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Searching for Inhibitors of the Protein Arginine Methyl Transferases: Synthesis and Characterisation of Peptidomimetic Ligands

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Searching for Inhibitors of the Protein Arginine Methyl Transferases: Synthesis and Characterisation of Peptidomimetic Ligands SEARCHING FOR INHIBITORS OF THE PROTEIN ARGININE METHYL TRANSFERASES: SYNTHESIS AND CHARACTERISATION OF PEPTIDOMIMETIC LIGANDS by ASTRID KNUHTSEN B. Sc., Aarhus University, 2009 M. Sc., Aarhus University, 2012 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2016 © Astrid Knuhtsen, 2016 UNIVERSITY OF COPENH AGEN FACULTY OF HEALTH A ND MEDICAL SCIENCES PhD Thesis Astrid Knuhtsen Searching for Inhibitors of the Protein Arginine Methyl Transferases: Synthesis and Characterisation of Peptidomimetic Ligands December 2015 This thesis has been submitted to the Graduate School of The Faculty of Health and Medical Sciences, University of Copenhagen ii Thesis submission: 18th of December 2015 PhD defense: 11th of March 2016 Astrid Knuhtsen Department of Drug Design and Pharmacology Faculty of Health and Medical Sciences University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Denmark and Faculty of Pharmaceutical Sciences University of British Columbia 2405 Wesbrook Mall BC V6T 1Z3, Vancouver Canada Supervisors: Principal Supervisor: Associate Professor Jesper Langgaard Kristensen Department of Drug Design and Pharmacology, University of Copenhagen, Denmark Co-Supervisor: Associate Professor Daniel Sejer Pedersen Department of Drug Design and Pharmacology, University of Copenhagen, Denmark Co-Supervisor: Assistant Professor Adam Frankel Faculty of Pharmaceutical Sciences, University of British Columbia, Canada Assessment Committee: Chairperson: Assistant Professor Anders Bach Department of Drug Design and Pharmacology, University of Copenhagen, Denmark Principal Scientist Christian Wenzel Tornøe, PhD Novo Nordisk A/S, Denmark Associate Professor Louis Lefebvre Department of Medical Genetics, University of British Columbia, Canada iii Abstract Within the last two decades research in the field of epigenetics has increased significantly as targeting the epigenetic enzymes has the potential to alter the transcription of genes. Aberrant regulation of transcription is seen in several disease states, and drugs targeting the epigenetic histone deacetylases and DNA methylases are already marketed for cancer treatment. The Protein Arginine Methyl Transferases (PRMTs) belong to an epigenetic enzyme family that is upregulated in several cancers. However, currently no inhibitors of the PRMTs have been marketed. In this thesis several peptidomimetic strategies were utilised to modify the tryptophan residues in two peptide leads in order to discover new inhibitors of the PRMTs. One of these strategies involved constraining the side chain indole of tryptophan to the peptide backbone, thus producing a seven- membered azepinone mimetic, Aia. The peptidomimetic efforts resulted in a structure-activity relationship study from which a constrained peptidomimetic containing two Aias was discovered to be a low micromolar inhibitor of several PRMTs. To characterise the inhibitor the conformation of the inhibitor was examined using solution-phase NMR and was shown to display an interesting turn-structure. The original peptide lead was fluorescently tagged and investigated in a cellular setting, but did not reveal any PRMT-specific localisation. In an effort to study the binding of the discovered inhibitor with the PRMTs, protein expression in E. coli and purification was performed. This resulted in the optimisation of PRMT6 purification in order to obtain highly pure PRMT6 for isothermal titration calorimetry (ITC) studies. Unfortunately these ITC studies were unsuccessful. Furthermore, as the constrained tryptophan mimetic had proven very useful in the peptidomimetic inhibitors of the PRMTs, we attempted to synthesise a lysine/arginine dipeptide mimetic using aziridine chemistry. iv Abstract in Danish (Dansk Resumé) Forskningen indenfor epigenetik er i de seneste to årtier øget betragteligt idet stoffer rettet imod modulering af effekten af epigenetiske enzymer har vist potentiale til at ændre i gentranskriptionen. Abnormal regulering af gentranskription ses i adskillige sygdomme, og lægemidler målrettet som hæmmere af de epigenetiske histon deacetylaser og DNA methylaser er allerede markedsført til kræftbehandling. Protein Arginin Methyl Transferaserne (PRMTerne) er en epigenetisk enzymfamilie der er overudtrykt i forskellige kræftformer. På trods af dette findes der dog ikke på nuværende tidspunkt markedsførte hæmmere af PRMTerne. I denne afhandling blev flere strategier benyttet til at lave peptid-lignende stoffer ved at modificere tryptophanerne i to basis peptider med det formål at finde nye hæmmere af PRMTerne. En af disse strategier involverede inkorporation af en ufleksibel tryptophan-lignende byggeblok, den 7-ledede azepinon, Aia, som har C2 i sidekæde indolen fra tryptophan bundet til peptidets rygrad. Disse peptid-modificerende strategier resulterede i et studie af struktur-aktivitet-forholdet for stoffernes effekt på PRMTerne. Således blev en rigid peptid-lignende forbindelse med to Aia’er identificeret som en lav mikromolær hæmmer af flere af PRMTerne. NMR blev brugt til at karakterisere strukturen af hæmmeren i opløsning, som viste sig at have en interessant drejnings-konformation. Det oprindelige peptid blev desuden mærket med en fluorofor og undersøgt i en cellekultur, men afslørede ikke nogen genkendelig PRMT-specifik lokalisering i cellerne. I et forsøg på at studere bindingen af den opdagede PRMT-hæmmer blev PRMT proteiner udtrykt i E. coli og oprenset. Dette resulterede i optimering af PRMT6 oprensningen med henblik på at producere yderst rent PRMT6 protein til brug i isotermal titrationskalorimetriske (ITC) studier. Disse ITC studier endte desværre uden resultat. Idet den ufleksible byggeblok Aia havde vist sig brugbar i udviklingen af de peptid-modificerede hæmmere af PRMTerne søgte vi ydermere at syntetisere en lysin/arginin dipeptid forbindelse ved hjælp af aziridin- kemi. v Preface This PhD thesis discloses the majority of work performed during my joint PhD studies from December 2012 to December 2015 at the Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen (UCPH), Denmark and at the Department of Pharmaceutical Sciences, University of British Columbia (UBC), Canada. During the time spent at UCPH the project was supervised by Assoc. Professor Jesper L. Kristensen (main supervisor) and Daniel Sejer Pedersen (co-supervisor). At UBC the project was supervised by Assistant Professor Adam Frankel (co-supervisor). Chapters 1 and 2 of the thesis provide a background to the project; Chapter 1 deals with epigenetics and the Protein Arginine Methyl Transferases (PRMTs) which were the targets for the medicinal chemistry efforts of the project and chapter 2 provides an overview of the use of peptides in drug discovery. Chapter 3 outlines the objectives of the project. Chapters 4 and 5 comprise the work performed at UCPH; Chapter 4 deals with the synthesis of peptides and peptidomimetics as inhibitors of the PRMTs and chapter 5 is concerned with synthetic strategies using aziridines towards dipeptide mimetic tool compounds for use in the synthesis of peptidomimetics. A manuscript on the work in chapter 4 concerning the peptidomimetic inhibitors may be found in Appendix 1. This manuscript will be submitted as soon as possible. Chapter 6 gives an account of the work with protein expression of the PRMTs and isothermal titration calorimetry (ITC) experiments which was performed at UBC. Chapter 7 provides a conclusion and perspectives to the PhD project. In addition, a review is under preparation in collaboration with researchers at Vrije Universiteit Brussel. The review is based on case studies involving constrained amino acid building blocks, such as the one used in the peptidomimetic inhibitors in chapter 4, and their use in biologically relevant peptidomimetic applications. The case studies are not included as an integral part of the thesis but may be found in Appendix 2. This dissertation is formatted in accordance with the regulations of the University of Copenhagen and submitted in partial fulfillment of the requirements for a PhD degree awarded jointly by the University of Copenhagen and the University of British Columbia. Versions of this dissertation will exist in the institutional repositories of both institutions. vi Acknowledgements The past three years have been such a journey; full of ups and downs, good and less good ideas, scientific creativity as well as hard work. It has been a fantastic experience to be involved in a multifaceted project and I have learnt much more than I ever expected to. To this end I want to express my deepest gratitude to my supervisors Jesper L. Kristensen and Daniel Sejer Pedersen for the endless scientific as well as moral support, and for cheering me on from the very first day. It has been an honour to have two such insightful and knowledgeable medicinal chemists as supervisors to challenge me, push me forward and help me develop into a proper scientist. I have truly loved to work at ILF, to interact with so many great, competent people and of course to have been a part of the Sejer and Kristensen groups. Over the years we have drunk countless cups of “coffee”, shared scientific endeavours and hardships, devoured copious amounts of delicacies and bake goods from around the world, listened to some great music as well as other sounds within the same classification,
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