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Development and application of NMR methods for challenges in drug discovery

dc.contributor.advisorGriesinger, Christian Prof.
dc.contributor.authorPilger, Jensde
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.titleDevelopment and application of NMR methods for challenges in drug discoveryde
dc.contributor.refereeGriesinger, Christian Prof.
dc.description.abstractengNMR spectroscopy plays an important role in all stages of the drug discovery process. Namely in the structure elucidation of natural products, synthetic ligands and metabolites, as well as a high-throughput screening technique. Yet, the application of NMR in structure-based drug design, e.g. in finding the binding mode of a small molecule drug to a macromolecular protein receptor is far from making the most from the opportunities available to it. Structure-based drug design is a powerful and widely used tool for the optimization of low molecular weight compounds that should be turned into highly efficient drugs. The method mainly relies on high-resolution crystal structures of the receptor-ligand complex to obtain the required information for optimizing target binding of small molecules. However, obtaining crystals and structures of sufficient quality cannot be achieved for nearly the half of pharmaceutically relevant protein targets. For those target proteins that cannot be crystallized, NMR spectroscopy is an alternative and structures of protein-ligand complexes can be determined, provided the protein can be labelled with stable isotopes such as 13C or 15N. However, pharmaceutically relevant non-crystallizable target proteins are often non-tractable by NMR, because they are too large and result in overcrowded spectra or they cannot be expressed in bacteria and therefore cannot be labelled with stable isotopes enabling heteronuclear NMR. In such cases one can employ INPHARMA (Inter-Ligand NOEs for PHARmacophore MApping). It utilizes two ligands that bind competitively to the same binding pocket of a protein. INPHARMA peaks in a NOESY spectrum emerge from the magnetization transfer from the protons of one ligand to the protons of the other ligand via the protein protons, provided the ligands dissociate from the protein several times during the NOESY mixing time. The method is further developed and it is investigated whether the methodology can be improved by inclusion of Saturation Transfer Difference (STD) restraints and transferred NOE (trNOE) restraints in addition to the INPHARMA restraints. STD is a frequently used technique in NMR spectroscopy and NMR-based screening for protein binders. The technique is developed and tested on protein kinase A, where crystal structures of the protein/ligand complexes are known. The results show that the combination of the NMR methods INPHARMA, tr- NOE and STD results in a precise scoring function for docking modes and therefore the determination of ligand binding modes. It is demonstrated that the method is superior to docking scoring functions alone and can lead to the correct result by using a molecular dynamics simulation driven refinement, even if the initial conformation of the protein side chains is not correct. Multiplexing of several ligands improves the reliability of the scoring function further. Then the technique is extended the G-protein coupled receptor GPR40, a membrane protein, for which only homology models exist and which is an interesting drug target in on-going research. For this system, the ligand binding mode found is supported by SAR data. The binding mode of epothilone to tubulin, an important interaction for cancer therapy is reinvestigated using STD data. The binding mode found by INPHARMA is confirmed and further optimized, while the electron crystallography derived structure does not fit to the experimental NMR data. The NMR-based ligand binding mode determination method is presented to derive binding modes of ligands based on simple NMR experiments (NOESY and STD). It is demonstrated on the examples of PKA, GPR40 and the tubulin-epothilone complex, that based on a crystal structure or homology model of the protein, binding modes can be determined that can be used for pharmacophore mapping and drug optimization. In the second part the drug metabolism of anle138b, a modulator of toxic protein oligomers in prion and Parkinson’s disease is investigated. A methodology is developed to extract the drug from organs and to determine its concentration in the brain. It was confirmed that anle138b is the only active compound in the brain, while metabolites are only formed in liver and kidney. With combined HPLC, mass spectrometry and NMR techniques, the structures of the metabolites were determined and the drug metabolism of anle138b in the mice and rat model was revealed. In the last part NMR spectroscopy is applied to reinvestigate the structural and stereochemical features of arthrofactin, a potentially antibiotic natural product. Arthrofactin was initially reported in 1993 as a bioactive cyclic lipopeptide from the bacterium Pseudomonas sp. The structure of arthrofactin and its derivatives was reassigned on the basis of extensive NMR experiments and chiral HPLC analysis. A new approach of phylogenetic structure prediction is tested and was successfully approved with NMR data. In conclusion, NMR spectroscopy is applied and further developed in this thesis to several challenges of the drug discovery
dc.contributor.coRefereeDiederichsen, Ulf Prof.
dc.subject.engNMR, medicinal chemistry, drug design, Inpharma, Noesyde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

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