Biochemical and drug targeting studies of Mycobacterium tuberculosis cholesterol oxidase P450 enzymes A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences. 2015 Cecilia Nwadiuto Amadi 1 Table of contents Title page 1 Table of contents 2 List of figures 10 List of tables 18 Abbreviations 20 Abstract 24 Declaration and copyright statement 25 Dedication 27 Acknowledgement 28 Chapter 1- Introduction 29 1.1 Tuberculosis: An Update 29 1.1.1 An ‘Ancient and Modern’ disease 29 1.1.2 The Tuberculosis Burden 31 1.1.3 Signs and Symptoms of Tuberculosis 32 1.1.4 Transmission of Tuberculosis: Latent TB Versus Active TB 33 1.2 Mycobacterium tuberculosis: A Description of a Debilitating 37 Human Pathogen 1.3 Tuberculosis Treatment: Past, Present and Future 40 1.3.1 The Past: Genesis of Anti-Tubercular Drug Discovery 40 2 1.3.2 The Present: Anti-Tubercular Drugs in Current Use 42 1.3.3. The Future: New Tuberculosis Drug Candidates in Development 48 1.3.4 Anti-Tubercular Drug Resistance: A Cause for Therapeutic Failures 59 1.4 The Cytochrome P450 Systems 60 1.4.1 Structure, Function and Mechanism 60 1.4.2 The P450 Catalytic Cycle 67 1.4.3. Cytochrome P450 Redox Partners 72 1.5 The Mycobacterium tuberculosis Cytochrome P450 77 Enzymes 1.5.1 Discovery of Mtb P450s and the Quest for their Physiological Roles 77 1.5.2 The Cholesterol Oxidase P450 Enzymes 81 1.5.2.1 CYP125A1(Rv3545c):Essential for Mtb Viability and 88 Infectivity 1.5.2.2 CYP142A1(Rv3518c): Functional Redundancy 91 1.5.2.3 CYP124A1 (Rv2266): A Methyl-Branched Lipid-Hydroxylase 96 1.5.2.4 Cholesterol Catabolism: A Promising Drug Target in 100 Mycobacterium tuberculosis 1.5.3. CYP51B1: The First Member of the CYP51 Family Identified in 103 Prokaryotes 1.5.4 CYP121A1: An Essential Gene for Mtb Viability 105 1.5.5 CYP130A1 (Rv1256c): Essential for Virulence in Mtb? 107 1.5.6 CYP126A1 (Rv0778) 108 1.5.7 CYP128A1: An Essential Enzyme with a Role in Hydroxylation of 109 Respiratory Menaquinone 1.5.8 Other Partially Characterized P450 Systems in Mycobacterium 111 tuberculosis 1.5.9 Azole Antibiotics: Non-Selective Inhibitors of Mtb Cytochrome P450 115 Enzymes 3 1.6 Novel Drug Discovery Approaches 118 1.6.1 Fragment Based Drug Discovery (FBDD): A Novel Approach to 118 Development of New P450 Inhibitor Scaffolds 1.6.2 High Throughput Screening (HTS) 123 1.7 Justification of Research 126 1.8 Aims of Research 128 Chapter 2 - Materials and Methods 129 2.1 Materials 129 2.2 Methods 129 2.2.1 Preparation of Plasmid DNA for Expression Constructs 129 2.2.1.1 Source and Description 129 2.2.1.2 Plasmid DNA Purification 130 2.2.2 Generation of Glycerol Stocks of E. coli Transformants 132 2.2.3 Expression Trials for CYP124A1 and CYP142A1 P450s 132 2.2.4 Scale up of the Expression of CYP142A1 134 2.2.5 Scale up of the Expression of CYP124A1 135 2.2.6 Protein Purification for CYP124A1 and CYP142A1 136 2.2.7 Assessment of P450 Concentration and Purity 138 2.2.8 Determination of P450 Extinction Coefficients Using the Pyridine 139 Hemochromagen Method 2.2.9 UV-Visible Spectroscopic Studies of Mtb P450s 140 4 2.2.9.1 Binding Assays with Substrates and Inhibitors 140 2.2.9.2 Formation of P450 Carbon Monoxide and Nitric Oxide 141 Adducts 2.2.10 Isothermal Titration Calorimetry (ITC) Studies on Mtb P450s 143 2.2.11 Guanidinium Chloride Denaturation of CYP142A1 144 2.2.12 Redox Potentiometry Studies on CYP124A1 and CYP142A1 144 2.2.13 Multi-Angle Laser Light Scattering (MALLS) Studies of Mtb P450s 146 2.2.14 Differential Scanning Calorimetry Analysis of Mtb P450s 147 2.2.15 Electron Paramagnetic Resonance (EPR) Spectroscopy of P450s 148 2.2.16 CYP124A1 Steady-State Kinetics 148 2.2.17 P450 Protein Crystallization and Structure Determination 149 2.2.18 CYP142A1 Nano-ESI Mass Spectrometry 151 Chapter 3 - Biochemical and Biophysical 153 Characterization of P450 CYP142A1: An Example of Functional Redundancy in the Mycobacterium tuberculosis cholesterol oxidases? 3.1 Introduction 153 3.2 Results and Discussion 158 3.2.1 Expression and Purification of CYP142A1 158 3.2.2 CYP142A1 Substrate Binding Assays 163 3.2.3 Inhibitor Binding Assays 170 3.2.4 Binding Analysis with CYP142A1 Fragment Hits 178 3.2.5 Binding Analysis with Compounds from CYP121A1 Fragment 182 Elaboration Hits 5 3.2.6 CYP142A1 Fe(II)-CO Adduct and NO Adduct Formation 187 3.2.7 Determination of an Extinction Coefficient for Mtb CYP142A1 191 Using the Pyridine Hemochromogen Method 3.2.8 Light Scattering (MALLS) Analysis of CYP142A1 194 3.2.9 Electron Paramagnetic Resonance (EPR) Analysis of CYP142A1 199 3.2.9.1 EPR Analysis with Selected CYP142A1 Ligands 199 3.2.9.2 EPR Analysis for CYP142A1 Fragments Hits 202 3.2.9.3 EPR Analysis of CYP142A1 Bound to MEK Compounds 205 3.2.10 Differential Scanning Calorimetry Studies of CYP142A1 207 3.2.11 Guanidinium Chloride Denaturation of CYP142A1 210 3.2.12 Isothermal Titration Calorimetry (ITC) Analysis of CYP142A1 214 3.2.13 Redox Potentiometry of CYP142A1 217 3.2.14 Nanoelectrospray Ionization Mass Spectrometric Analysis of Mtb 223 CYP142A1−Ligand Interactions 3.2.14.1 NanoESI Mass Spectra of Ligand-Free CYP142A1 224 3.2.14.2 Interaction of CYP142A1 with DTT 226 3.2.14.3 Interaction of CYP142A1 with Econazole 227 3.2.14.4 Analysis of the Interaction of CYP142A1 with 229 Cholestenone 3.2.14.5 Interaction of CYP142A1 with Solvents 232 3.3 Summary 233 Chapter 4 - Biochemical and Biophysical 240 characterization of CYP124A1: A promiscuous enzyme with broad substrate specificity in Mycobacterium tuberculosis 6 4.1 Introduction 240 4.2 Results and Discussion 243 4.2.1 Expression and Purification of CYP124A1 243 4.2.2 Spectroscopic Analysis of CYP124A1 251 4.2.2.1 The UV-Visible Spectrum of CYP124A1 251 4.2.2.2 CYP124A1 Optical Titrations with Substrates 252 4.2.2.3 CYP124A1 Inhibitor Binding Assays 259 4.2.2.4 CYP124A1 Fragment Binding Assays 266 4.2.3 CYP124A1 Heme Iron Coordination by Carbon Monoxide and Nitric 274 Oxide 4.2.4 Determination of the CYP124A1 Heme Extinction Coefficient 278 4.2.5 Steady-State Kinetic Analysis for CYP124A1 280 4.2.6 Multiangle Laser Light Scattering (MALLS) Analysis of CYP124A1 286 4.2.7 Thermostability Analysis of CYP124A1 by Differential Scanning 287 Calorimetry 4.2.8 Determination of the Heme Iron Redox potentials of Ligand-Free 293 and Ligand-Bound CYP124A1 4.2.9 Electron Paramagnetic Resonance (EPR) Analysis of CYP124A1 299 4.2.9.1 EPR Analysis with CYP124A1 Substrates and Azole 299 Inhibitors 4.2.9.2 CYP124A1 EPR Analysis with Fragments and MEK 305 Compounds 4.3 Summary 309 Chapter 5 - Structural Biology of Ligand-Bound 315 Complexes of the Cholesterol Oxidising P450s CYP142A1 and CYP124A1 7 5.1 Introduction 315 5.2 Results and Discussion 317 5.2.1 X-ray Crystallographic Studies and Structure Determination for 317 CYP142A1 and CYP124A1 5.2.1.1 Crystal Structure of the CYP142A1:Cholestenone Complex 321 5.2.1.2 Crystal structure of the CYP124A1:Cholestenone Complex 327 5.2.1.3 A Comparison of Cholestenone-Bound CYP124A1, CYP125A1 335 and CYP142A1 Structures 5.2.1.4 Crystal Structure of the CYP142A1:Econazole Complex 340 5.2.1.5 Crystal Structures of the CYP142A1 in Complex with Fragment- 348 Based Screening Hits 5.3 Summary 360 Chapter 6 - Conclusions and Future Directions 365 6.1 Conclusions 365 6.2 Future directions 372 References 375 8 Appendix 396 Word count: 84,129 9 List of Figures Chapter 1 Figure 1.1: World map showing tuberculosis high-burden countries 32 Figure 1.2: Tuberculosis transmission 36 Figure 1.3: An electron micrograph of Mycobacterium tuberculosis 37 Figure 1.4: The Mycobacterium tuberculosis cell wall 39 Figure 1.5: Structures of some selected anti-TB drugs in clinical use for 48 Mtb infections Figure 1.6: Chemical structure of PDKA 52 Figure 1.7: Chemical structures of HT1171 and GL5 53 Figure 1.8: Chemical structures of nitroimidazole compounds 56 Figure 1.9: Chemical structure of Bedaquiline - a diarylquinoline TB drug 57 Figure 1.10: Chemical structure of SQ109 58 Figure 1.11: Chemical structure of BTZ043 59 Figure 1.12: Heme B prosthetic group 62 Figure 1.13: Spectral features for cytochrome P450 and its ferrous– 63 carbon monoxide complex Figure 1.14: Typical topology of a cytochrome P450 67 Figure 1.15: Schematic representation of the d-orbital electron 68 configurations for low- and high-spin ferric heme iron Figure 1.16: A schematic representation of the P450 compound I 70 (oxyferryl radical cation species) Figure 1.17: Schematic representation of the catalytic cycle of a 71 cytochrome P450 enzyme Figure 1.18: Schematic representation of a variety of P450 redox systems 76 and P450 fusion proteins Figure 1.19: Evolutionary analysis of Mtb P450s 78 Figure 1.20: Genetic organization of cholesterol metabolising gene 83 clusters in Rhodococcus sp. RHA1 and Mtb: A comparison 10 Figure 1.21: The chemical structures of cholesterol and cholest-4-en-3- 87 one Figure 1.22: Cholesterol side chain oxidation reactions 87 Figure 1.23: Structural features of Mtb CYP125A1 in complex with diverse 90 substrates and inhibitor molecules. Figure 1.24: Structural features of CYP142 enzymes from Mtb and M.