TGT - a drug target to study pKa shifts, residual solvation & protein-protein interface formation [Elektronische Ressource] / vorgelegt von Tina Ritschel

Tina

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 24
• Poids de l'ouvrage: 8 Mo

Extrait

TGT a Drug Target to Study pKa Shifts,
Residual Solvation & Protein - Protein
Interface Formation




Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)


dem
Fachbereich Pharmazie
der PHILIPPS-UNIVERSITÄT MARBURG
vorgelegt von



Tina Ritschel
aus Wiesbaden



Marburg/Lahn 2009


























Vom Fachbereich Pharmazie der Philipps-Universität Marburg
als Dissertation angenommen am:

Erstgutachter: Prof. Dr. Gerhard Klebe
Zweitgutachter: Prof. Dr. Klaus Reuter

Tag der mündlichen Prüfung: 20. August 2009

2

Die Untersuchungen zur vorliegenden Arbeit wurden auf Anregung von Herrn Prof. Dr.
G. KLEBE am Institut für Pharmazeutische Chemie des Fachbereichs Pharmazie der
Philipps-Universität Marburg in der Zeit von Januar 2006 bis Juni 2009 durchgeführt.

3



4











„Wege entstehen dadurch, dass man sie geht“

Franz Kafka

5



6 Abbreviations

Abbreviations

-10Å Ångström ( 1Å = 10 m)
A absorption at 600 nm 600
CMC critical micellar concentration
CSD Cambridge Structural Database
DMSO dimethylsulfoxide
dNTP desoxynucleosidtriphosphate
DTT dithiothreitol
E. coli Escherichia coli
TyrECY2 unmodified E. coli tRNA
EDTA ethylenediamintetraacetate
ff force field
h hour
HEPES 2-[4-(2-hydroxyethyl)piperazino]ethansulfonic acid
IPTG isopropylthio- β-galactosid
K Klevin
kb kilo bases
kDa Dalton
K competitive inhibition constant i
K Michaelis Menten constant m
Km kanamycin
-1M molarity (mol ⋅ l )
MD Molecular dynamic
MES 2-morpholinoethansulfonic acid
min minute
NMR nuclear magnetic resonance
NPT isobar-isothermes Ensemble; N, p, and T constant
NTP nucleosidtriphosphate
PAGE polyacrylamide gel electrophoresis
PAMPA parallel artificial membrane permeability assays
PEG polyethylenglycol
PCR polymerase chain reaction

7 Abbreviations

PDB protein data bank
PMR Particle Mesh Ewald
preQ 7-cyano-7-deazaguanine 0
preQ 7-aminomethyl-7-deazaguanine 1
Q 7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)
methyl)-7-deazaguanosine
QueA S-adenosylmethionine:tRNA-ribosyltransferase-isomerase
QueTGT TGT involved in Q modification
SDS sodiumdodecylsulfate
S. flexneri Shigella flexneri
TCA trichloroacetic acid
TGT tRNA-guanine transglycosylase
TIM-barrel triose-phosphate isomerase (TIM) / (βα) barrel 8
T. maritima Thermotoga maritima
Tris tris-(hydroxymethyl)-aminomethane
w/v weight per volume
WT wild type
AspYadB glutamyl-queuosine tRNA synthetase
Z. mobilis Zymomonas mobilis


8 Table of contents


Abbreviations ....................................................................................................................... 7 
Table of contents .................................................................................................................. 9 
1  Introduction and Motivation ...................................................................................... 13 
1.1  Drug design and TGT ........................................................................................ 13 
1.2  Aim of the project .............................................................................................. 14 
1.3  Shigellosis .......................................................................................................... 15 
1.3.1  Disease and treatment .................................................................................... 15 
1.3.2  Shigella – Escherichia relationship ................................................................ 15 
1.3.3  Cellular and molecular pathogenicity ............................................................ 16 
1.3.4  The key role of VirF - regulation of pathogenicity ........................................ 17 
1.3.5  Procaryotic queuosin pathway ....................................................................... 18 
1.4  Mechanism of TGT ............................................................................................ 20 
1.5  Structure of TGT ................................................................................................ 21 
1.5.1  Folding and dimer formation ......................................................................... 21 
1.5.2  Active site of TGT ......................................................................................... 22 
2   Crystal structure analysis and in-silico pKa calculations suggest strong pKa shifts of
ligands as driving force for high affinity binding to TGT ................................................. 23 
2.1  Abstract .............................................................................................................. 23 
2.2  Introduction ........................................................................................................ 24 
2.3  Results and Discussion ...................................................................................... 29 
2.3.1  Binding mechanism and affinity data ............................................................ 29 
2.3.2  In-silico pK calculation ................................................................................. 31 a
2.3.3  Crystal structures ........................................................................................... 34 
2.3.4  Docking experiments ..................................................................................... 41 
2.4  Conclusion ......................................................................................................... 41 
3  Replace active site water molecules to achieve nanomolar inhibition of tRNA-
guanine transglycosylase ................................................................................................... 43 
3.1  Abstract .............................................................................................................. 43 
3.2  Introduction ........................................................................................................ 44 
3.2.1  The role of water molecules in structure-based drug design ......................... 44 
3.2.2  Target protein ................................................................................................. 45 

9 Table of contents

3.3  Results and Discussion ...................................................................................... 47 
3.3.1  Computational studies .................................................................................... 47 
3.3.2  Experimental characterization ....................................................................... 53 
3.4  Conclusion ......................................................................................................... 61 
4  Side chain variations .................................................................................................. 63 
4.1  Binding affinity 64 
4.2  Crystal structure ................................................................................................. 66 
4.3  Docking solutions .............................................................................................. 66 
4.4  MD simulation ................................................................................................... 68 
4.5  Conclusion ......................................................................................................... 72 
5  lin-Benzohypoxanthine-based inhibitors ................................................................... 74 
5.1  Introduction ........................................................................................................ 74 
5.2  Prediction of membrane permeability & modification of the parent skeleton ... 74 
5.3  Kinetic characterization ..................................................................................... 76 
5.5  Crystal structure ................................................................................................. 76 
5.6  Can new combinations of functional groups improve the PAMPA score? ....... 77 
5.7 79 
6  Suche nach neuartigen Fragmenten zur Inhibition von TGT mit Hilfe von
DrugScoreFP ...................................................................................................................... 80 
6.1&