Assessment of degradation rate constants for quantitative predictions of drug-drug interactions arising from CYP450 drug metabolising enzymes Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Christina Yik See Chan September 2017 1 This thesis is the result of my own work. The material contained within the thesis has not been presented, either wholly or in part, for any other degree of qualification. Christina Chan This research was carried out in the Liverpool HIV Pharmacology Group Department of Molecular and Clinical Pharmacology University of Liverpool UK 2 Table of Contents Acknowledgements 4 Abbreviations 5 Publications and communications 10 Abstract 11 Chapter 1 General Introduction 12 Chapter 2 Utility of single protein synthesis inhibitor agents for measuring 62 protein degradation rate constants; an analysis in hepatic primary cells and tumour cell lines Chapter 3 Utility of multiple protein synthesis inhibitor combinations for 92 measuring protein degradation rate constants Chapter 4 Using mRNA suppression to estimate CYP3A4 degradation 123 rate constant in primary human hepatocytes Chapter 5 Using mRNA suppression to estimate CYP2B6 degradation 173 rate constant in primary human hepatcytes Chapter 6 Exploring genetic polymorphisms in CYP3A4 protein 219 degradation machinery on substrate pharmacokinetics Chapter 7 Final Discussion 241 References 249 3 Acknowledgements The research presented in this thesis was carried out in the Department of Molecular and Clinical Pharmacology, University of Liverpool. I would like to express my gratitude to the MRC and Certara (Simcyp) for providing the funding and opportunity to carry out this project. The making of this thesis has been an incredible journey and I am thankful for everyone I met along the way who have supported and contributed to it. I would like to thank to my supervisors Prof. Andrew Owen, Dr Lisa Almond and Dr Marco Siccardi for their supervision and guidance throughout the project. Prof. Owen directed me throughout the PhD and supervised every aspect of my work - thank you you for your continued patience and for being so supportive during my many moments of ‘panic’. Dr Siccardi, thank you for all your inputs and advice, especially in all things analytical. Dr Almond and Dr Garner offered invaluable advice and interesting scientific discussions during the project. A big thank you to my colleagues (past and present) at the Liverpool HIV Pharmacology group: Owain, Louise, Rajith, Rohan, Chris, Megan, Rana, Paul, Neill, Jay, Beth, Lee, Darren, Justin, Ana, Hannah, Helen, Pedro, Henry, Alessandro, Adeniyi, Laura, Sara and Jo. Your friendship and professional wisdom (mainly imparted at the AJs) has made the last 4 years so much fun! It has been a real priveledge to work with such brilliant minds and in a culture of open knowledge sharing where no question is too silly and everyone’s willing to stop what they’re doing to help you in an instant. The lessons I have learnt here (not just scientifically) will stay with me for life. I would like to thank Rohan, Ky, Katie, Georgie, Izzi, flabbies and Gab for being there for me through the highs and lows and keeping me sane throughout. Finally, this thesis is dedicated to my family: Mum, Dad and Michael. Thank you for your unconditional love and support despite not really knowing what I’ve been up to in the last 4 years. Dad, you always have the words to put everything right again. 4 Abbreviations 3H Tritium µCi Microcurie(s) µg Microgram(s) µl Microliter(s) µM Micromolar ADME Absorption, distribution, metabolism, excretion ADR Adverse drug reaction AhR Aryl hydrocarbon receptor ALD Autophagic-lysosomal degradation AMFR Autocrine motility factor receptor ANOVA Analysis of variance AO Aldehyde oxidase ARNT Aryl hydrocarbon receptor nuclear translocator ATCC American type culture collection ATG Autophagy-related gene ATP Adenosine triphosphate ATV Atazanavir AUC Area under the curve BMI Body mass index BSA Bovine serum albumin CAR Constitutive active receptor CCRP Cytoplasmic CAR retention protein cDNA Complementary deoxyribonucleic acid oC Degree Celsius CHIP C-terminus of Hsc70-interacting protein CHRM Cryopreserved hepatocyte recovery medium CC50 Concentration causing 50% cytotoxicity cm Centimetre(s) Cmax Maximum plasma concentration CNS Central nervous system CO2 Carbon dioxide CPR Cytochrome P450 reductase 5 C(t) Cycle threshold CYP Cytochrome P450 Cyt b5 Cytochrome b5 DDI Drug-drug interaction DEST Aspartate (D), glutamine (E), serine (S), threonine (T) DME Drug metabolising enzyme DMEM Dulbecco’s modified eagle medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid E1 Ub-activating enzyme E2 Ub-conjugating enzyme E3 Ub-protein ligase enzyme EC50 Concentration at 50% efficacy ECL Enhanced chemiluminescence EGFR Epidermal growth factor receptor EMA European Medicines Agency Emax Maximum induction response ER Endoplasmic reticulum ERAD ER-associated degradation ERAD-C ER-associated degradation of cytosolic domain ERAD-L ER-associated degradation of lumenal domain ERAD-M ER-associated degradation of membrane domain ES Enzyme-substrate complex ESS Steady-state enzyme level e- Electron FBS Fetal bovine serum FDA Food and drug administration (US) FIC Fractional inhibitory concentration Fmcyp Fraction metabolised by CYP enzyme FMO Flavin monooxygenase GAPDH Glyceraldehyde 3-phosphate dehydrogenase gp78 Glycoprotein 78 GPSP Global protein stability profiling GSH Glutathione 6 GST Glutathione S-transferase h Hour(s) H+ Hydrogen ion HBSS Hank’s balanced salt solution HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid HPLC High performance liquid chromatography HPLC-UV High performance liquid chromatography-ultraviolet HIV Human immunodeficiency virus HIV-1 Human immunodeficiency virus subtype 1 HPRT1 Hypoxanthine-guanine phosphoribosyltransferase 1 HRP Horseradish peroxidase I Inhibitor / inactivator IL-6 Interleukin-6 Ind Inducer IC50 Concentration of inhibitor resulting in 50% inhibition iTRAQ Isobaric tag for relative and absolute quantification IVIVE In vitro – in vivo extrapolation kDa KiloDalton(s) kdeg Degradation rate constant kg Kilogram(s) KI Inactivation rate constant Kinact Time-dependent inhibition constant Km Michaelis-Menten constant ksyn Synthesis rate constant Kobs Apparent inactivation rate constant LC3 Microtubule-associated protein 1 light chain 3 LIR LC3-interacting region Lys Lysine MBI Mechanism-based inhibition MDI Metabolism-depedent inhibition MDZ Midazolam mg Milligram(s) MgCl2 Magnesium chloride min Minute(s) 7 ml Millilitre(s) mM Millimolar mRNA Messenger RNA MS Mass-spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide n Number of observations ng Nanogram(s) nM Nanomolar nt Nucleotide NADH Reduced nicotinamide adenine dinucleotide NADPH Reduced nicotinamide adenine dinucleotide phosphate NAT N-acetyltransferase NBR1 Neighbour of BRCA1 gene1 NFDM Non-fat dried milk NME New molecular entity NNRTI Non-nucleoside reverse transcriptase inhibitor NQO1 NADPH quinone oxidoreductase 1 nt Nucleotide NTC Non-targeting control O2 Molecular oxygen PB Phenobarbital PBPK Physiologically-based pharmacokinetic PBREM Phenobarbital-responsive enhancer module PCR Polymerase chain reaction PD Pharmacodynamic pH -Log10 hydrogen ion concentration PI Protease inhibitor PK Pharmacokinetic PKA Protein kinase A PKC Protein kinase C PRH Primary rat hepatocytes PXR Pregnane X receptor QC Quality control qPCR Real-time polymerase chain reaction 8 R2 Correlation coefficient RIF Rifampicin RIS Relative induction score RISC RNA-induced silencing complex RNA Ribonucleic acid RNAi RNA interference ROS Reactive oxygen species RT Room temperature RT-PCR Reverse-transcription polymerase chain reaction RTV Ritonavir s Second(s) S Substrate SD Standard deviation siRNA Small-interfering ribonucleic acid SILAC Stable isotope labelling by amino acid SNP Single nucleotide polymorphism SQSTM1 Sequestosome-1 SULT Sulfotransferase t1/2 Half-life TBT Thiazolyl blue tetrazolium TDI Time-dependent inhibition TPMT Thiopurine S-methyltransferase T-TBS Tween-tris buffered saline Ub Ubiquitin UBC7 Ubiquitin-conjugating enzyme E2 7 UBE2G1 Ubiquitin-conjugating enzyme E2 G1 UGT UDP-glucuronosyltransferase UPD Ubiquitin-proteasome degradation V Volt v/v Volume per volume VCP Valosin-containing protein Vmax Maximal rate of enzyme reaction XRE Xenobiotic response element XREM Xenobiotic response enhancer module 9 Publications Chan, C., Roberts, O., Rajoli, R., Siccardi, M., Almond, L. and Owen, A. (2017) Derivation of CYP3A4 and CYP2B6 degradation rate constants in primary human hepatocytes: A siRNA-silencing-based approach. Drug Metabolism & Pharmacokinetics. In production. Chan, C., Martin, P., Liptrott, N. J., Siccardi, M., Almond, L. and Owen, A. (2017) Incompatibility of chemical protein synthesis inhibitors with accurate measurement of extended protein degradation rates. Pharmacology Research & Perspectives. DOI: 10.1002/prp2.359. Communications Chan, C. Y., Liptrott, N. J., Martin, P., Siccardi, M., Almond, L. and Owen, A. The lack of utility of pharmacological interference for the study of protein degradation. Presented at the 13th European meeting for International Society for the Study of Xenobiotics. Glasgow, UK, June 2015. Chan, C. Y., Roberts, O., Hassan, N., Liptrott, N. J., Siccardi, M., Almond, L. and Owen, A. Use of mRNA suppression to estimate CYP3A4 protein degradation rate constant in primary human hepatocytes. Drug Metabolism