In Silico Strategies to Modulate DNA Damage Response.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2015)
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|Item Type:||Ph.D. Thesis|
|Title:||In Silico Strategies to Modulate DNA Damage Response|
The objective of this work was the investigation of DNA damage response in irradiated tumor cells at molecular level. Using different computational (in silico) approaches three proteins and protein complexes relevant to radiation biology were analyzed in terms of structural-dynamic and evolutionary aspects. Inhibition of the 20S proteasome was shown to selectively sensitize tumor cells to radiation-induced DNA damage in contrast to surrounding healthy tissue. Thus proteasome inhibitors have a great potential as radiosensitizing agents. Simulation of the enzyme inhibition (protein-ligand docking) allows to investigate a ligand’s conformation inside the β5 active site of the proteasome thereby supporting proteasome inhibitor optimization. In this study inhibitors are considered that form a covalent bond to the β5 active site. Here modeling of the covalent interaction presented a major challenge. In collaboration with medicinal chemists of the lab of Prof. Schmidt (GrK 1657 – Project 2E) it was possible to establish a general procedure for the docking of covalently bound ligands using the software MOE. In an extended cooperation with crystallographers (Prof. Groll, TU München) and biochemists (Prof. Kloetzel, Charité Berlin) we have succeeded in developing potent and highly selective α-keto-phenylamide proteasome inhibitors. These are characterized through a unique binding mode to the primed sites of the substrate binding channel. In a follow-up project of this collaboration we focused on the elucidation of the natural proteolysis mechanism in the β5 subunit. It was hypothesized that substrates with large hydrophobic P1 residues interact with the Met45 side chain located in the β5 active site thus accelerating the cleavage mechanism. This trigger mechanism is considered to be analogous to that of a mouse trap. However, biochemical experiments have shown that the cleavage mechanism is also triggered by small residues which contradicts the hypothesis. Our results of extensive molecular dynamics (MD) simulations confirmed the Met45 side chain dynamics to be directly related to the substrate’s residue size. This shows that the β5 binding pocket accommodates to a variety of differently-sized substrate residues. These findings allow for future rational design of proteasome inhibitors. ow for future rational design of proteasome inhibitors. The DNA-dependent protein kinase (DNA-PK) consisting of the protein Ku (with subunits Ku70 and Ku80) and its catalytic subunit (DNA-PKcs) is the key complex responsible for DNA double-strand break (DSB) repair at early stages of the non- homologous recombination pathway (NHEJ). For the development of DNA-PK inhibitors which modulate the NHEJ pathway, structural knowledge of the DNA-PK complex is mandatory. This work addresses the question if molecular coevolution can provide information on the yet unknown three-dimensional architecture of the iiiDNA-PK complex. Molecular coevolution defines the mutual evolution of interacting amino acid residues located at interaction interfaces in order to ensure residue recognition and complex stability. The mutual information (MI), an information-theoretical measure, was applied to coevolutionary signals in sets of homologous sequences. Different MI correction procedures were evaluated with respect to their ability to predict interacting residues in the Ku70/Ku80 complex of which the crystal structure is known. It turned out that prediction quality is limited. Results show that the procedures tested must be further enhanced to extract those signals revelant to protein-protein interactions from other background signals. In order to interfere with the homologous recombination pathway (HR), Rad54 is a straightforward target since this protein plays a substantial role in this DNA repair pathway. This project was developed in collaboration with radiation biologists of the lab of Prof. Löbrich (GrK 1657 - Project 1A) who discovered a specific phosphorylation reaction to be necessary for Rad54 activation. Using a reduced biophysical network model, putative phosphorylation-induced structural changes were investigated. Instead of the expected local response, dynamics intrinsic to the protein domain were observed. Most probably, these are related to the principal function of Rad54, namely the translocation along double-stranded DNA. This result led to a more intensive study on the structural basis of the translocation cycle which might be reproduced by currently available crystal structures. Based on these findings we were able to construct a three-dimensional model of the Rad54 structure from the zebrafish which serves as a starting structure for further studies.
|Place of Publication:||Darmstadt|
|Classification DDC:||000 Allgemeines, Informatik, Informationswissenschaft > 004 Informatik
500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
500 Naturwissenschaften und Mathematik > 540 Chemie
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
|Divisions:||10 Department of Biology
10 Department of Biology > Computational Biology and Simulation
10 Department of Biology > Radiation Biology and DNA Repair
|Date Deposited:||18 Sep 2015 11:30|
|Last Modified:||25 Sep 2015 12:25|
|Referees:||Hamacher, Prof. Dr. Kay and Thiel, Prof. Dr. Gerhard|
|Refereed:||6 October 2014|