P. aerguinosa is a serious human bacterial pathogen. This thesis describes attempts to use structural biology to identify new starting points for drugs against P. aerguinosa .A number of fragment-based screening techniques were used in order to identify potential inhibitors to P. aerguinosa RmlA protein, the first enzyme in the L-Rhamnose pathway. A 500 “Rule of 3” Fragment Library (Maybridge) was investigated. The first approach was the application of Differential Scanning Fluorimetry (DSF) approach to detect ligands that bind and stabilize RmlA protein. The stabilisation of RmlA was determined by thermal unfolding in the presence of each of the 500 compounds. 21 of those compounds were found to increase the protein stability. The library was then screened by NMR spectroscopy for binding to RmlA. Two techniques were evaluated STD and WaterLOGSY. 106 compounds gave positive results in both NMR experiments. These hits were then tested by a simple STD competition binding with dTTP, a natural RmlA substrate, in order to identify those binding at the active or allosteric site. 21 out of the 106 compounds were observed to compete with dTTP. The results were compared to the results of the DSF screening. Compounds that tested positive in the dTTP competition binding STD experiment and in the DSF screening were tested for their ability to inhibit RmlA in a biological assay. A coupled enzyme assay was used to monitor RmlA activity. Only one compound, 3-pyridin-3-ylaniline, showed significant inhibition of the enzyme activity. The PA1645 protein from P. aerguinosa has been identified as essential. The protein was overexpressed, purified and crystallised. Data were collected at Diamond on beamline IO3 and phases were determined by S-SAD at a wavelength of 1.6Å. Final coordinates have been deposited in the protein data bank under entry code 2XU8. The structure has 3 molecules in the asymmetric unit. There is some ambiguity as to the validity of the proposed trimeric arrangement, with results from solution and crystal disagreeing. Fragment-based screening approach has been applied to RmlA protein, using the DSF technique, a number of ligand-based NMR experiments and a coupled enzyme biological assay. 3-pyridin-3-ylaniline was the only compound that showed significant inhibition of the enzyme activity. The structure of PA1645 from P. aerguinosa has been solved. This work will help to design new drugs to combat multi-drug resistant P. aerguinosa and MTB.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595615 |
| Date | January 2013 |
| Creators | Boulkeroua, Wassila Abdelli |
| Contributors | Naismith, James |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/4477 |
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