Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki Finland The DAG-responsive C1 domain as a drug target: structure- activity and in vitro pharmacology of isophthalate derivatives Virpi Talman ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Pharmacy, University of Helsinki, for public examination in auditorium 1041, Viikki Biocenter 2, on November 22nd 2013, at 12 noon. Supervisors Professor Raimo K. Tuominen Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki Finland Dr Elina Ekokoski Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki Finland Current address: Finnish Safety and Chemicals Agency Helsinki, Finland Reviewers Professor Jyrki Kukkonen Department of Veterinary Biosciences Faculty of Veterinary Medicine University of Helsinki Finland Adj. professor Päivi Koskinen Department of Biology University of Turku Finland Opponent Professor Kid Törnquist Department of Biosciences/Cell Biology Åbo Akademi University Finland © Virpi Talman ISBN 978-952-10-9408-8 (paperback) ISBN 978-952-10-9409-5 (PDF) ISSN 1799-7372 Unigrafia - Helsinki University Print Helsinki 2013 “Research is the process of going up alleys to see if they are blind.” - Marston Bates - CONTENTS 1. INTRODUCTION .......................................................................................................................................... 1 2. REVIEW OF THE LITERATURE ...................................................................................................................... 3 2.1 DIACYLGLYCEROL (DAG) SIGNALLING ......................................................................................................... 3 2.2 PROTEIN KINASE C (PKC), THE PRIMARY DAG EFFECTOR............................................................................ 4 2.2.1 PKC family members and domain structure ........................................................................................ 4 2.2.2 Structure and function of PKC C1 domains .......................................................................................... 5 2.2.3 Regulation of PKC activity .................................................................................................................... 7 2.2.4 PKC-mediated signal transduction ....................................................................................................... 9 2.3 OTHER DAG EFFECTORS ............................................................................................................................ 11 2.3.1 Protein kinase D (PKD) ....................................................................................................................... 11 2.3.2 Diacylglycerol kinases (DGKs) ............................................................................................................ 13 2.3.3 Myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) ................................................ 14 2.3.4 Munc13 proteins ................................................................................................................................ 15 2.3.5 Chimaerins ......................................................................................................................................... 17 2.3.6 Ras guanyl-releasing proteins (RasGRPs) .......................................................................................... 18 2.4 POTENTIAL THERAPEUTIC INDICATIONS FOR DAG EFFECTOR-TARGETED DRUGS ................................... 18 2.4.1 Cancer ................................................................................................................................................ 19 2.4.2 Alzheimer’s disease (AD).................................................................................................................... 21 2.4.3 Other therapeutic indications ............................................................................................................ 23 2.5 C1 DOMAIN LIGANDS ................................................................................................................................ 25 2.5.1 Phorbol esters and phorbol derivatives ............................................................................................. 25 2.5.2 Bryostatins and their synthetic analogues ........................................................................................ 26 2.5.2 Ingenol derivatives ............................................................................................................................. 27 2.5.3 DAG lactones ..................................................................................................................................... 28 2.5.4 Indolactams and benzolactams ......................................................................................................... 28 2.5.5 Other C1 domain ligands ................................................................................................................... 29 3. AIMS OF THE STUDY ................................................................................................................................. 30 4. MATERIALS AND METHODS ...................................................................................................................... 31 4.1 DRUGS ....................................................................................................................................................... 31 4.2 PRODUCTION AND PURIFICATION OF RECOMBINANT PROTEINS ............................................................ 32 4.2.1 Production of PKCα, PKCδ and β2-chimaerin in Sf9 insect cells (I, III)................................................ 32 4.2.2 Preparation of Sf9 cell homogenates (I, III) ....................................................................................... 32 4.2.3 Production of the C1 domain of MRCKα in E. coli (III) ........................................................................ 32 4.2.4 Purification the C1 domain of MRCKα (III) ......................................................................................... 32 4.3 PHORBOL ESTER DISPLACEMENT ASSAYS ................................................................................................. 33 4.3.1 Filtration method for [3H]PDBu binding (I, III, IV) .............................................................................. 33 4.3.2 Centrifugation method for [3H]PDBu binding (I, III) ........................................................................... 33 4.4 CELL CULTURE (I-IV) .................................................................................................................................. 33 4.4.1 RNA interference (III) ......................................................................................................................... 34 4.5 CELL VIABILITY AND PROLIFERATION ASSAYS ........................................................................................... 34 4.5.1 LDH assay (II) ..................................................................................................................................... 34 4.5.2 MTT assay (II, III) ................................................................................................................................ 35 4.5.3 Thymidine incorporation assay (II) .................................................................................................... 35 4.5.4 Cell proliferation and morphology analysis with Cell-IQ® (II, III, IV) ................................................... 35 4.6 WESTERN BLOTTING ................................................................................................................................. 36 4.6.1 Sample preparation (I-IV) .................................................................................................................. 36 4.6.2 Protein separation and detection (I-IV) ............................................................................................. 36 4.7 IMMUNOCYTOCHEMISTRY AND FLUORESCENCE MICROSCOPY (III) ........................................................ 37 4.8 GENOME-WIDE GENE EXPRESSION ANALYSIS (III, IV) ............................................................................... 38 4.9 STATISTICAL ANALYSIS (I-IV) ..................................................................................................................... 38 5. RESULTS ................................................................................................................................................... 39 5.1 STRUCTURE-ACTIVITY RELATIONSHIP STUDIES ON ISOPHTHALATE DERIVATIVES (I, III) .......................... 39 5.1.1 Binding to the C1 domain of PKC (I) ................................................................................................... 39 5.1.2 Binding affinity constants for PKCα and PKCδ (I) ............................................................................... 39 5.1.3 Binding to other DAG effectors (III) ................................................................................................... 40 5.1.4 Effects on ERK1/2 signalling in HeLa cells (I) ..................................................................................... 41 5.2 EFFECTS OF ISOPHTHALATE DERIVATIVES IN HELA CELLS (II, III) .............................................................. 42 5.2.1 Viability and proliferation (II)............................................................................................................