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Studies on Bilirubin and Steroid Glucuronidation

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Studies on Bilirubin and Steroid Glucuronidation Centre for Drug research Division of Pharmaceutical Chemistry Faculty of Pharmacy University of Helsinki Finland UDP-GLUCURONOSYLTRANSFERASES IN SICKNESS AND HEALTH STUDIES ON BILIRUBIN AND STEROID GLUCURONIDATION Nina Sneitz ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki, for public examination in Auditorium 1, Korona Information Centre, on 25 October 2013, at 12 noon. Helsinki 2013 Supervisor: Dr. Moshe Finel Centre for Drug Research (CDR) Faculty of Pharmacy University of Helsinki Finland Reviewers: Professor Peter Mackenzie Department of Clinical Pharmacology School of Medicine Flinders University Australia Professor Hannu Raunio Faculty of Health Sciences School of Pharmacy University of Eastern Finland Finland Opponent: Professor Philip Lazarus Department of Pharmaceutical Sciences College of Pharmacy Washington State University College of Pharmacy USA © Nina Sneitz 2013 ISBN 978-952-10-9292-3 (Paperback) ISBN 978-952-10-9293-0 (PDF) ISSN 1799-7372 http://ethesis.helsinki.fi/ Helsinki University Printing House Helsinki 2013 CONTENTS Abstract 5 Acknowledgements 7 List of original publications 8 Abbreviations 9 1 Introduction 10 2 Review of the Literature 12 2.1 UDP-glucuronosyltransferases in brief 12 2.2 Bilirubin metabolism and Familial hyperbilirubinemias 15 2.2.1 Bilirubin: properties and metabolism 15 2.2.2 Gilbert syndrome 17 2.2.3 Crigler-Najjar syndromes 18 2.2.3.1 Current therapy for Crigler-Najjar syndrome 20 2.2.3.2 Gene therapy in treatment of Crigler-Najjar syndrome 20 2.3 Steroid hormones and their metabolism 22 2.3.1 Glucuronidation of steroid hormones 24 2.3.2 Steroids and health 26 2.4 Steroid glucuronidation and UGT structure 28 3 Aims of the study 30 4 Materials and Methods 31 4.1 Materials 31 4.1.1. Chemicals 31 4.1.2. Microsomes and RNA sources 33 4.2 Patients 34 4.3. Animal experiments 35 4.4 Methods 36 4.4.1 Molecular biology 36 4.4.1.1 Cloning 36 4.4.1.2. UGT expression in Sf9 cells 36 4.4.1.3. UGT quantification and visualisation 36 4.4.2 Glucuronidation assays 37 4.4.3 Analytical methods and data analysis 37 4.4.4 Molecular modeling 39 5 Results and discussion 40 5.1. UGT1A1 and Crigler-Najjar syndrome 40 5.1.1 UGT1A1 mutations in CN patients in the Netherlands 40 5.1.2 Bilirubin glucuronidating activity of ten UGT1A1 missense mutants 42 3 5.1.3 Activity of UGT1A1 missense mutants toward other substrates 43 5.1.4 Biomarker for efficacy of gene therapy for Crigler-Najjar syndrome 44 5.2 Expression and characterisation of UGT2A2 47 5.3 Glucuronidation of selected steroids 50 5.3.1. Glucuronidation of estradiol, ethinylestradiol and estriol by UGT2A1 and UGT2A2 50 5.3.2. Estrogen glucuronidation and UGT1A10 substrate binding site 50 5.3.3. Glucuronidation of estriol and estradiol stereoisomers and enantiomeric steroids 52 6 Summary and Conclusions 59 References 61 Appendix: Original publications I-VI 75 4 ABSTRACT UDP-glucuronosyltransferase (UGT) enzymes are responsible for the metabolism of many xenobiotics, as well as several endogenous compounds like bilirubin and certain steroids. This thesis involves three major themes: UGT1A1 and Crigler-Najjar syndrome; expression and characterisation of a novel UGT, UGT2A2; and the glucuronidation of various endogenous and synthetic steroids. Fourteen different UGT1A1 mutations were identified in 19 CN patients, four of which were novel. The mutant proteins were expressed in baculovirus infected Sf9 insect cells and their activity towards bilirubin and other substrates was determined. Residual glucuronidation activity and the severity of hyperbilirubinemia were found to correlate well in most cases. In addition, a major advancement to aid the development of gene therapy for CN was achieved by in discovering that ezetimibe glucuronidation correlates with reduction of serum bilirubin in gene therapy treated Gunn rats. This compound might be useful in examining the efficacy of gene therapy in humans in the future. When the novel UGT2A2 was cloned, expressed, and studied alongside UGTs 2A1 and 2A3, results indicated that UGT2A2 is a functional enzyme with broad substrate specificity. The previously suggested exon sharing between UGT2A1 and UGT2A2 was also confirmed, and UGT2A2 was found to be expressed mainly in nasal mucosa, similar to UGT2A1. In addition, UGT2A1 and UGT2A2 were found to differ largely in their regioselectivity towards estrogens, and this encouraged us to further investigate the glucuronidation of steroids and the differences in how they are glucuronidated by the different human UGT enzymes. The glucuronidation of estriol, 16-epiestriol, 17-epiestriol, 13-epiestradiol, ent-estradiol, ent-androsterone, and ent-etiocholanolone by different UGT enzymes was studied and analytical methods for analysing these compounds were developed. UGTs 1A10 and 2B7 were found to be the most active UGTs in the glucuronidation of almost every steroid studied. Several patterns in the regio- and stereoselectivity of steroid glucuronidation were observed for the first time. The effects of phenylalanines 93 and 90 on human UGT1A10 on glucuronidation were also studied, exploiting site directed mutagenesis. The results revealed that F93 is involved in the interactions of the enzyme with estrogens, but the interactions with smaller molecules were found to be more complex and less drastic. A homology model of UGT1A10 was created based on the results. Knowledge of glucuronidation of endogenous compounds is important, since it may be directly or indirectly linked to clinical conditions and also because certain drugs that inhibit UGTs may inhibit the metabolic pathways of endogenous substrates that are metabolised by UGTs. Understanding the glucuronidation of steroids could be used to 5 make better predictions on the role of glucuronidation in the metabolism of newly designed drugs that resemble steroids. Detailed information on steroid glucuronidation may also be used in determining UGT structure via function and the results obtained during this PhD work may be used in the development of more detailed homology models of other UGT enzymes. 6 ACKNOWLEDGEMENTS This study was carried out in the years 2008-2013 at the Faculty of Pharmacy, University of Helsinki. Besides the Faculty of Pharmacy, financial support from Magnus Ehrnrooth foundation, Finnish Chemical Society, Graduate School of Pharmaceutical Research, Orion-Farmos Research Foundation and Finnish Cultural Foundation are gratefully acknowledged. My deepest thanks go to my supervisor, docent Moshe Finel who has always given me help and encouragement when I needed it. I am and I will always be inspired by your enthusiasm and dedication to science. I am also deeply grateful to Dr. Piter Bosma and Docent Liisa Laakkonen and all my other co-authors for their help and contribution. I am also sincerely grateful for the pre-examiners, Professor Peter Mackenzie and Professor Hannu Raunio, for all the effort they made in reviewing and improving this thesis. My past and present colleagues at pharmaceutical chemistry deserve my sincerest thanks for the unreserved and friendly atmosphere of the department: Anna, Gusse, Hongbo, Inkku, Johanna T., Katriina, Linda, Mika, Mikko, Nenad, Nina, Niina, Paula, Päivi, Raisa, Sanna S., Taina, Tiina S., Titti, and all others – Thank you! Very special thanks go to my roommate and great friend Johanna M. I have enjoyed working with this PhD project almost every moment of it and much of that I owe to you. You made it fun to come to work even on the rainy days. All my wonderful friends deserve my thanks as well! The most fun I have ever spent has been with you guys. Especially I want to thank Ilkka, Jussi, Janne, Eevi, Jere, Pate, Stefu, Salla, Teemu, Tomppa, Tuomas, and Valtteri: Thank you so much for everything! Special thanks go to Sini, my oldest and dearest friend, for being there no matter what. You are the best! My dear friend Sakari also deserves special thanks: your sense of humour has always been a great relief from weekday routines. My dear parents Ronald and Kaisu have my very special thanks. You brought me up believing in my skills and always supported and encouraged me to study. Thank you so much for being such great parents. And, although I know this is an utter waste, I would like to thank my cat Sissi for always being happy to see me and forcing me to stay off the computer from time to time. Finally, I want to thank my loving husband Antti. You have been by my side during this whole project and I can not imagine a better person for that. I´m eagerly waiting for the next big “project” in our lives when our soon-to-be-born child comes into the world. Helsinki, July 2013 Nina Sneitz 7 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following publications: I Sneitz N, Court MH, Zhang X, Laajanen K, Yee KK, Dalton P, Ding X, Finel M (2009) Human UDP-glucuronosyltransferase UGT2A2: cDNA construction, expression, and functional characterization in comparison with UGT2A1 and UGT2A3. Pharmacogenet Genomics 19:923-934. II Sneitz N, Bakker CT, de Knegt RJ, Halley DJ, Finel M, Bosma PJ (2010) Crigler-Najjar syndrome in The Netherlands: identification of four novel UGT1A1 alleles, genotype-phenotype correlation, and functional analysis of 10 missense mutants. Hum Mutat 31:52-9. III Höglund C1, Sneitz N1, Radominska-Pandya A, Laakkonen L, Finel M (2011) Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens. Steroids 76:1465-73. IV Sneitz N, Krishnan K, Covey DF, Finel M (2011) Glucuronidation of the steroid enantiomers ent-17ȕ-estradiol, ent-androsterone and ent- etiocholanolone by the human UDP-glucuronosyltransferases. J Steroid Biochem Mol Biol 127:282-8. V Montenegro-Miranda1 PS, Sneitz N1, de Waart DR, Ten Bloemendaal L, Duijst S, de Knegt RJ, Beuers U, Finel M, Bosma PJ (2012) Ezetimibe: A biomarker for efficacy of liver directed UGT1A1 gene therapy for inherited hyperbilirubinemia.
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