Target-Fishing entzündungsrelevanter Arzneistoffe sowie biochemisch- molekularpharmakologische Charakterisierung der Arzneistoff-Target Interaktion [Elektronische Ressource] = Target-fishing of drugs relevant to inflammation and biochemical molecular pharmacological characterisation of the drug-target interaction / vorgelegt von Felix Behnke

Hugo Lhotel

Sujets

• Naturwissenschaften

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2011
• Nombre de lectures: 25
• Poids de l'ouvrage: 7 Mo

Extrait

Target-Fishing entzündungsrelevanter Arzneistoffe
sowie biochemisch-molekularpharmakologische Charakterisierung der
Arzneistoff-Target Interaktion




Target-fishing of drugs relevant to inflammation
and biochemical molecular pharmacological characterisation of the
drug-target interaction






Dissertation
der Mathematisch-Naturwissenschaftlichen Fakultät
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)



vorgelegt von
Felix Behnke
aus Vaihingen/Enz



Tübingen
2011






































Tag der mündlichen Qualifikation: 28.04.2011
Dekan: Prof. Dr. Wolfgang Rosenstiel
1. Berichterstatter: Prof. Dr. O. Werz
2. Berichterstatter: Prof. Dr. S. Laufer
TABLE OF CONTENTS III
TABLE OF CONTENTS
TABLE OF CONTENTS ............................................................................................ III
ABBREVIATIONS.......................................................................................................VI
1 INTRODUCTION ................................................................................................. 1
1.1 Importance of drug target identification and validation..........................................................1
1.2 The target-fishing approach .......................................................................................................2
1.2.1 Ligand immobilisation.............................................................................................................. 3
1.2.2 Fishing matrices........................................................................................................................ 4
1.2.3 Linker molecules ...................................................................................................................... 5
1.2.3.1 Photoreactive linker systems........................................................................................... 5
1.2.4 Cell lysates................................................................................................................................ 7
1.2.5 Elution conditions..................................................................................................................... 8
1.2.6 Analysis of target-fishing experiments ..................................................................................... 8
1.2.6.1 Electrophoretic separation of fished proteins .................................................................. 9
1.2.7 Target validation and characterisation .................................................................................... 12
1.2.8 Importance of the target-fishing approach.............................................................................. 13
1.3 Inflammation .............................................................................................................................13
1.4 Synthetic and natural anti-inflammatory drugs as candidate ligands for the fishing
approach ...................................................................................................................................................14
1.4.1 Acetaminophen (Paracetamol)................................................................................................ 14
1.4.2 Resveratrol.............................................................................................................................. 16
1.4.3 Indirubin-3´-monoxime .......................................................................................................... 17
1.4.4 Myrtucommulone A................................................................................................................ 18
1.4.5 Betulinic Acid......................................................................................................................... 18
1.5 Gender bias in inflammation ....................................................................................................19
1.6 The steroid hormones testosterone and progesterone as ligands for target-fishing
experiments ..............................................................................................................................................20
2 AIM OF THE STUDY......................................................................................... 22
3 MATERIALS AND METHODS ........................................................................ 23
3.1 Materials.....................................................................................................................................23
3.1.1 Chemicals ............................................................................................................................... 23
3.1.2 Primary Antibodies................................................................................................................. 27
3.1.3 Secondary Antibodies............................................................................................................. 28
3.1.4 Recombinant proteins ............................................................................................................. 28
3.2 Methods ......................................................................................................................................29
3.2.1 Isolation of primary human blood cells (PMNL, PBMC, monocytes and platelets) .............. 29
3.2.2 Culture of immortalised cells.................................................................................................. 30
3.2.3 Preparation of cell lysates ....................................................................................................... 30
3.2.3.1 Preparation of whole cell lysates................................................................................... 30
3.2.3.2 Isolation of soluble proteins .......................................................................................... 31
3.2.3.3 Preparation of E. coli lysate .......................................................................................... 31
3.2.4 Immobilisation of small molecule ligands to insoluble matrices (beads) ............................... 31
3.2.4.1 Immobilisation strategies for ligands ............................................................................ 32 IV TABLE OF CONTENTS
3.2.5 Fishing experiments / pull-down assays ................................................................................. 35
3.2.5.1 Pull down assay of proteins from cell lysates................................................................ 35
3.2.5.2 Pull down of recombinant proteins................................................................................ 35
3.2.5.3 Elution of fished proteins .............................................................................................. 35
3.2.6 Protein analytics...................................................................................................................... 36
3.2.6.1 One dimensional gel electrophoresis (SDS-PAGE) ...................................................... 36
3.2.6.2 Western blot analysis..................................................................................................... 36
3.2.6.3 Silver staining................................................................................................................ 38
TM3.2.6.4 CyDye labelling for differential gel electrophoresis (DIGE)..................................... 39
3.2.6.5 Isoelectric focussing (IEF) ............................................................................................ 39
3.2.6.6 Acquisition of results..................................................................................................... 40
3.2.7 Functional enzyme assays....................................................................................................... 41
3.2.7.1 Determination of phosphorylated proteins .................................................................... 41
3.2.7.2 Serine protease activity assays ...................................................................................... 42
4 RESULTS..............................................................................................................43
4.1 Development of methods to improve fishing approaches.......................................................43
4.1.1 Evaluation of different matrix types: ...................................................................................... 43
4.1.2 Generation of carboxymethoxylamine beads for immobilisation of ligands via keto- or
aldehyde groups..................................................................................................................................... 43
4.1.3 Evaluation of fishing conditions ............................................................................................. 45
4.1.4 Evaluation of elution conditions ............................................................................................. 45
4.1.5 Improvement of the target-fishing appro