The application of the fragment-based screening approach to RmlA protein and PA1645 structure
Abstract
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.
Type
Thesis, PhD Doctor of Philosophy
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