Untersuchung von p38α MAP-Kinase/Inhibitor-Komplexen in Lösung mittels Kernresonanzspektroskopie

Zur Entwicklung potentieller Arzneistoffe ist eine genaue Kenntnis des Bindungsverhaltens eines Inhibitors im Zielprotein notwendig. Einige Proteine lassen sich im Komplex mit potentiellen Inhibitoren kristallisieren und deren Bindungsweise erforschen. In Lösung dagegen findet man das Protein eher...

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Bibliographic Details
Main Author: Honndorf, Valerie Simone
Contributors: Klebe, Gerhard (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2008
Subjects:
RDC
p38
Online Access:PDF Full Text
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Table of Contents: The development of potential drugs requires knowledge of the binding behavior of the inhibitor/activator to the target protein. To this end, some proteins can be crystallized in complex with their small molecules. The method for investigating the structure and additionally the dynamic behavior of proteins is by solution-state NMR. p38α MAP kinase plays an important role in cellular pathways including differentiation, proliferation and apoptosis. p38α is an important target protein for the development of inhibitors aiming to act as drugs against cancer, inflammatory diseases or autoimmune diseases. On this account the structure, as well as the dynamic behavior of p38α in complex with two different inhibitors, was investigated by liquid-state NMR. SB203580 was chosen for the investigation as it is a prototype of p38α inhibitors and NR243 as it belongs to a new group of highly selective p38α inhibitors developed and synthesized by the group of Prof. Laufer (University of Tübingen). The great number of alpha helices in the C-terminus of the protein displaying relatively similar chemical shifts in the 2D HSQC spectra complicated the assignment. Thus the method of selective labeling was applied. In this manner 60% of the backbone resonances of the p38α/inhibitor complexes and 71% of the backbone resonances of the uncomplexed p38α were assigned. Additionally, measurements of residual dipolar couplings (RDCs) of the two complexed and uncomplexed forms of the protein were performed. The overall structure of the three forms agree with the available uncomplexed crystal structure. An important difference was detected between the crystal structure and the solution structure in uncomplexed p38α. The hinge region in solution exhibits a conformation, the so called peptide flip, that is only seen in p38α crystal structures complexed with quinazolinone and pyridole-pyrimidine inhibitors. According to this finding the peptide flip seems not to be induced by special inhibitors but is the natural conformation of the hinge region in solution. The measurement of distances by nuclear Overhauser enhancement – spectroscopy (NOESY) using perdeuterated protein samples with selectively protonated amino acids of the binding pocket of p38α provided an insight into the interactions of the inhibitors with the protein. The p38α/SB203580 complex served as control of these experiments. The measurement of NOE-distances between the inhibitors and selectively protonated leucine, threonine and tyrosine samples of p38α proved to be successful. After analysis, the gathered information was used to model the complex structure of the p38α/NR243 complex in HADDOCK. The docking program provided an unambiguous result. In conclusion one can say that it is possible to solve a protein/inhibitor complex structure by liquid-state NMR when lacking a complete assignment. The focus lies on the interaction of the amino acids of the protein with the inhibitor together with the collection of backbone RDC data. This method can be used for inhibitor screening tests and as fast alternative to crystallographic studies.