High-value oxy-pharmaceuticals from P450 BM3 ‘gatekeeper’ mutations A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering 2018 Laura N. Jeffreys School of Chemistry BLANK PAGE 2 Table of Contents Figures……. ………………………………………………………………………………..8 Tables……………………………………………………………………………………...10 Supplementary Figures………………………………………………………………….. 11 List of Abbreviations…………………………………………………………………….. 13 Abstract…………………………………………………………………………………... 15 Acknowledgements ………………………………………………………………………16 Declaration……………………………………………………………………………….. 17 Copyright Statement…………………………………………………………………….. 18 Preface to the Journal Format Thesis………………………………………………….. 19 Author contributions……………………………………………………………………. 21 Chapter 1: General Introduction……………………………………………………... 23 1.1. An Overview of Cytochromes P450……………………………………... 23 1.1.1. The Evolution and Nomenclature of Cytochromes P450…………….23 1.1.2. The History of Cytochrome P450 Research ………………………….28 1.1.3. The P450 Catalytic Cycle …………………………………………….32 1.1.4. The Structure of P450 Enzymes ……………………………………...38 1.1.5. Unusual P450 Proteins ……………………………………………….43 1.2. The Natural Fusion Protein P450 BM3 (CYP102A1) ……………………47 1.2.1. The Structure of P450 BM3 ………………………………………….48 1.2.2. Electron Transfer Within P450 BM3 ………………………………...54 1.2.3. P450 BM3 Mutagenesis and the Gatekeeper Mutants ……………….58 1.3. Real-World Applications of P450 Enzymes ……………………………...61 1.3.1. Using P450 BM3 in the Pharmaceutical Industry ……………………63 1.3.2. Using other P450 Enzymes in Industry ………………………………65 1.4. Project Aims ……………………………………………………………...67 1.5. References ………………………………………………………………..68 3 Chapter 2: Characterization of the Structure and Interactions of the Active form of P450 BM3 using Hybrid Mass Spectrometry Approaches……………………………. 82 2.1. Abstract …………………………………………………………………...82 2.2. Introduction……………………………………………………………….82 2.3. Materials and Methods …………………………………………………...86 2.3.1. P450 BM3 expression and purification ………………………………86 2.3.2. Native MS and IM-MS ……………………………………………….88 2.3.3. Collision-induced unfolding (CIU) …………………………………..89 2.3.4. Calculating CCS values from X-ray crystallographic structures …….89 2.3.5. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) ……..89 2.4. Results…………………………………………………………………….91 2.4.1. Native mass spectrometry ……………………………………………91 2.4.2. Collision-induced unfolding and ion-mobility mass spectrometry …..91 2.4.3. Hydrogen-deuterium exchange mass spectrometry ………………….93 2.5. Discussion ……………………………………………………………….100 2.6. Conclusions ……………………………………………………………..105 2.7. References……………………………………………………………….107 2.8. Supplementary Information ……………………………………………..110 Chapter 3: The Promiscuous Nature of P450 BM3 and its Ability To Bind Pharmaceutical Compounds Using a Novel Library Screen………………………… 115 3.1. Abstract ………………………………………………………………….115 3.2. Introduction……………………………………………………………...115 3.3. Materials and Methods ………………………………………………….119 3.3.1. Protein expression and purification …………………………………119 3.3.2. Screening of the FDA-approved compound library ………………...120 3.3.3. Binding affinity determination of pharmaceutical compounds ……..122 3.4. Results …………………………………………………………………..123 3.4.1. FDA-approved compound library screening ……………………….123 3.4.2. Determination of binding affinities for WT and DM BM3 variants ..125 4 3.5. Discussion ……………………………………………………………….128 3.6. Conclusions ……………………………………………………………..131 3.7. References……………………………………………………………….132 3.8. Supporting Information …………………………………………………135 Chapter 4: Novel Insights into P450 BM3 Interactions with FDA-approved Antifungal Azole Drugs…………………………………………………………………157 4.1. Abstract ………………………………………………………………….157 4.2. Introduction……………………………………………………………...157 4.3. Materials and Methods ………………………………………………….161 4.3.1. Expression and purification of WT and A82F/F87V (DM) BM3 heme domain proteins ……………………………………………………………………..161 4.3.2. UV-Visible spectroscopic assays of azole drug binding to WT and DM BM3 heme domains …………………………………………………………………162 4.3.3. EPR spectroscopy studies of WT and DM BM3 heme domains bound to azole drugs..………………………………………………………………………163 4.3.4. X-ray crystallography of the DM BM3 heme domain with azole compounds………………………………………………………………………….. 163 4.4. Results…………………………………………………………………...164 4.4.1. Ligand binding assays of azole drugs to the WT and A82F/F87V (DM) BM3 heme domains …………………………………………………………………164 4.4.2. Electron paramagnetic resonance (EPR) studies of WT and A82F/F87V (DM) BM3 heme domains bound to azole drugs ……………………...168 4.4.3. X-ray crystallography of azole-bound DM heme domain complexes……………………………………………………………………………170 4.5. Discussion ……………………………………………………………….173 4.6. Conclusions……………………………………………………………...177 4.7. References……………………………………………………………….179 4.8. Supporting Information …………………………………………………184 5 Chapter 5: Screening Antidiabetic Binding to P450 BM3 and the Production of Human Metabolites…………………………………………………………………….. 187 5.1. Abstract ………………………………………………………………….187 5.2. Introduction……………………………………………………………...187 5.3. Materials and Methods ………………………………………………….190 5.3.1. P450 BM3 protein expression and purification …………………….190 5.3.2. Antidiabetic binding affinity determination... ………………………191 5.3.3. Electron paramagnetic resonance (EPR) spectroscopy…………….. 192 5.3.4. Structural determination by X-ray crystallography …………………192 5.3.5. Product determination by HPLC, LCMS, and LCMS/MS………….193 5.3.6. Product determination by NMR …………………………………….195 5.4. Results …………………………………………………………………..195 5.4.1. Binding affinity determination for antidiabetic compounds ………..195 5.4.2. EPR spectroscopy of the troglitazone-bound DM heme domain …...197 5.4.3. The X-ray crystal structure of the troglitazone-bound DM heme domain……………………………………………………………………………….197 5.4.4. Production of human metabolites …………………………………...199 5.5. Discussion ……………………………………………………………….200 5.6. Conclusions……………………………………………………………...204 5.7. References……………………………………………………………….205 5.8. Supporting Information …………………………………………………208 Chapter 6: Binding of Fibrates to P450 BM3 Reveals Novel Changes to the P450 BM3 Landscape………………………………………………………………………… 215 6.1. Abstract ………………………………………………………………….215 6.2. Introduction……………………………………………………………...215 6.3. Materials and Methods ………………………………………………….219 6.3.1. Protein expression and purification of P450 BM3 ………………….219 6.3.2. Binding affinity determination ……………………………………...221 6.3.3. EPR analysis of ligand binding to WT and DM BM3 ……………...221 6 6.3.4. Native MS studies …………………………………………………..222 6.3.5. Collision-induced unfolding (CIU) analyzes ……………………….222 6.3.6. HDX-MS characterization ………………………………………….223 6.3.7. Product determination by HPLC, LCMS and LCMS/MS ………….224 6.3.8. Product determination by NMR …………………………………….225 6.4. Results …………………………………………………………………..226 6.4.1. Fibrate drug binding affinity determination ………………………...226 6.4.2. EPR spectroscopy of WT and DM BM3 heme domain in complex with fibrate drugs…………………………………………………………………………228 6.4.3. Production of human metabolites from fibrate drugs………….........229 6.4.4. Native MS and CIU studies …………………………………………229 6.4.5. HDX-MS analysis of substrate binding to WT and DM BM3 heme domains……...………………………………………………………………………231 6.5. Discussion………………………………………………………………. 233 6.6. Conclusions ……………………………………………………………..239 6.7. References……………………………………………………………….240 6.8. Supporting Information …………………………………………………244 Chapter 7: Conclusions and Future Work………………………………………….. 248 7.1. Summary ………………………………………………………………...248 7.2. Conclusions ……………………………………………………………..252 7.3. Future Work ……………………………………………………………..253 7.4. References……………………………………………………………….255 Word Count: 73,921 7 Figures Figure 1: An overview of xenobiotic metabolism ............................................................ 25 Figure 2: Different P450 redox class systems .................................................................. 27 Figure 3: Binding of carbon monoxide to P450 BM3 elicits a Soret shift to 450 nm ... 30 Figure 4: Electron rearrangement in the d-orbitals of the heme iron atom upon substrate binding ....................................................................................................... 33 Figure 5: An overview of the cytochrome P450 catalytic cycle ..................................... 37 Figure 6: The structures of the three most common heme prosthetic groups found in hemoproteins .............................................................................................................. 38 Figure 7: The core of a P450 protein and the P450 fold................................................. 41 Figure 8: The primary and secondary structure of P450 BM3 ..................................... 50 Figure 9: The structures of the P450 BM3 heme and CPR domains ............................ 53 Figure 10: The proposed CPR conformational changes that occur during electron transfer to a cytochrome P450 .................................................................................. 57 Figure 11: A comparison of the flexibility of the P450 BM3 active site in the WT and DM proteins ................................................................................................................ 62 Figure 12: Collision-induced unfolding of the P450 BM3 domains and the full-length P450 BM3 enzyme ...................................................................................................... 92 Figure 13: HDX-MS of the surface of the isolated BM3 domains in comparison to the full-length dimeric protein .......................................................................................