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In search of human cytochrome P450 1A1 enzyme inhibitor By Supriya Kaduskar Thesis submitted to De Montfort University as a partial fulfilment of the requirements for the degree of Doctor of Philosophy April 2015 Page | 1 Abstract Human cytochrome P450 1 (CYP1) family consists of three members; they are CYP1A1, CYP1A2 and CYP1B1. The mRNAs of CYP1A1 and CYP1B1 genes are known to be expressed in extra-hepatic tissues while that of the CYP1A2 gene is found in the liver amongst other tissues. Under normal circumstances, rarely has CYP1A1 protein expression been seen in human tissues. All three CYP1A enzymes have been widely studied for different reasons. The CYP1A1 enzyme is known to be induced by xenobiotics and poly- aromatic hydrocarbons (PAHs). Induction of CYP1A1 results in its involvement in Phase 1 biotransformation of xenobiotics and in the metabolism of pro-carcinogens, such as PAHs, to carcinogenic substances that eventually lead to cancer. Natural products such as flavonoids, chalcones and stilbenes have attracted a great deal of attention since these compounds have an ability to modulate the activity of CYPs by inhibiting CYP-specific enzymatic activity. Several compounds have been found which potently inhibit CYP1 family of enzymes. They are resveratrol, rhaphontigenin, isorhaphontegenin, α- napthoflavone, tetramethoxystilbene. Some of these compounds are available over the counter in pharmacies in the UK, mainland European countries and the US with proclamation of health benefits. Until now, no potent CYP1A1-specific inhibitor has been identified. Compounds that inhibit CYP1A1 can potentially act as chemo-preventive agents in the treatment of cancer. Page | 2 In this thesis, at first, a yeast strain that co-expresses CYP1A1 with a genetically engineered variant of human P450 reductase (ΔhRDM) was successfully grown; microsomes containing active CYP1A1 enzyme were prepared. A 200-compound library (available at DMU’s School of Pharmacy) consisting of chemically synthesised compounds belonging to chalcone flavonoid and stilbene classes were screened on the CYP1A1 microsomal enzyme to find a potent inhibitor of CYP1A1; later, CYP1A2 and CYP1B1 microsomal enzymes were used to determine specificity towards CYP1A1. Various inhibitors were identified with differing degrees of potency and specificity. The lead inhibitory molecules obtained from the initial in vitro screen were assessed in live yeast cells that expressed active CYP1A1 enzyme. The results interestingly indicated that there is a strict correlation between IC50s obtained in microsomal and cellular assays. We were curious to know whether the activation of a CYP by the novel reductase ΔhRDM created an active site geometry which is identical to the active sites formed by coupling of the wild-type human and yeast P450 reductases (hRD and yRD). Since currently there is no way of determining the active site of a membrane-bound protein complex using X-ray crystallography or 2-D NMR, we decided to use chemical tools (i.e. inhibitors of the CYP1 enzymes) to find out if the IC50s of inhibitory molecules could be used instead as a way of determining active site geometry. It was quite fascinating to see that the IC50 of a compound with a CYP activated by ΔhRDM is more or less the same as a CYP activated by a wild-type reductase (yRD or a hRD) indicating that the active site geometries of a CYP are the same irrespective of the coupled reductase. Page | 3 DMU 2157 (a heterocyclic chalcone) and DMU 110 (a flavonoid) were found to be potent inhibitors of CYP1A1 enzyme (IC50, 72nM and 397nM, respectively). Structural analysis of DMU 2157 suggested that the positions of the methoxy groups in ring ‘A’ and the position of the <N in the pyrido ‘B’ ring play important roles in facilitating inhibition of CYP1A1 enzyme. Therefore DMU 2157 analogues, modelled on ‘chalcone’ and ‘stilbene’ scaffolds, were chemically synthesized to better understand the structural features of DMU 2157 that contribute to specific inhibition of CYP1A1 and with the aim that more potent inhibitors could ultimately be designed. Intriguingly, besides obtaining CYP1A1-specific inhibitors, these chemical studies have led to compounds that are also exquisitely specific to CYP1B1 and CYP1A2. In conclusion, the body of data generated in this thesis has created the basic foundations for further studies that could eventually lead to entities with strong anticancer potential. Page | 4 Acknowledgment I take this opportunity to express my appreciation to all those people who have been helpful in the successful completion of my research. I hereby take pleasure in expressing my sincere thanks and deep sense of gratitude to Prof. Bob Chaudhuri for sharing his knowledge and providing continuous support. This research would not have materialised without his direction and encouragement. I would also like to thank him for having faith in me and recognising my talent for the scholarship. I am extremely grateful to Dr. Neill Horley for his continuous support and guidance throughout my research. I would like to express the deepest appreciation to Glen McCann for his technical assistance in my experimental requirements. I would like to thank Dr. Ken Beresford and Dr. Ketan Ruparelia for the guidance in compound synthesis and the chemistry aspects of my research. Page | 5 fÑxv|tÄ g{tÇ~á gÉ f{Ü| fãtÅ| ftÅtÜà{ `ç `ÉÅ fÅÜâà| ^twâá~tÜ? Wtw f{Ü|Ñtw ^twâá~tÜ? f|áàxÜ f{Üxçt ^twâá~tÜ 9 UÜÉà{xÜ ftÜà{ ^twâá~tÜ Page | 6 Abbreviations AhR Aryl hydrocarbon receptor AP-1 Activated protein ADH2 Alcohol dehydrogenase II Arnt Aryl hydrocarbon nuclear translocator BSA Bovine Serum Albumin cDNA Complementary deoxyribonucleic acid CEC 3-cyano-7-ethoxycoumarin CHC 3-cyano-7-hydroxycoumarin CO Carbon Monoxide CPR NADPH-P450 reductase CYP Cytochrome P450 DBF Dibenzylfluorescein DMSO Dimethyl sulfoxide DMU De Montfort University DTT Dithiothreitol E. coli Escherichia coli Page | 7 EDTA Ethylene diamine tetra acetic acid EOMCC 7-ethoxymethyl-3-cyanocoumarin ER Endoplasmic reticulum 7-ER 7-ethoxyresorufin eq Equivalent G6P Glucose-6-phosphate G6DPH Glucose-6-phosphate dehydrogenase H1NMR Hydrogen Nuclear Magnetic resonance hRD Human NADPH-P450 reductase ∆hRDM Truncated human NADPH-P450 reductase IC50 The half maximal inhibitor concentration kDa kilo Dalton Kpi Potassium phosphate buffer KH2PO4 Potassium phosphate monobasic K2HPO4 Potassium phosphate dibasic LB Luria-Bertani LiOH Lithium hydroxide Page | 8 NaOH Sodium hydroxide NC-IUB Nomenclature committee of the International Union of Biochemistry NMR Nuclear Magnetic resonance nM Nanomole nm Nanometre MW Molecular Weight mg Milligram µg Microgram µl Microliter µM Micromole MTT 3-(4,5-dimethlylthiazol-2-yl)-2,5-diphenyltetrazolium bromide OD Optical density PAH Poly aromatic hydrocarbon PBS Phosphate buffered saline SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel Page | 9 electrophoresis TAE Tris/EDTA/Glacial acetic acid buffer TE Tris-HCL/EDTA TLC Thin layer column chromatography UV Ultraviolet yRD Yeast NADPH-P450 reductase Page | 10 Contents Abstract ......................................................................................................... 2 Acknowledgment .......................................................................................... 5 Abbreviations ................................................................................................ 7 Contents ....................................................................................................... 11 Chapter 1 Introduction ................................................................................ 23 1.1 Biotransformation of xenobiotics ............................................... 23 1.2 Phase I and II of Biotransformation ............................................ 23 1.3 Cancer and Carcinogenesis .......................................................... 24 1.4 What are Cytochrome P450s and their role? ............................... 25 1.5 NC-IUB and Cytochrome P450 .................................................. 26 1.6 NADPH-Cytochrome P450 reductase (CPR) ............................. 28 1.7 Cytochrome b5 ............................................................................ 29 1.8 Biochemistry of Cytochrome P450 enzymes .............................. 30 1.9 CYP450 role in medical therapeutics in human ......................... 31 1.10 CYP1 family ............................................................................... 37 1.11 CYP1A1 ...................................................................................... 39 1.12 Induction of CYP1A1 mediated by the AhR receptor ................ 41 1.13 Flavonoids ................................................................................... 43 1.14 Chalcones .................................................................................... 45 1.15 Stilbenes ...................................................................................... 48 1.16 Reactive oxygen species ............................................................. 50 Page | 11 1.17 cDNA expression of CYP450s ..................................................... 52 1.18 Prokaryotic expression system ....................................................... 53 1.18.1 Bacterial expression system ................................................... 53 1.19 Eukaryotic expression system ........................................................ 54 1.19.1 Yeast expression system ........................................................ 54 1.19.2 Insect expression

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