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Validation of New Casein Kinase 1 (CK1) Small Molecule Inhibitor Compounds and Characterization of Inhibitors of Wnt Production (Iwps) As Inhibitors of Ck1δ

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Validation of New Casein Kinase 1 (CK1) Small Molecule Inhibitor Compounds and Characterization of Inhibitors of Wnt Production (Iwps) As Inhibitors of Ck1δ Ulm University Hospital Surgery Center Department of General and Visceral Surgery Medical Director: Prof. Dr. med. Doris Henne-Bruns Validation of new Casein Kinase 1 (CK1) small molecule inhibitor compounds and characterization of Inhibitors of Wnt Production (IWPs) as inhibitors of CK1δ Dissertation submitted in partial fulfillment of the requirements for the Doctoral Degree of Human Biology (Dr. biol. hum.) at the Medical Faculty of Ulm University submitted by Balbina García Reyes born in San José, Costa Rica 2017 Acting Dean: Prof. Dr. Thomas Wirth First reviewer: Prof. Dr. Uwe Knippschild Second reviewer: PD Dr. Timo Burster Day of Graduation: 05.05.2017 Table of Contents List of abbreviations ........................................................................................................... iii 1. Introduction ..................................................................................................................... 1 1.1 Protein phosphorylation and signal transduction ......................................................................1 1.2 The CK1 family ........................................................................................................................2 1.3 CK1 substrate-specificity and consensus sequence ..................................................................3 1.4 Regulation of CK1 activity .......................................................................................................7 1.5 Functions of CK1 ......................................................................................................................8 1.6 CK1 role in the Wnt pathway .................................................................................................11 1.7 CK1 and diseases ....................................................................................................................13 1.8 CK1 isoforms as drug targets ..................................................................................................16 1.9 Aim of the study .....................................................................................................................21 2. Materials and Methods ................................................................................................. 22 2.1 Materials .................................................................................................................................22 2.2 Methods ..................................................................................................................................30 2.2.1 Molecular biological methods ...................................................................................................... 30 2.2.2 Bacteria methods .......................................................................................................................... 32 2.2.3 Protein methods ............................................................................................................................ 33 2.2.4 Cell culture methods ..................................................................................................................... 37 2.2.5 Statistical analysis ........................................................................................................................ 39 3. Results ............................................................................................................................. 40 3.1. Validation of new CK1-specific small molecule inhibitor compounds .................................40 3.1.1 Biological activity of the new compounds ..................................................................................... 40 3.1.2 Efficacy of compounds in cell culture ........................................................................................... 46 3.2 IWP derivatives as inhibitors of CK1δ ...................................................................................55 3.2.1 Characterization of IWPs as CK1 inhibitors in vitro .................................................................... 56 3.2.2 Efficacy of IWP compounds in cell culture ................................................................................... 63 3.2.3 Development of novel IWP-2 and IWP-2-V2 derivatives .............................................................. 66 4. Discussion ....................................................................................................................... 68 4.1 Validation of new CK1-specific small molecule inhibitor compounds ..................................68 4.1.1 Several compounds inhibit CK1δ and ε in the nanomolar range .................................................. 68 4.1.2 Compounds 118-125 inhibit the growth of various tumor cell lines ............................................. 69 4.2 IWPs as inhibitors of CK1δ ....................................................................................................72 i 4.2.1 IWP compounds inhibit CK1δ ....................................................................................................... 72 4.2.2 Effect of IWP derivatives on cell culture ....................................................................................... 73 4.2.3 Newly developed IWP derivatives as CK1δ-specific inhibitors .................................................... 74 5. Summary ........................................................................................................................ 76 6. References....................................................................................................................... 78 Acknowledgements .......................................................................................................... 107 List of Publications .......................................................................................................... 108 ii List of abbreviations A adenine AD Alzheimer’s disease ALS amyotrophic lateral sclerosis AML acute myelogenous leukemia APC adenomatous polyposis coli APS ammonium persulfate Arg arginine ATP adenosine triphosphate BCA bicinchoninic acid BSA bovine serum albumin C cytosine Ci curie CK casein kinase CMGC kinase family including cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, and CDK-like kinases DNA deoxyribonucleic acid DCIS ductal carcinomas in situ DDX3 DEAD-box RNA helicase 3 dH2O demineralized water DMEM Dulbecco's Modified Eagle's Medium DMSO dimethyl sulfoxide dNTP deoxyribonucleoside triphosphate DTT dithiothreitol DVL dishevelled E. coli Escherichia coli EC50 half maximal effective concentration ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetraacetic acid ePK eukaryotic protein kinase FACS fluorescence assisted cell sorting iii FAP familial adenomatous polyposis coli FCS fetal calf serum for forward FP fusion protein FZD Frizzled G guanine g gram GLI glioma-associated oncogene G/LOF Gain/Loss of function GSK3β glycogen synthase kinase 3 beta GST glutathione-S-transferase Hh hedgehog HPF high power field HPRT hypoxanthine-guanine phosphoribosyltransferase HRP horse-radish peroxidase hrs hour(s) IC50 half maximal inhibitory concentration IgG immunoglobulin G IPTG isopropyl-β-D-thiogalactopyranoside IWP Inhibitor of Wnt Production kcat turnover number KD kinase domain kDa kilodalton l liter LATS large tumor suppressor LEF lymphoid enhancing factor Leu leucine Lys lysine M molar MDM2 mouse double minute 2 homolog min minute(s) mRNA messenger ribonucleic acid MST mammalian STE20-like protein kinase iv mut mutant MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid MW molecular weight NLS nuclear localization signal OD optical density PCR polymerase chain reaction PD Parkinson’s disease PER period pH negative decimal logarithm of the hydrogen ion concentration PKA protein kinase A PTCH Patched PVDF polyvinylidene fluoride qRT-PCR quantitative real-time PCR rev reverse RNA ribonucleic acid rpm revolutions per minute RPMI Roswell Park Memorial Institute RQ relative quantification RT room temperature S. cerevisiae Saccharomyces cerevisiae SDM site-directed mutagenesis SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis sec seconds SMAD SMA/mothers against decapentaplegic SMO smoothened STPK Serine/Threonine Protein Kinases T thymine TAZ tafazzin TBS tris-buffered saline TCF T cell factor TEAD transcription factor TEA domain TEMED N,N,N',N'-tetramethylethylenediamine Thr threonine v TPK Tyrosine Protein Kinase TV transcript variant UICC Union Internationale Contre le Cancer V volt v/v volume per volume Vmax maximum velocity w/v weight per volume Wnt Wingless/Int-1 wt wild type YAP Yes-associated protein β-TrCP beta-transducin repeat containing E3 ubiquitin protein ligase µ micro °C degree Celsius vi 1. Introduction 1. Introduction 1.1 Protein phosphorylation and signal transduction Posttranslational modifications such as phosphorylation, glycosylation, ubiquitination, nitrosylation, acylation, methylation, lipidation, and proteolysis affect the physiological and cellular processes that occur within organisms in many ways (Walsh et al. 2005). Among them, reversible phosphorylation is one of the most studied modifications. It is performed by protein kinases, which transfer the gamma phosphate of ATP onto different substrates, a process that in turn is reversed by phosphatases. Eukaryote protein kinases (ePKs) phosphorylate either tyrosine (TPKs, Tyrosine Protein Kinases), serine/threonine (STPKs, Ser-/Thr Protein
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