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Characterisation of Equine Cytochrome P450s Catherine Orr

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Characterisation of Equine Cytochrome P450s Catherine Orr, BSc, MRes Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy October 2015 Abstract Cytochrome P450s (CYPs) are a superfamily of enzymes involved in the phase I metabolism of endogenous and exogenous substances. They are present in almost all forms of life and have been studied extensively, particularly in relation to human medicine, where knowledge of their activities is essential for predicting drug-drug interactions. In the horse, little is currently known about CYP-specific drug metabolism, which holds importance for animal welfare and for doping control within the horseracing industry where drug-specific metabolites are tested for on race days. Recently the first recombinant equine CYPs have been produced, allowing specific data on equine P450 activity to be gathered for the first time. During the current study,46 full-length P450 sequences were identified from the equine genome. RT- PCR analysis was then carried out on equine liver in order to detect hepatic expression of P450s across various families. After this, cold-induction (pCold) E. coli were used for production of recombinant P450 proteins for subsquent functional testing. Four recombinant equine P450s were successfully expressed (CYP1A1, CYP2A13, CYP2C92 and CYP2D50). Due to being the isoforms most likely to be involved in drug metabolism, rCYP2D50 and rCYP2C92 were selected to be screened against ten of the most commonly used horse drugs to identify potential substrates. rCYP2C92 appeared to metabolise all four NSAIDs tested (flunixin, ketoprofen, phenylbutazone and diclofenac), however presence of the known hydroxylated metabolites of diclofenac and phenylbutazone (4-hydroxydiclofenac and oxyphenbutazone, respectively) could not be confirmed despite being present within equine liver microsome and human recombinant CYP2C9 samples. In spite of the apparant acivity displayed by rCYP2C92 towards all four NSAIDs, no 1 conclussions can be made about this enzyme’s role in NSAID metabolism due to a lack of known hydroxylated metabolite production. 2 Acknowledgements I would like to thank everyone whose help and support made this thesis possible. Firstly, my supervisors, Stuart and Lisa, who made this fascinating and diverse project come to life and allowed me to gain such a wide range of knowledge and skills. Without your guidance and encouragement throughout I would not have made it through the challenges that doing a PhD brings! I would also like to thank the British Horseracing Authority (BHA) for providing funding for the project and the Veterinary school for providing the labs I spent most of my time in. Additionally I have to say thank you to Freya and Kamila, without whom I would have struggled to get past the many problems I had during the project! I am also grateful to Susan in South lab, Rob in food sciences and Michael at the QMC for collaborating and allowing me to access the resources I needed to get some of my most essential results. I also have to thank Richard Emes, who got me started on the project by providing support for the equine genome analysis. Finally, a big thank you to my family for giving me the financial help to relocate to Nottingham in the first place and being there for me throughout these past four years – you may not fully know what this thesis is about but I know you are as happy and proud as I am that it is finally over! 3 Contents Abstract ................................................................................................................................................... 1 Acknowledgements ................................................................................................................................. 3 Chapter 1: Introduction .......................................................................................................................... 9 1.1: Overview ...................................................................................................................................... 9 1.2: The liver ..................................................................................................................................... 10 1.2.1: Liver architecture ................................................................................................................ 10 1.3: Xenobiotic metabolism .............................................................................................................. 13 1.4: Cytochrome P450s ..................................................................................................................... 15 1.4.1: Mechanism of CYP-based metabolism ............................................................................... 16 1.5: Other phase I metabolism enzymes .......................................................................................... 17 1.5.1: Flavin monoxygenase .......................................................................................................... 18 1.5.2: Alcohol dehydrogenases ..................................................................................................... 18 1.5.3: Aldehyde dehydrogenase ................................................................................................... 19 1.5.4: Monoamine oxidases .......................................................................................................... 19 1.5.5: Xanthine oxidase ................................................................................................................. 20 1.6: Phase II metabolism (conjugation) ............................................................................................ 20 1.7: Evolution of the P450 superfamily ............................................................................................. 21 1.8: P450 structure, function and conservation ............................................................................... 23 1.9: P450 nomenclature convention................................................................................................. 26 1.10: Expression and tissue distribution of P450 enzymes ............................................................... 26 1.11: Human P450s ........................................................................................................................... 29 1.11.1: The CYP1 family................................................................................................................. 29 1.11.2: The CYP2 family................................................................................................................. 33 1.11.3: The CYP3 family................................................................................................................. 38 1.13: CYPs in non-mammals.............................................................................................................. 41 1.14: CYPs in mammals ..................................................................................................................... 41 1.15: Equine drug metabolism .......................................................................................................... 42 1.15.1: Equine P450s ..................................................................................................................... 44 1.15.2: Equine P450 expression levels .......................................................................................... 45 1.15.3: In vivo equine studies ....................................................................................................... 46 1.15.4: Recombinant equine P450s .............................................................................................. 46 1.16: Methods used to study hepatic metabolism ........................................................................... 48 1.17: Rational for current study ........................................................................................................ 51 4 1.17.1: Aims of study .................................................................................................................... 51 Chapter 2: Materials and methods ....................................................................................................... 52 2.1: Bioinformatics analysis .............................................................................................................. 52 2.11: Alignments of known horse and human sequences ............................................................ 52 2.12: Searching of equine genome ............................................................................................... 52 2.13: Searching for automatically annotated equine sequences ................................................. 53 2.14: Generation of phylogenetic trees ........................................................................................ 54 2.15: Identification of conserved haem-binding motif ................................................................. 54 2.2: Cloning, sequencing and protein structure predictions ............................................................ 54 2.21: Isolation of RNA from equine liver ....................................................................................... 54 2.22: Synthesis of cDNA ................................................................................................................ 55 2.23: RT-PCR analysis ...................................................................................................................
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