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Production of High Value Drug Metabolites Using Engineered Cytochromes P450

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Production of high value drug metabolites using engineered cytochromes P450 A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2013 Christopher Butler Table of Contents Title Page 1 Table of Contents 2 List of Figures 8 List of Tables 15 Abbreviations 17 Abstract 20 Declaration and Copyright statement 21 Acknowledgements 22 Preface to the alternate format thesis 23 Chapter 1 – Introduction 26 1.1 Drug metabolites 26 1.1.1 What are they? 26 1.1.2 How are they produced? 26 1.1.3 Why are they important? 27 1.1.4 The enzymes involved 28 1.1.5 Regulatory guidance 29 1.1.6 Reasons for regulation 30 1.1.7 Genetics 30 1.1.8 Current methods of metabolite synthesis 31 1.2 Cytochromes P450 32 1.2.1 Overview 32 1.2.2 Physiological roles 33 1.2.3 Heme proteins 36 1.2.4 The P450 catalytic cycle 37 1.2.5 Active species in the P450 catalytic cycle 38 1.2.6 The P450 heme iron spin state 41 1.2.7 Cytochrome P450 structures 42 1.2.8 Redox partners 43 1.2.9 P450 electron transfer reactions 46 2 1.3 P450 BM3 from Bacillus megaterium 51 1.3.1 Overview 51 1.3.2 P450 BM3 structure 53 1.4 Biotechnology 57 1.5 References 62 Chapter 2 – Title page 83 “Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias” 2.1 Summary 84 2.2 Introduction 85 2.3 Experimental Procedures 87 2.3.1 Generation, expression and purification of WT and 87 variant P450 BM3 proteins 2.3.2 Quantification of P450 BM3 enzymes and 88 determination of their substrate affinity and steady- state kinetic properties 2.3.3 Omeprazole and 5-OH omeprazole turnover and 89 analysis by LC-MS 2.3.4 Omeprazole turnover and analysis by NMR 90 2.3.5 Examination of hemoprotein stability by differential 90 scanning calorimetry 2.3.6 Crystallization of P450 BM3 heme domains and 91 determination of protein structures 2.4 Materials 92 3 2.5 Results 93 2.5.1 Characterization of omeprazole binding properties of 93 BM3 variants 2.5.2 Steady-state kinetics of P450 BM3 variants with 96 omeprazole 2.5.3 Oxidation of omeprazole by WT and variant P450 96 BM3 enzymes 2.5.4 Structural analysis of omeprazole-binding P450 BM3 100 variants 2.6 Discussion 112 2.7 Supplemental Data 116 2.7.1 Characterization of substrate and oxidized products 116 by NMR spectroscopy 2.7.2 Differential Scanning Calorimetry (DSC) 121 2.8 References 124 2.9 Footnotes 130 Chapter 3 – Title Page 131 “Human P450-like oxidative transformations of proton pump inhibitor drugs by a P450 BM3 variant that induces conformational reconfiguration of the enzyme” 3.1 Abstract 132 3.2 Introduction 133 3.3 Experimental Procedures 135 3.3.1 Mutagenesis and expression of WT and variant P450 135 BM3 enzymes 3.3.2 Purification of WT and variant intact P450 BM3 and 135 heme domains 4 3.3.3 P450 quantification 3.3.4 Substrate binding and kinetic properties of WT and 136 variant intact BM3 with fatty acids and PPI substrates 136 3.3.5 EPR spectroscopy 3.3.6 Enzymatic oxidation of substrates and product 137 characterization 137 3.3.7 Crystallization of BM3 heme domains and determination of protein structures 139 3.4 Materials 140 3.5 Results 3.5.1 Spectral binding studies 141 3.5.2 Steady state kinetics 147 3.5.3 EPR spectroscopy 151 3.5.4 Oxidation of PPI’s by WT and variant P450 BM3 157 enzymes using Liquid Chromatography Mass Spectrometry (LCMS) 3.5.5 Oxidation of PPI’s by WT and Variant P450 BM3 165 enzymes using NMR 3.5.6 X-ray crystallography 180 3.6 Discussion 184 3.7 References 187 Chapter 4 – Title Page 193 “Oxidation of diverse drug molecules by P450 BM3 gatekeeper variants” 4.1 Abstract 194 4.2 Introduction 195 5 4.3 Materials and methods 198 4.3.1 Mutagenesis and expression of WT and variant P450 198 BM3 enzymes 4.3.2 Purification of WT and variant P450 BM3 and heme 198 domains 4.3.3 P450 quantification 199 4.3.4 Thermofluor binding assay 199 4.3.5 EPR Spectroscopy 200 4.3.6 Analysis of the kinetics of substrate-dependent 200 NADPH oxidation by WT and variant intact BM3 enzymes 4.3.7 Drug turnover and analysis by LCMS 201 4.4 Materials 202 4.5 Results 203 4.5.1 Thermal unfolding of WT and variant P450 BM3 203 heme domains using a Thermofluor assay 4.5.2 Electron Paramagnetic Resonance spectrometry 205 (EPR) 4.5.3 Steady state kinetic analysis of drug-dependent 209 NADPH oxidation 4.5.4 Product identification from P450 BM3 turnover of drug 213 substrates by LCMS 4.6 Discussion 222 4.7 References 229 Chapter 5 – Summary, conclusions and 235 further work 5.1 Summary 235 6 5.2 Conclusions 245 5.3 Further Work 246 5.4 References 247 Appendix 251 7 List of Figures Chapter 1 Figure 1.1 - Simplified reaction pathway for 33 cytochromes P450 Figure 1.2 - Common P450 reaction types 34 Figure 1.3 - The major forms of heme prosthetic groups 36 Figure 1.4 - The P450 catalytic cycle 38 Figure 1.5 - The P450 radical rebound mechanism 39 Figure 1.6 - The ferric heme iron d-orbital electron state 41 in low spin and high spin configurations Figure 1.7 - General P450 structure 42 Figure 1.8 - Structures of the P450 reductase enzyme 44 cofactors Figure 1.9 - Schematic representation of the 3 major 45 P450 classes Figure 1.10 - Diagrammatic representation of flavin 46 oxidation states Figure 1.11 - The electron transfer process in type I 47 P450 systems Figure 1.12 - Electron transfer in type II P450 redox 49 systems that use CPR 8 Figure 1.13 - P450 electron transfer in type III redox 50 systems Figure 1.14 - The crystal structure of the heme domain 54 of P450 BM3 with N-palmitoylglycine (NPG) bound (PDB code 1PJZ) Chapter 2 Figure 2.1 - The structure of omeprazole 93 Figure 2.2 - Binding and oxidation of omeprazole by 95 P450 BM3 variants Figure 2.3 - LC-MS analysis of products derived from 97 omeprazole oxidation by the P450 BM3 F87V/A82F (DM) double mutant enzyme Figure 2.4 - Optical binding titration for the BM3 DM with 99 5-OH OMP Figure 2.5 - Time course of substrate oxidation and 100 product formation in the reaction of the P450 BM3 DM enzyme with omeprazole Figure 2.6 - Structures of P450 BM3 enzymes and their 101 omeprazole binding sites Figure 2.7 - Interactions of omeprazole in the active site 105 of the A82F BM3 heme domain Figure 2.8 - Stereoviews of structural overlays of 107 substrate-bound forms of the BM3 A82F- containing variant heme domains with WT BM3 9 Figure 2.9 - Structural overlay of the omeprazole-bound 109 A82F variant with the WT BM3 heme domain Figure 2.10 - Conformational equilibria and the 111 relationship with structural stability in P450 BM3 Chapter 2 – Supplemental Data Figure 2.S1 - 1H NMR spectrum of omeprazole 117 Figure 2.S2 - HMBC spectrum of omeprazole 118 Figure 2.S3 - 1H NMR spectrum of turnover products 119 from omeprazole oxidation Figure 2.S4 - HMBC spectra of turnover products from 120 omeprazole oxidation Figure 2.S5 - Graphical overlay of DSC data for WT and 123 variant BM3 heme domains Chapter 3 Figure 3.1 - Proton pump inhibitor (PPI) drug structure 141 and functional groups Figure 3.2 - Esomeprazole binding to the P450 BM3 DM 142 enzyme Figure 3.3 - Pantoprazole binding to the P450 BM3 DM 143 enzyme Figure 3.4 - Lansoprazole and rabeprazole binding to 143 the DM P450 BM3 enzyme 10 Figure 3.5 - Esomeprazole binding to the P450 BM3 144 A82F enzyme Figure 3.6 - Pantoprazole binding to the P450 BM3 145 A82F enzyme Figure 3.7 - Lansoprazole and rabeprazole binding to 145 the A82F P450 BM3 enzyme Figure 3.8 - PPI-dependent steady state kinetic analysis 148 for the A82F P450 BM3 variant Figure 3.9 - PPI-dependent steady state kinetic analysis 149 for the F87V P450 BM3 variant Figure 3.10 - PPI-dependent steady state kinetic 150 analysis for the F87V/A82F (DM) P450 BM3 Figure 3.11 - PPI-dependent steady state kinetic 151 analysis for the WT P450 BM3 with lansoprazole Figure 3.12 - EPR analysis of WT P450 BM3 heme 153 domain Figure 3.13 - EPR analysis of the A82F P450 BM3 154 heme domain Figure 3.14 - EPR analysis of the F87V BM3 heme 155 domain Figure 3.15 - EPR analysis of the DM P450 BM3 heme 156 domain Figure 3.16 - Reactions schemes outlining pathways of 157 P450 metabolism of PPI drugs 11 Figure 3.17 - LC-MS traces of esomeprazole turnover 158 by the P450 BM3 DM enzyme Figure 3.18 - LC-MS traces for lansoprazole turnover by 160 the P450 BM3 DM enzyme Figure 3.19 - LC-MS traces showing pantoprazole 161 turnover from the P450 BM3 DM enzyme Figure 3.20 - LC-MS traces for rabeprazole turnover by 163 BM3 enzymes Figure 3.21 - Proportions of PPI turnover products 165 identified by LCMS Figure 3.22 - 1H NMR spectrum of esomeprazole 167 Figure 3.23 - HMBC spectrum of esomeprazole 168 Figure 3.24 - 1H NMR spectrum of turnover products 169 from esomeprazole oxidation Figure 3.25 - HMBC spectra of turnover products from 170 esomeprazole oxidation Figure 3.26 - 1H NMR spectrum of lansoprazole 172 Figure 3.27 - 1H NMR spectrum of the lansoprazole 173 sulfone standard Figure 3.28 - 1H NMR spectrum of turnover products 174 from lansoprazole oxidation Figure 3.29 - 1H NMR spectrum of rabeprazole 176 Figure 3.30 - COSY NMR spectrum of rabeprazole 177 12 Figure 3.31 - HMBC spectrum of rabeprazole 178 Figure 3.32 - 1H NMR spectrum of turnover products 179 from rabeprazole (100 µM) oxidation Figure 3.33 - 1H NMR spectrum of turnover products 180 from rabeprazole (50 µM) oxidation Figure 3.34 - Overlay of the DM BM3 heme domain 182 esomeprazole crystal structure with the previously reported DM omeprazole structure (4KEY) Figure 3.35 - Figure 3.35 Density overlay of the 183 pantoprazole-bound DM heme domain crystal structure with the esomeprazole-bound DM heme domain structure.
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