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Investigations on the Human Myt1 Kinase: Substrate Studies, Assay Development and Inhibitor Screening Alexander Rohe

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Investigations on the Human Myt1 Kinase: Substrate Studies, Assay Development and Inhibitor Screening Alexander Rohe Investigations on the Human Myt1 Kinase: Substrate Studies, Assay Development and Inhibitor Screening Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I - Biowissenschaften Martin-Luther-Universität Halle-Wittenberg von Alexander Rohe geboren am 06.11.1985 in Saarbrücken Gutachter: 1. Prof. Dr. W. Sippl 2. Prof. Dr. M. Schutkowski 3. Prof. Dr. M. Jung Halle (Saale), 28.August 2013 Tag der Verteidigung: 15. Januar 2014 II III Durchgeführt am Institut für Pharmazie, Abteilung Medizinische Chemie, Martin-Luther-Universität Halle-Wittenberg Gutachter: Prof. Dr. W. Sippl, Martin-Luther-Universität Halle-Wittenberg Zweitgutachter: Prof. Dr. M. Schutkowski, Martin-Luther-Universität Halle-Wittenberg Drittgutachter: Prof. Dr. M. Jung, Albert-Ludwigs-Universität Freiburg i. Br. IV Table of Contents V Table of Contents Table of Contents .................................................................................................... V Abbreviations and Symbols .................................................................................. IX List of Figures ...................................................................................................... XV List of Tables..................................................................................................... XVII 1. Introduction ...................................................................................................... 1 2. Theoretical Background ................................................................................... 3 2.1. Kinases ..................................................................................................... 3 2.1.1. From Structure to Molecular Function .............................................. 3 2.1.2. Kinase Inhibition ............................................................................... 4 2.1.3. Selectivity in Kinase Inhibition ......................................................... 8 2.1.4. Kinase Inhibitor Drug Resistance ................................................... 11 2.2. Myt1, a Wee Family Kinase ................................................................... 11 2.2.1. Structural Features .......................................................................... 11 2.2.2. Myt1 and its Physiological Role ..................................................... 13 2.2.2.1. Cell Cycle in General and G2/M in Particular ......................... 13 2.2.2.2. Myt1 in Regulation of Mitotic Entry ....................................... 16 2.2.2.3. Intracellular Membrane Dynamics and Mitotic Exit ............... 17 2.2.2.4. Checkpoint Recovery and Maintaining G2-Arrest .................. 18 2.2.2.5. Meiosis ..................................................................................... 19 2.2.3. Myt1 as a Potential Drug Target in Cancer Therapy ...................... 19 2.3. Target-Based Drug Discovery and Virtual Screening ............................ 21 3. Aim of the Work ............................................................................................ 25 4. Materials and Methods................................................................................... 27 4.1. Devices and Equipment .......................................................................... 27 4.2. Consumables .......................................................................................... 28 4.3. Reagents ................................................................................................. 29 4.4. Buffers and Solutions ............................................................................. 34 VI Table of Contents 4.5. Kinase Preparation .................................................................................. 35 4.5.1. Myt1 (full-length) ............................................................................ 35 4.5.1.1. Cell Culture .............................................................................. 35 4.5.1.2. Vital Stain and Cell Counting .................................................. 35 4.5.1.3. Transfection and Harvesting .................................................... 36 4.5.1.4. Isolation of Protein ................................................................... 36 4.5.2. Myt1 (kinase domain) ...................................................................... 37 4.6. Statistics and General Data Analysis ...................................................... 37 4.7. BCA-Assay ............................................................................................. 38 4.8. Peptide Quantitation ............................................................................... 39 4.9. SDS-PAGE ............................................................................................. 39 4.10. Western Blot ........................................................................................ 40 4.11. Dot Blot ............................................................................................... 41 4.12. TCA Precipitation ............................................................................... 41 4.13. In vitro Kinase Reactions Utilizing Protein Substrates ....................... 41 4.14. Fluorescence Polarization: Background and Analysis ........................ 42 4.15. Fluorescence Polarization Immunoassay (FPIA) ................................ 43 4.16. pTyr FP Immunoassay (FPIA II) ........................................................ 44 4.17. LanthaScreen Binding Assay .............................................................. 44 4.17.1. Instrumentational Setup and Data Analysis ................................. 44 4.17.2. Tracer Titrations .......................................................................... 45 4.17.3. Inhibitor Studies ........................................................................... 45 4.18. Synthesis and Characterization of DasAFITC .................................... 46 4.19. CMC Determination Assays................................................................ 47 4.20. DasAFITC Assay Procedure ............................................................... 48 4.20.1. Kinase Concentration and Incubation Time ................................ 48 4.20.2. Kinase Binding Assays ................................................................ 48 4.20.3. Anisotropy Data Analysis ............................................................ 49 Table of Contents VII 4.21. Microarray Assays .............................................................................. 49 4.22. Solid Phase Peptide Synthesis ............................................................ 50 5. Results and Discussion .................................................................................. 53 5.1. Substrate Studies .................................................................................... 53 5.2. TR-FRET Based Binding Assay: LanthaScreen .................................... 60 5.2.1. Assay Development: LanthaScreen (Adaption to Myt1) ................ 60 5.2.2. LanthaScreen Inhibition Data ......................................................... 63 5.2.3. LanthaScreen: Limitations and Drawbacks .................................... 66 5.2.4. Docking Studies .................................................................................. 68 5.3. FP Based Kinase Binding Assay: DasAFITC-Assay ............................. 70 5.3.1. Synthesis and Characterization of a Suitable Tracer....................... 71 5.3.2. Assay Development: DasAFITC Assay .......................................... 73 5.3.2.1. CMC Determination and Assay Buffer ................................... 73 5.3.2.2. Microplates and Tracer Concentration .................................... 77 5.3.2.3. Construction of Binding Isotherms .......................................... 77 5.3.2.4. Time Dependence and Assay Performance ............................. 79 5.3.2.5. Assay Validation ...................................................................... 81 5.3.3. Inhibitor Screening .......................................................................... 83 5.3.3.1. Identifying False-Postives: Flavonoids as Examples............... 83 5.3.3.2. Virtual Screening ..................................................................... 85 5.3.3.3. Glycotriazoles .......................................................................... 89 5.3.4. DasAFITC Assay: Conclusion, Limitations, Drawbacks ............... 89 5.3.5. Affinity of ATP to Myt1 ................................................................. 90 5.4. Glycoglycerolipids as Myt1 Inhibitors? ............................................. 92 5.5. Advanced Substrate Studies ................................................................... 96 5.5.1. Peptide Microarray Studies ............................................................. 96 5.5.2. A Homogenous FP Based pTyr Assay (FPIA II) .......................... 101 5.5.3. Solution Phase Verification of Peptidic Substrates ....................... 104 VIII Table of Contents 6. Summary and Perspectives........................................................................... 113 Bibliography ........................................................................................................ 115 Appendix ............................................................................................................. 137 Sequence Alignment and Modeling ................................................................
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