AbstractThe alarming increase in antibacterial drug resistance indicates an urgent need to develop new drugs. This project aimed to assess and select potential antibacterial targets and carry out initial biochemical characterisation concentrating on enzymes from biowarfare agents Bacillus anthracis, Burkholderia pseudomallei, Francisella tularensis and Yersinia pestis. The overall objective is to combine genetic and chemical studies to validate, or not, targets for early stage drug discovery.In collaboration with the Dstl (Defence Science and Technology Laboratory), a series of potential targets were selected. This was carried out using essentiality prediction data from the Dstl labs, information at AEROPATH and ChEMBL databases, and the literature. Once ten different targets were selected, recombinant protein production was carried out to support structural and biochemical characterisation. Seven proteins were successfully purified and four of them prioritised for further studies. These are kynurenine formamidase (KynB), D-alanine—D-alanine ligase (Ddl), caseinolytic protease subunit P (ClpP), and the dihydrofolate synthase:folyl-poly-glutamate synthase (bifunctional protein FolC). X-ray crystallography was used to determine protein structures of KynB, Ddl and ClpP from various bacteria. Additionally, different enzymatic and binding assays were applied to assess kinetic parameters of KynB, Ddl and FolC, and compound library screenings were carried out for Ddl. In parallel, the genetic validation of these targets was being carried out by the Dstl.KynB is an important enzyme in tryptophan metabolism and predicted to be essential in Pseudomonas aeruginosa. The B. anthracis, B. cenocepacia and P. aeruginosa KynB structures showed an amidase fold not previously described, with a distinctive binuclear Zn2+ catalytic site that indicated a distinct reaction mechanism. Whilst the characterisation of the enzyme was ongoing, the Dstl lab reported the gene as non-essential and so no additional chemical validation was pursued. Ddl generates a precursor for the peptidoglycan layer and appears to be an essential protein in several Gram-negative bacteria. The structure of B. pseudomallei Ddl (BpDdl) in the presence of the co-factor and the reaction product D-alanyl-D-alanine (1.5 Å resolution) gives information about the substrate-binding site. Biolayer interferometry (BLI) and high-throughput (HTP) assay protocols were developed and applied. Despite testing around 22,000 compounds no inhibitors or suitable hits were found. This suggests BpDdl is a challenging target and a different approach for drug discovery might have to be considered. Fourteen ClpP subunits form a proteolytic complex which presents two different conformations; open and compressed. Structures of F. turalensis ClpP (FtClpP) with open and compressed conformations have been determined indicating this major conformational change is caused by a loop rearrangement at the proteasome inner canal that leads to the protease active site. FolC is an essential protein for the synthesis of folyl-poly-glutamates, a reference pathway for drug development. A new enzymatic assay, using malachite green, has been identified and used to confirm Y. pestis FolC activity. Such an assay could be used to determine kinetic parameters and to develop a HTP assay.The studies carried out informed about potential antibacterial target structures and biochemical properties. Protocols have been developed for protein recombinant expression, purification, crystallisation and structure determination as well as enzymatic assay development and compound library screens.
|Date of Award||2016|
|Add any sponsors of the thesis research||Defence Science and Technology Laboratory|
|Supervisor||Bill Hunter (Supervisor)|
Assessment of potential antibacterial drug targets
Diaz Saez, L. (Author). 2016
Student thesis: Doctoral Thesis › Doctor of Philosophy