Drug Research Program Division of Pharmaceutical Chemistry and Technology Faculty of Pharmacy University of Helsinki Finland Julius Sipilä Sulfonation and methylation in silico - modelling studies on SULT1A3 and COMT Academic Dissertation To be presented for public discussion, with the permission of the Faculty of Pharmacy of the University of Helsinki, in Auditorium Porthania PII, on May 25th, 2020, at 5 PM Helsinki 2020 Supervisors Professor Jyrki Taskinen D.Sc. (Tech.) 2003-2006 Division of Pharmaceutical Chemistry Faculty of Pharmacy University of Helsinki Professor Jari Yli-Kauhaluoma PhD Division of Pharmaceutical Chemistry and Technology Faculty of Pharmacy University of Helsinki Reviewers Docent Maija Lahtela-Kakkonen School of Pharmacy University of Eastern Finland UEF Finland Professor Olli Pentikäinen Institute of Biomedicine University of Turku UTU Finland Opponent Professor Gerhard Wolber PhD Department of Biology, Chemistry and Pharmacy Institute of Pharmacy Freie Universität Berlin Germany ISBN 978-951-51-6064-5 (paperback) ISBN 978-951-51-6065-2 (PDF) Helsinki 2020 Abstract Phenolic compounds are ubiquitously encountered in all living organisms. Due to the versatile chemical properties of phenols, they can form various types of specific interactions with biological macromolecules and participate in different types of reactions. In humans, endogenous phenols act as important neurotransmitters, hormones, and building blocks of proteins. A multitude of potentially bioactive phenols is ingested from different sources every day. To modulate the activities of endogenous and xenobiotic phenols, several families of Phase II metabolic enzymes have evolved, which can eliminate phenolic compounds through conjugation. In humans, the most important Phase II enzymes for phenol metabolism are UDP-glucuronosyltransferases (UGTs), cytosolic sulfotransferases (SULTs) and catechol O-methyltransferase (COMT). These enzymes increase the solubility of phenolic substrates, making them less active and easier to excrete. Because many clinically applied drugs also possess phenolic functionalities, UGTs, SULTs, and COMT are potentially important for the pharmacokinetics, exposure, and efficacy of therapeutics. In this thesis, the substrate specificity of human SULT1A3 and COMT were studied computationally, using comparative molecular field analysis (CoMFA), which is a widely used 3-dimensional (3D) quantitative structure-activity relationship (QSAR) method, based on the molecular interaction fields of known substrate molecules and their activities. The CoMFA fields describe the shape, size, and electrostatic properties of the substrates, which are the most important determinants of molecular recognition by enzymes. In our CoMFA models, variations in the substrate fields were statistically correlated with changes in enzyme kinetic parameters such as the Michaelis-Menten constant (Km) or maximum velocity (Vmax), allowing the structural elements that are most important for activity to be extracted. The derived CoMFA models can be used to assess the probability that a new and unknown phenolic drug candidate may be a substrate of SULT1A3 or COMT. In the SULT1A3 models, we found a clear preference for structural elements typically found in catecholamines, in agreement with the results of site-directed mutagenesis studies, X-ray crystal structures and molecular dynamics (MD) simulations that have been published after our studies. When describing the electrostatic effects in the CoMFA models for COMT, semi-empirical atomic partial charges were preferred over empirically parametrized charges. In addition, the acid dissociation constant (pKa)value of the reacting catecholic hydroxyl was a critical factor that affected the affinities of the ligands. Replacing the electrostatic field in the CoMFA model with the predicted pKa value facilitated the development of less acidic COMT ligands that are capable of central nervous system (CNS) permeation. To improve the prediction of pKa values for certain privileged COMT ligand scaffolds, we developed a modified Hammett equation, by synthesizing a series of p-vinylphenols and measuring their pKa values. As increasing amounts of X-ray structural information, covering whole protein families, have become publicly available, we have attempted to extract relevant knowledge from the binding sites of several related proteins, to inspire ligand design. We created an automated data processing workflow, designed to process, combine, and analyze the electrostatic and knowledge-based contact preference fields of aligned binding sites. This analysis was performed and validated using the nuclear receptor (NR) family of proteins and was later tested for the prediction of SULT isoenzyme substrate affinities. The field- based protein binding pocket analysis has proven to be a useful tool, however, the intrinsic flexibility that has been observed among the cytosolic SULT family can be challenging to model without considering the dynamics of the system. In summary, the computational models that were developed in this study could be used in combination with other in silico approaches, especially MD simulations, to provide a better picture of the probable enzymes that may be relevant for the metabolism of a new phenolic drug or active metabolite. These models could be also used to design compounds with an improved affinity towards the studied enzymes, which may be clinically interesting due to the important roles played by SULT1A3 and COMT in the catecholamine-mediated neurotransmission pathways. Acknowledgements The research presented in this thesis was performed at the current Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki and at the Research and Development unit of Orion Pharma, Espoo. Computers and chemistry, especially medicinal chemistry, have always fascinated me. My deepest gratitude belongs to the late Professor Jyrki Taskinen, who agreed to supervise my Master’s thesis and, later, my doctoral studies, which combined these two topics. These studies have represented a turning point in my professional life, after which I knew what I really wanted to do. I am still following that path, with passion. Professor Taskinen told me that the best thoughts usually do not appear when sitting in front of the computer in your office; on the contrary, they appear outdoors while doing something enjoyable. Even today, when I am puzzled by a difficult problem, I remember and follow this great advice. I cannot express enough nice words to describe my respect for the other supervisor of my thesis, Professor Jari Yli-Kauhaluoma. Without your patience, responsiveness, and support, this study would have been very difficult for me to finalize. Sincerely, I want to thank Docent Maija Lahtela-Kakkonen and Professor Olli Pentikäinen for reviewing this thesis and improving it, through their constructive comments and suggestions. I want to thank all of my former and current colleagues and co-authors at the Faculty of Pharmacy and Orion R&D, especially Docent Martti Ovaska, for all their support and guidance. During the years, I have really enjoyed the discussions with my colleagues Mika Kurkela, Olli Aitio, Gerd Wohlfahrt, Lars-Olof Pietilä, Peteris Prusis, Heikki Käsnänen, and Tuomo Kalliokoski. I would like to thank my supervisors at Orion R&D, Leena Otsomaa, for giving me a great example to follow, and Christer Nordstedt, for all the inspiring scientific discussions and, of course, for making it crystal clear that getting a PhD is a very wise thing to do. I want to thank the Finnish Cultural Foundation, the Sigrid Juselius Foundation, and the Graduate School in Pharmaceutical Research, for funding. I want to express my greatest gratitude to my parents, for all their support during the years of my studies. My kids Astrid, Alvar and Viktor, I truly thank you all for the patience, and for constantly reminding me about the most important stuff in life. Finally, I want to thank you, Anna, for helping me with this project and for everything else. Julius Sipilä February 2020 Espoo, Finland Contents Abstract Acknowledgements Contents List of publications ..............................................................................................11 Abbreviations.......................................................................................................13 1 Introduction....................................................................................................15 2 Review of the literature .................................................................................19 2.1 Relevance of phenolic compounds in biology ........................................19 2.2 Phase II enzymes in the metabolism of phenolic compounds.................19 2.3 Glucuronidation by UGTs.......................................................................22 2.4 Methylation by COMT............................................................................23 2.4.1 Clinical relevance of COMT.......................................................23 2.4.2 The human COMT gene, protein expression and localization....25 2.4.3 Structure and function of COMT................................................26 2.5 Sulfonation by SULTs.............................................................................28 2.5.1 SULT isoforms............................................................................29 2.5.2 Clinical relevance of SULTs.......................................................29 2.5.3 Structure