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

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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.
Publié le : samedi 1 janvier 2011
Lecture(s) : 23
Source : D-NB.INFO/1013219929/34
Nombre de pages : 155
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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 approach........................................................................... 46
4.1.5.1 Development of a photoreactive linker system.............................................................. 46
4.1.5.2 Development of an immobilisation strategy for UV light cleavable linker systems ..... 48
4.1.5.3 Fishing conditions for UV light cleavable linker systems ............................................. 49
4.1.5.4 Elution conditions for UV light cleavable linker systems ............................................. 50
4.1.5.5 Establishment of 2D-DIGE for pull down assays.......................................................... 52
4.1.5.6 Elution conditions for 2D-DIGE ................................................................................... 52
4.1.5.7 Labelling conditions for 2D-DIGE:............................................................................... 52
4.1.5.8 IEF conditions for 2D-DIGE:........................................................................................ 53
4.1.5.9 Development of the second dimension for 2D-DIGE ................................................... 54
4.1.5.10 2D-DIGE result evaluation:........................................................................................... 54
4.2 Target-fishing experiments .......................................................................................................55
4.2.1 Acetaminophen (ACAP) ......................................................................................................... 55
4.2.2 Indirubin-3-monoxime (IR3MO)............................................................................................ 58
4.2.2.1 Immobilisation of IR3MO............................................................................................. 58
4.2.2.2 Target-fishing experiments with IR3MO ...................................................................... 60
4.2.3 Myrtucommulone A................................................................................................................ 61
4.2.3.1 Immobilisation of Myrtucommulone A......................................................................... 61
4.2.3.2 Target-fishing experiments with Myrtucommulone ...................................................... 61
4.2.4 Betulinic acid .......................................................................................................................... 63
4.2.4.1 Immobilisation of betulinic acid.................................................................................... 63
4.2.4.2 Fishing experiments with BA........................................................................................ 64
4.2.4.3 Western blot confirmation............................................................................................. 66
4.2.4.4 CG and PR3 enzyme activity assay............................................................................... 66
4.2.5 Resveratrol .............................................................................................................................. 68
4.2.5.1 Derivatisation and immobilisation of Resveratrol ......................................................... 68
4.2.5.2 Target-fishing experiments with RV ............................................................................. 69
4.2.5.3 Western blot confirmation............................................................................................. 75
4.2.6 Testosterone / Progesterone .................................................................................................... 75
4.2.6.1 Immobilisation of Testosterone and Progesterone......................................................... 75
4.2.6.2 Immobilisation of the C3 derivatised testosterone and progesterone ............................ 76
4.2.6.3 Immobilisation of the C17 derivatised testosterone and progesterone .......................... 76
4.2.6.4 C7 derivatised testosterone and progesterone................................................................ 76
4.2.6.5 Target-fishing in PMNL cell lysates using C3- and C17-derivatised testosterone........ 79
4.2.6.6 2D-DIGE analysis of testosterone and progesterone fished proteins ............................ 81
4.2.6.7 Testosterone and progesterone fishing in PMNL cell lysates........................................ 81
4.2.6.8 Testosterone and progesterone fishing in PBMC cell lysates........................................ 89 TABLE OF CONTENTS V
4.2.6.9 Testosterone and progesterone fishing in platelet lysates.............................................. 95
4.2.6.10 Confirmation via Western blot analysis ........................................................................ 98
4.2.6.11 Evaluation of Vimentin as target structure.................................................................. 100
4.2.6.12 Evaluation of Serpin B1 as target structure................................................................. 100
4.2.6.13 Evaluation of HSP27 as target structure...................................................................... 100
4.2.6.14 Evaluation of visfatin as target structure..................................................................... 102
4.2.6.15 Comparison of different testosterone and progesterone derivatives............................ 104
5 DISCUSSION..................................................................................................... 107
5.1 Methodological improvements of the target-fishing approach............................................107
5.1.1 Matrix types for target-fishing experiments.......................................................................... 107
5.1.2 Generation of carboxymethoxylamine beads for immobilisation of ligands via keto- or
aldehyde groups .................................................................................................................................. 108
5.1.3 Evaluation of fishing conditions ........................................................................................... 109
5.1.4 Evaluation of elution condition for target-fishing experiments ............................................ 109
5.1.5 Development of a photoreactive linker system..................................................................... 110
5.1.6 Establishment of the 2D-DIGE approach for pull down assays............................................ 111
5.1.7 Target-fishing experiments with specific bioactive agents................................................... 113
5.1.7.1 Acetaminophen (ACAP) ............................................................................................. 113
5.1.7.2 Indirubin-3-monoxime ................................................................................................ 114
5.1.7.3 Myrtucommulone (MC) .............................................................................................. 114
5.1.8 Betulinic acid (BA)............................................................................................................... 115
5.1.9 Resveratrol............................................................................................................................ 116
5.1.10 Testosterone and progesterone......................................................................................... 117
6 CONCLUSION .................................................................................................. 125
7 ZUSAMMENFASSUNG................................................................................... 127
8 LITERATURE................................................................................................... 129
9 PUBLICATIONS............................................................................................... 144
9.1 Poster presentations ................................................................................................................144
9.2 Patents/ Invention disclosures ................................................................................................144
9.3 Others .......................................................................................................................................144
10 ACKNOWLEDGEMENTS .............................................................................. 145
VI Abbreviations
Abbreviations

2D-DIGE Two dimensional difference gel electrophoresis
2D-EL 2-dimensional gel electrophoresis
5-LO 5-lipoxygenase
AA Arachidonic acid
ABHD14B Abhydrolase domain-containing protein 14B
ABPP Activity-based protein profiling
ACAP Acetaminophen (= Paracetamol)
ADP Adenosine diphosphate
Akt Ak-transformed kinase
AM404 N-arachodinyl-phenolamine
AR Androgen receptor
ATM Ataxia telangiectasia mutated kinase
ATP Adenosine triphosphate
ATR Ataxia telangiectasia-Rad3-related kinase
BA Betulinic acid
BCiP 5-bromo-4-chloro-3-indolyl phosphate
Boc tert-butoylxycarbonyl
BSA Bovine serum albumin
CCD Charge-coupled Device,
CDK Cyclin-dependend kinases
c-fos Cellular proto-oncogene encoded by the FOS gene
CG Cathepsin G
Chk1 Checkpoint kinase 1
c-jun Protein encoded by the JUN oncogene
CMO Carboxymethoxylamine
COX Cyclooxygenase
c-Raf See Raf
c-Src Cellular-sarcoma
Ctrl Control
DCC N,N-dicyclohexylcarbidiimide
DHT Dihydrotestosterone
DIC N,N-diisopropylcarbodiimide Abbreviations VII
DIGE Difference gel electrophoresis
DMAP 4-Dimethylaminopyridine
DMEM Dulbecco´s Eagle Medium
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
EC Half maximal effective concentration 50
ECL Chemoluminescence
E. coli Escherichia coli
EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
EDTA Ethylenediaminetetraacetic acid
EGF Epidermal growth factor
eNOS Endothelian NO synthase
ErbB2 Erythroblastic leukaemia viral oncogene homolog 2
ERK Extracellular-signal-regulated kinase
ERK1/2 Extracellular signal-regulated kinases 1 and 2
ESI Electrospray ionization
FBP Far upstream binding protein
FCS Fetal calf serum
fig Figure
FK-506 Synonym for rapamycin
fMLP N-Formylmethionine leucyl-phenylalanine
FUBP Far upstream binding protein
GnRH Gonadotropin-releasing hormone
GTP Guanosine triphosphate
hAR-A Human androgen receptor isoform A
hAR-B Human androgen receptor isoform B
HeLa Henrietta Lacks, cervical cancer cell line
Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Her2/neu Human Epidermal growth factor Receptor 2
HIV Human immunodeficiency virus
HLE Human leukocyte elastase VIII Abbreviations
HRE Hormone responsive elements
HRP Horseradish peroxidase
HSA Human serum albumin
HSP B1 Heat shock protein beta-1
HSP27 Heat shock protein 27
IC Half maximal inhibitory concentration 50
IEF Isoelectric focussing
IEP Isoelectric pint
IL Interleukin
iNOS Inducible NO synthase
IPG Immobilised pH gradient
IR3MO Indirubine-3´-monoxime
IB- Inhibitor of NF-B
KHSRP KH-type splicing regulatory protein
KPN- Karyopherin
LB (medium) Lysogeny broth (medium)
LC Liquid chromatography
LNCap Lymph Node Carcinoma of the Prostate
LO Lipoxygenase
MAPK Mitogen-activated protein kinase
MC Myrtucommulone A
Mek Mitogen-activated protein kinase kinase kinase 1
MeOH Methanol
MK2 MAPK-activated Protein Kinase-2
MNEI Monocyte/neutrophil elastase inhibitor
Mops 3-(N-morpholino)propanesulfonic acid
mPGES1 Microsomal prostaglandin E2 synthase 1
mRNA Messenger RNA
MS Mass spectrometry
mTOR Mammalian target of rapamycin
NAMPT Nicotinamide phosphorybosyltransferase
NBT Nitro blue tetrazolium chloride
NFB Nuclear factor kappa-light-chain-enhancer of activated B
cells/ nuclear factor B Abbreviations IX
NHS N-hydroxysuccinimide
No. Number
NSAID Non-steroidal anti-inflammatory drug
OGX-427 HSP27 antisense drug
p(Ser82)HSP27 HSP27 phosphorylated on serine No. 82
p38 MAPK p38 mitogen-activated protein kinases
P8340 Protease inhibitor cocktail
PAA Polyacrylic acid
PARP Poly (ADP-ribose) polymerase
PBA Phenoxybutyric acid
PBEF Pre-B-cell colony enhancing factor = visfatin
PBMC Peripheral blood mononuclear cell
PBS Phosphate buffered saline
PC12 Pheochromocytoma cell line
PCT Pressure cycling technology
PDGF Platelet-derived growth factor
pERK Phosphorylated ERK (see ERK)
PG buffer PBS containing glucose
PGC PBS containing glucose and calcium
PI3K Phosphatidylinositol 3-kinases
PKA Protein kinase A
PMNL Polymorphonuclear leukocyte
PMSF Phenylmethanesulfonyl fluoride
PR Progesterone receptor
PR3 Proteinase 3
PR-A Progesterone receptor isoform A
PR-B Progesterone receptor isoform B
prog. Progesterone
qTOF Quadrupole time-of-flight
Raf Rapidly growing fibrosarcoma
Ras Rat Sarcoma
RNA Ribonucleic acid
RNAi RNA interference
RPMI 1640 Roswell Park Memorial Institute medium X Abbreviations
RT Room temperature
RTK Receptor tyrosine kinase
RV Resveratrol
SDS Sodium dodecyl sulfate
SDSb SDS buffer (see SDS)
SDS-PAGE SDS polyacrylamide gel electrophoresis
Ser Serine
Serpin B1 Serine protease inhibitor B1
SHBG Sex hormone binding globulin
SHP-2 Member of the protein tyrosine phosphatase (PTP) family
Src Sarcoma
Tbl Table
TBS Tris buffered saline
TBST Tris buffered saline containing Tween
test. Testosterone
THF Tetrahydrofuran
TNF Tumor necrosis factor alpha
TOF Time-of-flight
Tris Tris(hydroxymethyl)aminomethane
TTR-RBPs Turnover and translation regulatory mRNA-binding proteins
UV Ultra violett
v/v Volume per volume
VIM Vimentin
VIS Visfatin
Vol Volume
VSMC Vascular smooth muscle cells
w/v Weight per volume

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