University of Dundee DOCTOR of PHILOSOPHY Evaluation Of

University of Dundee DOCTOR OF PHILOSOPHY Evaluation of Glycogen Synthase Kinase 3 as a drug target in African trypanosomes Grimaldi, Raffaella Award date: 2014 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 11. Oct. 2021 Evaluation of Glycogen Synthase Kinase 3 as a drug target in African trypanosomes Raffaella Grimaldi PhD Thesis December 2014 Supervisor: Professor Alan H. Fairlamb University of Dundee To my daughter Gaia I Table of Contents List of abbreviations…………………………………………………………………VIII Acknowledgements…………………………………………………………………….XI Declaration…………………………………………………………………………….XII Abstract………………………………………………………………………………XIII Chapter 1 Introduction ............................................................................................................. 1 1.1 Human African trypanosomiasis ................................................................................... 2 1.1.1 Epidemiology and distribution ............................................................................... 2 1.1.2 Trypanosome cell cycle in vector and in human host ............................................. 4 1.1.3 Trypanosome cell architecture ................................................................................ 6 1.1.4 Symptoms and diagnosis ........................................................................................ 7 1.2 Chemoteraphy of sleeping sickness ............................................................................... 8 1.2.1 T. b. rhodesiense infection ...................................................................................... 8 1.2.2 T. b. gambiense infection ...................................................................................... 10 1.2.3 New drug candidates for HAT.............................................................................. 14 1.3 Target assessment and target product profile............................................................... 16 1.4 Protein kinases ............................................................................................................. 18 1.4.1 The T. brucei kinome ........................................................................................... 19 1.4.2 Protein kinases in T. brucei as drug targets .......................................................... 21 1.5 Glycogen synthase kinase: role in diseases and inhibitors development ..................... 25 1.6 Glycogen synthase kinase as a drug target in kinetoplastida ....................................... 30 1.7 Aims of the project ...................................................................................................... 34 Chapter 2 Materials and methods .......................................................................................... 35 2.1 Materials ...................................................................................................................... 36 2.2 General molecular biology .......................................................................................... 36 2.2.1 Isolation of genomic DNA from bloodstream form T. brucei .............................. 36 2.2.2 DNA amplification ............................................................................................... 37 2.2.3 Agarose gel electrophoresis and extraction of DNA ............................................ 37 2.2.4 Cloning of PCR products ...................................................................................... 38 2.2.5 Transformation of competent E. coli cell lines ..................................................... 38 2.2.6 Purification and digestion of plasmid DNA ......................................................... 39 2.2.7 DNA sequencing .................................................................................................. 39 2.2.8 Ligation ................................................................................................................ 40 2.2.9 Southern analysis of genomic DNA ..................................................................... 40 2.3 Recombinant protein expression and purification ....................................................... 41 2.3.1 Sodium dodecyl sulphate polyacrimide gel electrophoresis (SDS-PAGE) .......... 41 2.3.2 Protein quantification ........................................................................................... 42 2.3.3 Recombinant protein expression .......................................................................... 42 2.3.4 Immobilised metal ion affinity chromatography .................................................. 43 II 2.3.5 TEV cleavage of hexahistidine-tag ....................................................................... 44 2.3.6 Anion-exchange chromatography ......................................................................... 45 2.3.7 Gel filtration chromatography .............................................................................. 45 2.3.8 Protein characterization by mass spectrometry .................................................... 46 2.3.9 Protein characterization by Western blotting........................................................ 46 2.4 Enzymatic and kinetic studies ..................................................................................... 47 2.4.1 TbGSK3 biochemical characterization ................................................................. 47 2.4.2 Determination of the TbGSK3 short Km for the substrates ................................... 47 2.4.3 Determination of mechanism of inhibition ........................................................... 48 2.4.4 Surface Plasmon Resonance ................................................................................. 48 2.4.5 Isothermal Titration calorimetry ........................................................................... 50 2.5 Screening ..................................................................................................................... 50 2.5.1 Compound library ................................................................................................. 50 2.5.2 Screening workflow.............................................................................................. 50 2.5.3 Primary screening assay – KinaseGlo format ....................................................... 51 2.5.4 Potency screening assay – Flashplate assay ......................................................... 52 2.5.5 Screening batch – pass criteria ............................................................................. 53 2.5.6 Mammalian kinase profiling ................................................................................. 53 2.5.7 Data analysis......................................................................................................... 54 2.5.8 Proliferation assay of MRC5 and BSF T. brucei cells .......................................... 54 2.6 T. brucei cell culture and genetic manipulation ........................................................... 55 2.6.1 Bloodstream form T. brucei cell culture ............................................................... 55 2.6.2 Cell density ........................................................................................................... 55 2.6.3 Generation of TbGSK3 short single knock-outs cell lines.................................... 56 2.6.4 Generation of transgenic T. brucei cell lines ........................................................ 56 2.6.5 Cloning of genetic modified T. brucei lines ......................................................... 57 2.6.6 Growth inhibition studies of T. brucei .................................................................. 58 2.6.7 Cell lysis for Western blotting .............................................................................. 59 2.7 Chemical-proteomics ................................................................................................... 59 2.7.1 Kinobeads method ................................................................................................ 59 2.8 Molecular modelling .................................................................................................... 60 2.8.1 Homology modelling ............................................................................................ 60 2.8.2 Generation of binding modes ............................................................................... 60 Chapter 3 Results ................................................................................................................... 61 3.1 Identification of small molecules inhibitors ................................................................ 62 3.1.1 Biochemical characterization of TbGSK3 ...........................................................

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