms of azole resistance ................................................................... 54 1.4.4.1.1 Increase of drug efflux .......................................................................... 54 1.4.4.1.2 Target mutation ..................................................................................... 55 1.4.4.1.3 Target expression deregulation ............................................................. 55 1.4.4.1.4 Ergosterol biosynthesis pathway alteration ........................................... 55 1.5 Protein Kinases are fundamental for all living organisms. ........................................ 58 1.5.1. Background ........................................................................................................ 58 1.5.1 Classification of protein kinase ........................................................................... 62 1.5.1.1 Conventional protein kinases ....................................................................... 62 1.5.1.2 Atypical protein kinase ................................................................................ 63 1.5.2 Human protein kinase ......................................................................................... 64 1.5.3 Kinases as drug target ......................................................................................... 66 1.6 Project overview ........................................................................................................ 69 1.6.1 Hypothesis ............................................................................................................... 69 1.6.2 Study plan ............................................................................................................... 70 1.6.3 Aims of the project .................................................................................................. 70 Chapter 2 : Materials and Methods ...................................................................................... 71 2.1 Bioinformatics ............................................................................................................ 71 2.1.1 Data search to identify the predicted kinases ...................................................... 71 2.1.2 Phylogenetic analysis .......................................................................................... 72 2.1.3 Kinases cluster and heat map analysis of kinases ............................................... 72 2.2 Gene knockout ........................................................................................................... 72 2.2.1 A. fumigatus strain, culture and growth conditions ............................................. 72 2.2.1.1 Handling of samples ..................................................................................... 73 2.2.1.2 A. fumigatus strains used in this study ......................................................... 73 2.2.1.3 Growth of A. fumigatus, spore harvesting and storage ................................ 73 3 2.2.1.4 Preparing PK mutants’ pool for competitive fitness study .......................... 74 2.2.1.5 Harvesting of fungal mycelia from pooled growth ...................................... 74 2.2.1.6 Growth of A. fumigatus A1160+ and PK mutants for total RNA extraction .................................................................................................................................. 74 2.2.1.7 Growth of A. fumigatus A1160+ and PK mutants for genomic DNA extraction .................................................................................................................. 75 2.2.2 Extraction of nucleic acids .................................................................................. 75 2.2.2.1 DNA extraction ............................................................................................ 75 2.2.2.1.1 DNA extraction from fungal spores using Cetyl Trimethyl Ammonium Bromide and glass beads ...................................................................................... 75 2.2.2.1.2 DNA extraction from fungal mycelia ................................................... 76 2.2.2.1.3 DNA extraction from infected lungs tissues ......................................... 76 2.2.2.1.4 DNA extraction from infected macrophage .........................................