BlaC is the β-lactamase of Mycobacterium tuberculosis. We show that it can recover from inhibition by clavulanic acid and that phosphate helps it do so. We also show that in solution, BlaC is a... Show moreBlaC is the β-lactamase of Mycobacterium tuberculosis. We show that it can recover from inhibition by clavulanic acid and that phosphate helps it do so. We also show that in solution, BlaC is a rigid protein on the pico-nanosecond timescale but shows dynamics around the active site on the catalytic timescale. These dynamics become more pronounced upon inhibitor binding. Lastly, we show that two mutations that both provide BlaC with inhibitor resistance have very different effects on the dynamic behaviour. Show less
The studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a... Show moreThe studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a degree of ambiguity, this approach allows to iteratively refine and validate the model and interpret its meaning for the molecular basis of protein function. Often all three points at the same time. This dynamic nature makes the use of structural models in the design of therapeutic compounds especially useful since the inhibition of a certain protein function might not require a structure to be accurate down to the last atom but rather highlight key interactions or structural features that can be addressed in context of small molecule or peptide inhibitors. Presented are the use of strucutral biochemistry techniques to investigate the mechanism of how the ubiquitine ligase PSIP1 obtains its target specificity. Furthermore, another epigenetic effector protein PSIP1 is investigated with the aim to develop a workflow for the design of potential peptide-based inhibitors. Show less
The research presented in the thesis describes the development and implementation of solution based NMR methods that provide 3D structural information on the protein-small molecule complexes. These... Show moreThe research presented in the thesis describes the development and implementation of solution based NMR methods that provide 3D structural information on the protein-small molecule complexes. These methods can be critical for structure based drug design and can be readily applied in the early stages of preclinical drug discovery and fragment based drug discovery Show less
By utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and... Show moreBy utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and P450cam, while the results of PRE demonstrated the presence of a transient encounter complex. Furthermore, the significant insights of the interaction in the interface were uncovered by X-ray crystallography. Currently, the nature of Pdx effector activity is under debate. Since paramagnetic NMR experiments are applicable to solution studies at ambient temperature, PCS, RDC and PRE methods can further resolve the molecular mechanism of P450cam in the future. Show less
This thesis describes the application of a variety of different classical and paramagnetic NMR to different protein system. To this end cytochrome P450cam was assigned using classical techniques... Show moreThis thesis describes the application of a variety of different classical and paramagnetic NMR to different protein system. To this end cytochrome P450cam was assigned using classical techniques and paramagnetic NMR was used to investigate the solution state of this enzyme in complex with its binding partner, putidaredoxin. The solution structure of Avr2 was also solved using classical NMR spectroscopy techniques. Additionally, an algorithm for the assignment of protein nuclei on the basis pseudocontact shifts, PARAssign, is presented. An investigation of the dynamic behaviour of a paramagnetic tag attached to T4 lysozyme using relaxation dispersion is described. Show less
Receptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer.... Show moreReceptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer. The overexpression of EphA4, a member of the RTK family, has been observed in a variety of malignant carcinomas. The aim of the research project associated with this thesis was to develop high affinity inhibitors of the tyrosine kinase EphA4. Ligand discovery was based on two complementary approaches, a computational screen and an NMR based screen using Target Immobilized NMR Screening (TINS). In addition, orthogonal biophysical methods including Surface Plasmon Resonance (SPR) and protein observed NMR were employed to analyse fragment binding. The crystal structure of the EphA4 kinase domain was solved and the structure of the kinase domain in complex with dasatinib, a well-known kinase inhibitor, was also elucidated. The in silico approach discovered a potent inhibitor of EphA4 for which the binding mode was elucidated via X-ray crystallography. Moreover, the TINS approach identified two compounds that may constitute starting points for the generation of more potent EphA4 inhibitors. Show less
Membrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug... Show moreMembrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. Using the recently developed Target Immobilized NMR Screening (TINS) approach, we screened 1,200 fragments for binding to the enzyme Disulphide bond forming protein B. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 uM, which could be categorized as cofactor binding inhibitors or mixed model inhibitors of both cofactor and substrate protein interaction. Our results establish the utility of fragment-based methods in the development of inhibitors of membrane proteins, making a wide variety3of important membrane bound pharmaceutical targets amenable to such an approach. We first tested the immobilization procedure on G protein coupled receptors and ion channels. Furthermore, we used Nanodiscs, an alternative solubilization strategy, to solubilize teh protein without detergents. This resulted in less broad NMR signals, less non-specific binding issues, and identification of the binders from the original screen, proving that the nanodisc solubilization technique is compatible with TINS. Show less
The design of novel anticancer agents is one of the most active fields in Medicinal Chemistry, as the number of effective drugs for treatment of cancer is still very limited. This demand for new... Show moreThe design of novel anticancer agents is one of the most active fields in Medicinal Chemistry, as the number of effective drugs for treatment of cancer is still very limited. This demand for new drugs is even higher, considering the high cancer-prevalence rate in our society. The therapeutic application of metal complexes is an under-developed area of research and basic principles in the development of metallopharmaceuticals are lacking, or at least just recently being discovered. Metal-containing agents may offer unique therapeutic opportunities. However, significant obstacles, including potential metal accumulations and toxicities, require further research before a promising metal compound may be introduced in the clinic. In particular several ruthenium and gold coordination compounds have shown promising application as anticancer agents. In these terms, this thesis project, performed in the Coordination and Bioinorganic Chemistry group in the Leiden Institute of Chemistry, comprises the design, synthesis, detailed characterization (i.e. elemental analysis, UV-Vis spectroscopy, IR, far-IR, NMR, mass spectroscopy and X-ray single-crystal structure determination) and also the biological evaluation of novel gold compounds and ruthenium compounds. The promising cytotoxic activity developed for several of these compounds and the findings from this research are providing a better understanding of chemistry of Ru(III) and Ru(II) and Au(III) coordination compounds and their structure-activity relationships and may lead to the development of improved ruthenium and gold-chemotherapeutic drugs. Show less
The perfect anticancer drug would kill cancer cells without causing any harm to the surrounding healthy tissues. That ideal medicine is searched for in this thesis, in the form of a ruthenium... Show moreThe perfect anticancer drug would kill cancer cells without causing any harm to the surrounding healthy tissues. That ideal medicine is searched for in this thesis, in the form of a ruthenium coordination complex.Metal complexes are known to interact with DNA in several different modes, amongst which are coordination, intercalation of the aromatic part of the molecule between the DNA base-pairs and binding of the complex to one of the DNA grooves.Is there a correlation between the ruthenium–DNA interactions and the cytotoxicity of these complexes? Three ruthenium polypyridyl complexes (1a-c) were synthesized and characterized, which can coordinate to guanine. The structure-related 1e and 1f, as well as the dinuclear complex 1g, were also chosen for these studies. The interactions between each ruthenium complex and DNA were studied; the activities of the compounds in different cancer cells were tested.The results of these tests suggest that 1a-c and 1e coordinate to DNA, while 1g probably fits in the DNA groove. These complexes display moderate activity in some cancer cell lines. The inactive complex 1f can coordinate to guanine and bind to DNA via intercalation or groove-binding. 1f is the only one of the studied compounds that lacks an azo group, which suggests that this group is essential for cytotoxicity.The interactions between metal complexes and other biological molecules are also mentioned. Show less
The work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in... Show moreThe work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in initiating and regulating new DNA synthesis. The first half of the thesis describes region of the RFC and structural determinants of DNA required for productive protein-DNA interaction. The second half describes three-dimensional structure determination of the protein-DNA complex, which consists of the BRCT region of the RFC and doubled stranded DNA. The resulting structure based on the data from NMR and mutagenesis reveals structural conservations of few amino acids among the members of BRCT domain superfamily, which are known to bind either phosphorylated peptide or DNA. The work may help us to identify other potential DNA binding BRCT domains. Show less
