http://hdl.handle.net/1842/3414 2013-06-19T19:05:31Z http://hdl.handle.net/1842/6504 Title: Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors Authors: Blackburn, Elizabeth Anne Abstract: The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors. 2010-06-26T00:00:00Z http://hdl.handle.net/1842/6267 Title: Ecological and molecular investigation of wheat bulb fly (Delia coarctata, Fallén, Diptera: Anthomyiidae) for the advancement of population monitoring and control methodologies Authors: Rogers, Craig David Abstract: Wheat bulb fly (WBF) (Delia coarctata, Fallén, Diptera: Anthomyiidae) is a pest of commercial importance in cereal crops. Control is dependent on organophosphates some of which are restricted in the UK, while current oviposition monitoring techniques are labour intensive and subjective. Eggs are not laid in association with a host-plant, therefore, prompt location of a suitable host is critical to the survival of the newly hatched larvae. Wheat bulb fly larvae have been shown to exhibit a positive chemotactic response to wheat and other host-plant seedlings and their root exudates. The objective of this study was to improve the control and population monitoring methodology associated with WBF, by investigating the ecology and specifically the chemical ecology of the WBF. Bioassays were used to investigate the behavioural response of WBF to known chemical constituents of host-plant exudates. Four secondary metabolites were found to be attractive while CO2 was found to alter the behaviour of larvae. Wheat bulb fly oviposition was assessed in field situations to describe egg laying spatially and through time. Geostatistical and ecological techniques were used to observe the spatial dependence and dispersion of oviposition and construct contour maps or scale-sized dot graphs of oviposition density. The traditional single line transect sampling pattern was compared against a more intensive sampling regime. Oviposition monitoring was conducted over a three year period to ascertain the time of peak egg density of this fly. A molecular based diagnostic test to assess WBF egg populations for damage forecasting was developed. A real time polymerase chain reaction (PCR) protocol was produced to estimate field populations of WBF eggs through the quantification of eggs from field samples. In addition endpoint PCR was used to identify the presence or absence of eggs from samples. This study gives the potential to advance current control methodology by providing the basis for the development of a lure and kill or confusion/disruption strategy, while offering a more accurate sampling system and a molecular diagnostic test, for improvement of the management of WBF. 2012-06-22T00:00:00Z http://hdl.handle.net/1842/6258 Title: Intermediates of DNA double strand break repair in escherichia coli Authors: Mawer, Julia Sofia Pamela Abstract: A DNA double-strand break (DSB) is a severe form of DNA damage. In fastgrowing cells, DSBs are commonly repaired by homologous recombination (HR) and in E. coli they are exclusively repaired by this mechanism. Failure to accurately repair DSBs can lead to genomic instability. Characterising the DNA intermediates formed during DSB repair by HR is key to understanding this process. A system for inducing a site-specific DSB in the E. coli chromosome has previously been described (Eykelenboom et al., 2008). Here, this system has been used to determine the nature of the intermediates of the repair. It was shown that in a Rec+ background the repair process is rapid and efficient. By contrast, in a ruvAB mutant, which is defective for the Holliday junction (HJ) migration and cleavage complex, RuvABC, HJs are accumulated on both sides of the breakpoint. Replication forks also accumulate at defined positions from the DSB, indicating that unresolved HJs are a barrier to efficient replication that is associated with the repair. This suggests that the resolution of HJs needs to occur prior to the establishment of DNA synthesis. Despite the accumulation of HJs in a ruvAB mutant, cell survival occurs when DSBs are induced for short periods, suggesting that HJs can be resolved in a RuvAB-independent manner. In contrast, the RecG helicase is essential for survival. In a recG mutant, replication forks but not HJs are detected in the region of DSB repair. In a ruvAB recG mutant, intermediates in this region are lost. These observations are consistent with a role of RecG in the stabilisation and maturation of D-loops and not the resolution of Holliday junctions. Nevertheless, an additional role for RecG in later stages of repair cannot yet be excluded. This work provides a solid framework for the further study of DSB repair in E. coli. 2012-06-22T00:00:00Z http://hdl.handle.net/1842/6237 Title: Effects of metal ions on the structural and biochemical properties of trypanosomatid phosphoglycerate mutases Authors: Fuad, Fazia Adyani Ahmad Abstract: Flagellate protozoa from the order Trypanosomatida have developed a range of strategies to survive in their mammalian hosts. A consequence is that the glycolytic pathway has assumed an important role, especially in bloodstream-form Trypanosoma brucei, where it is essential as the sole producer of ATP. The seventh enzyme in the pathway, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shares no common properties with the corresponding enzyme in humans. This enzyme catalyses the conversion of 3PGA to 2PGA, with the requirement for metal ions to assist the catalytic function. In this study, two important biochemical and structural aspects of the enzyme were investigated: i) The in vitro and in vivo requirements for biologically relevant metal ions to support the activity of iPGAM, and ii) The ability of trypanosomatid iPGAM to exist in multiple conformations and oligomeric states in solution. The maximum activity of iPGAM in vitro requires Co2+, but this cannot be the case in vivo where ICP-OES analyses confirmed that Co2+ was essentially undetectable in T. brucei cytosolic fractions. The activity of iPGAM in vivo is therefore one of the lowest among the glycolytic enzymes. By contrast, Mg2+ and Zn2+ were found to be the most abundant metals in both cytosolic fractions and in purified bacterially expressed iPGAM. Our newly-developed multimode-plate reader discontinuous assay further revealed that of the biologically relevant metals, only Mg2+ can support iPGAM activity, but at less than 50% of the level of Co2+. By contrast, Zn2+ strongly inhibits iPGAM. This assay which was developed with minimal metal interference on the coupling enzymes, also showed that in solution, the ratio of the concentrations of 3PGA:2PGA (substrate:product) at equilibrium is not 1:1 as observed in the crystal structure, but is in fact 12:1, which may be due to the tighter binding of 2PGA to the enzyme. A series of biophysical analyses, notably by SEC-MALS showed that iPGAM from Leishmania mexicana, another trypanosomatid protozoan parasite exists in different forms and oligomeric states in solution, either as the closed-form monomer, openiii form monomer, or closed/open-form dimer which can be successfully separated by ion-exchange chromatography. The open-form LmiPGAM is particularly relevant for drug development, as the catalytic site in the closed-form structure is poorly inaccessible. Both virtual and high-throughput screening approaches were used to identify novel potential inhibitors. Out of a collection of 11 compounds tested at 1 mM, two showed substantial inhibition with 49% and 14% remaining activity. Taken together, the findings from this study demonstrated the potential of iPGAM to be a key modulator in controlling glycolytic flux in trypanosomes, and thus further validated it as an important drug target. 2012-06-22T00:00:00Z