Thermodynamic and NMR structural studies on the DNA binding of bifunctional drugs with alkylating activity [Elektronische Ressource] / vorgelegt von Michael Rettig

Thermodynamic and NMR Structural Studies on the DNA Binding of Bifunctional Drugs with Alkylating Activity I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Michael Rettig geboren am 06.09.1980 in Berlin Greifswald, Januar 2010 Dekan: .................................................................................................................... 1. Gutachter : .................................................................................................................... 2. Gutachter: .................................................................................................................... Tag der Promotion: Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde. Ferner erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als die darin angegebenen Hilfsmittel benutzt habe. Unterschrift des Promovenden Parts of this thesis were published: Rettig, M., Kamal, A., Ramu, R., Mikolajczak, J.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 35
Source : D-NB.INFO/1002425433/34
Nombre de pages : 153
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Thermodynamic and NMR Structural Studies on the DNA Binding
of Bifunctional Drugs with Alkylating Activity


I n a u g u r a l d i s s e r t a t i o n

zur

Erlangung des akademischen Grades

doctor rerum naturalium (Dr. rer. nat.)

an der Mathematisch-Naturwissenschaftlichen Fakultät

der

Ernst-Moritz-Arndt-Universität Greifswald


vorgelegt von Michael Rettig
geboren am 06.09.1980
in Berlin


Greifswald, Januar 2010















Dekan: ....................................................................................................................


1. Gutachter : ....................................................................................................................

2. Gutachter: ....................................................................................................................


Tag der Promotion:






Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch-
Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen
wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde.
Ferner erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als die darin
angegebenen Hilfsmittel benutzt habe.




Unterschrift des Promovenden





Parts of this thesis were published:


Rettig, M., Kamal, A., Ramu, R., Mikolajczak, J., and Weisz, K. (2009) Spectroscopic and
calorimetric studies on the DNA recognition of pyrrolo[2,1-c][1,4]benzodiazepine
hybrids, Bioorganic & Medicinal Chemistry 17, 919-928.

Rettig, M., Weingarth, M., Langel, W., Kamal, A., Kumar, P. P., and Weisz, K. (2009)
Solution Structure of a Covalently Bound Pyrrolo[2,1-c][1,4]benzodiazepine-
Benzimidazole Hybrid to a 10mer DNA Duplex, Biochemistry 48, 12223-12232.

Rettig, M., Langel, W., Kamal, A. and Weisz, K. (2010) NMR Structural Studies on the
Covalent DNA Binding of a Pyrrolobenzodiazepine-Naphthalimide Conjugate, Organic &
Biomolecular Chemistry (submitted).


Table of Contents
Table of Contents
ACKNOWLEDGEMENTS ........................................................................................................................... 1
1 INTRODUCTION ................................................................................................................................ 2
2 THEORY ............................................................................................................................................. 11
2.1 DNA MELTING CURVES .................................................................................................................... 11
2.2 CD SPECTROSCOPY OF NUCLEIC ACIDS ............................................................................................. 12
2.3 FLUORESCENCE TITRATIONS ............................................................................................................. 13
2.4 ISOTHERMAL TITRATION CALORIMETRY ........................................................................................... 14
2.5 DNA RESONANCE ASSIGNMENT STRATEGIES .................................................................................... 16
312.6 P NMR OF DNA ............................................................................................................................ 17
2.7 MOLECULAR MODELING .................................................................................................................. 18
2.7.1 Molecular Modeling using AMBER........................................................................................ 18
2.7.2 Explicit and implicit solvent ................................................................................................... 20
2.7.3 Determination of partial charges ........................................................................................... 21
2.8 NMR RESTRAINTS IN MOLECULAR MODELING .................................................................................. 22
2.9 CLUSTERING ..................................................................................................................................... 23
3 RESULTS ............................................................................................................................................ 25
3.1 SPECTROSCOPIC AND CALORIMETRIC STUDIES ON THE DNA-DRUG INTERACTION ............................ 25
3.1.1 UV melting experiments ......................................................................................................... 25
3.1.2 CD spectroscopy ..................................................................................................................... 29
3.1.3 UV titration ............................................................................................................................. 33
3.1.4 Fluorescence titration ............................................................................................................ 35
3.1.5 Isothermal titration calorimetry ............................................................................................. 36
3.2 NMR STRUCTURAL STUDIES ON THE DNA-DRUG COMPLEX ............................................................. 38
3.2.1 NMR titration ......................................................................................................................... 38
3.2.2 Resonance assignment of the DNA-drug complex .................................................................. 41
3.2.3 Evaluation of drug-DNA interactions ..................................................................................... 53
3.2.4 Stereochemistry at C11 ........................................................................................................... 58
3.2.5 NOE restraints ........................................................................................................................ 61
3.3 MOLECULAR MODELING .................................................................................................................. 62
3.3.1 Restraint MD simulations ....................................................................................................... 62
4 DISCUSSION ...................................................................................................................................... 69
4.1 MODE OF BINDING ............................................................................................................................ 69
4.2 KINETICS OF BINDING ....................................................................................................................... 69
4.3 STRUCTURAL ANALYSIS OF THE DNA-DRUG COMPLEXES ................................................................ 70
4.3.1 Stereochemistry at C11 and orientation of the drug ............................................................... 70
4.3.2 PBD-naphthalimide DNA adduct ........................................................................................... 72
I Table of Contents
4.3.2.1 General characterization of the refined structure .......................................................................... 72
4.3.2.2 Drug-DNA interactions ................................................................................................................ 75
4.3.2.3 Ring flipping of the naphthalimide chromophore ......................................................................... 78
4.3.3 PBD-benzimidazole DNA adduct ............................................................................................ 78
4.3.3.1 General characterization of the refined structure78
4.3.3.2 Drug-DNA interact....... 81
4.3.3.3 Conformation of the N-methylpiperazine ring .............................................................................. 83
4.4 SEQUENCE SELECTIVITY .................................................................................................................... 84
4.5 THERMODYNAMICS OF BINDING ........................................................................................................ 88
5 CONCLUSIONS .................................................................................................................................. 95
6 MATERIALS AND METHODS ........................................................................................................ 97
6.1 MATERIALS ....................................................................................................................................... 97
6.2 UV MELTING EXPERIMENTS ............................................................................................................... 97
6.3 CIRCULAR DICHROISM ....................................................................................................................... 98
6.4 FLUORESCENCE MEASUREMENTS ...................................................................................................... 98
6.5 ISOTHERMAL TITRATION CALORIMETRY ............................................................................................ 99
6.6 NMR ................................................................................................................................................. 99
6.7 NMR DERIVED DISTANCE RESTRAINTS101
6.8 MOLECULAR MODELING ................................................................................................................. 102
6.8.1 Starting structures ................................................................................................................. 102
6.8.2 Force field and parameterization .......................................................................................... 103
6.8.3 Simulated annealing .............................................................................................................. 104
6.8.4 Production run ...................................................................................................................... 105
6.8.5 Data analysis ......................................................................................................................... 106
7 ABBREVIAT IONS ............................................................................................................................ 108
8 LITERATURE ................................................................................................................................... 110
9 CURRICULUM VITAE ................................................................................................................... 122
S SUPPLEMENTARY MATERIAL .................................................................................................. 123
S.1 NMR RESTRAINTS ........................................................................................................................... 123
S.2 CUSTOM RESIDUES .......................................................................................................................... 135
S.3 PROTON CHEMICAL SHIFTS .............................................................................................................. 143
II Acknowledgements
Acknowledgements

First of all I want to thank Prof. Dr. Weisz for giving me the opportunity to do research
and to write this dissertation in his group. Especially, I would also like to thank him for
introducing me into the exciting topic of drug-DNA interactions and for providing me
with his expert guidance throughout the whole project. Additionally, I want to thank him
for his support in writing the DAAD proposal, for the possibility to join the ACS meeting
in Philadelphia and for many useful and interesting discussions reaching far beyond the
current scientific topic.
I would also like to thank Prof. Dr. Langel for a great and close collaboration. It would not
have been possible to solve the structures of the DNA-drug complexes without his
support. Additionally, I want to thank him for being a referee of my DAAD proposal. My
special thanks go to Armin, Basti, Daniel, and Wenke who helped me to proceed through
the challenges of computational chemistry.
Furthermore I would like to thank Dr. Ahmed Kamal and his group for kindly providing
me with the PBD hybrids used throughout this work.
I would also like to thank Markus for introducing me into force field methods and for
providing me with necessary information.
In particular, I thank Steffen for giving me additional insight into physical chemistry and
Petra, who competently took care of organizational issues and personally contributed to a
pleasant atmosphere within the group.
Also, I would like to thank Andrea and Fanny. Thanks for your help and the good time we
had in Greifswald.
Finally, I would like to thank my family for being there and supporting me on my way.

This work is dedicated to my friend Sebastian.
He put up a good fight but was finally defeated by cancer.
1 1 Introduction
1 Introduction
Chemical modifications of known drugs are usually employed to enhance their biological
potential. As a compound has to fulfill several criteria to be therapeutically active, the
chemist’s challenging task is to modify existing or to introduce new functionalities
without altering beneficial attributes of the drug. Clearly, knowledge of the drug chemical
properties and the basis of drug-target interactions are essential for such a rational drug
design approach. Allowing for both the determination and breakdown of the overall free
energy into its components to reveal major driving forces of binding, a complete
thermodynamic characterization provides a starting point and may help to guide
subsequent drug optimizations. As an example, strong binding is usually expected to be
beneficial for a drug, thus, in a series of drugs one may select the one with the highest
binding constant or may try to increase the affinity of a selected candidate. However, if
specificity for a certain target is low, competing binding to other molecules may lead to
unwanted side effects. As specific drug-target interactions are expected to positively
influence binding enthalpy ∆H rather than the binding entropy ∆S, analysis of the
thermodynamic parameters may help to select a drug with favorable ∆H even if the overall
binding constant appears to be ordinary. This scheme can nicely be followed in the
development of HIV-1 protease inhibitors. First generation drugs were associated with
high association constants and favorable ∆S upon binding. In contrast, a trend towards
more favorable ∆H and thus potentially higher specificity has emerged for second
generation HIV-1 protease inhibitors (1) as can be seen in Figure 1.1.
The affinity and specificity of a drug may additionally be increased, e.g. by the selective
introduction of potential hydrogen-bond donors or acceptors. Clearly, the structure of the
drug-target complex has to be known before such specific modifications can be
successfully carried out. Taken together, knowledge about the thermodynamics of drug-
target interactions and the structure of the drug-target complex complement each other
thus paving the way for a rational optimization of the drug.
2

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