Inhibition of oxidative protein folding in gram-negative bacteria : fragment-based design of DsbA inhibitors.
2017-02-03T04:05:09Z (GMT) by
The disulfide-dithioloxidoreductase enzyme DsbA is an oxidative folding catalyst found in the bacterial periplasm and is a key determinant of virulence in Gram-negative pathogens. DsbA has a wide spectrum of substrate specificity and has been demonstrated to catalyse the formation of disulfides in numerous secreted proteins. DsbA operates at a central point in the production of virulence-determinants because most virulence factors are proteins that require disulfide bonds to be active. DsbA knockout mutants in E. coli, have been shown to be impaired in a range of processes related to virulence. Significantly, DsbA mutations in many pathogenic bacteria attenuate their virulence, demonstrating the value of targeting DsbA to develop anti-bacterial agents to counteract virulence. DsbA is a structurally as well as functionally well characterized protein which has no direct homologue in eukaryotes. To date there are no known small molecule inhibitors of this protein. Hence DsbA represents an attractive antibacterial target and is the protein of interest for this project. The current thesis reports the identification of scaffolds that are suitable starting points for designing inhibitors of DsbA. These scaffolds were identified using a fragment-based drug design (FBDD) approach. This study describes a structure assisted-FBDD process performed to validate DsbA as an antibacterial drug target. A broad range of complementary techniques were employed to identify inhibitors of DsbA from two different Gram negative bacterial species; Escherichia coli and Vibrio cholerae. NMR based screening of a fragment library was carried out in two rounds. Theprimary screen employed STD (Saturation Transfer Difference) experiments to identify candidate hits and 1H-15N HSQC (Heteronuclear Single Quantum Coherence) experiments were used to confirm binding. A number of strong hits were identified and the binding locations were identified from the HSQC data. The binding efficiency of the fragments was determined by calculating NMR-based dissociation constants.Several of these initial binding fragments also show inhibitory activity in a phenotype assaysin E.coli. X-ray crystallographic studies revealed that the most potent fragment hits bind in the hydrophobic groove of EcDsbA in adjacent or overlapping positions. Charactisation of the structures of complexes of EcDsbA with theoriginal NMR hits was followed by fragment elaboration through structure-guided medicinal chemistry efforts. Preliminary structure-activity relationships(SAR) were identified for several series. This SAR will facilitate the development of novel DsbA inhibitors that specifically target functionally important protein surface sites. In conclusion, this study presentsthe discovery of novel small molecule inhibitors of DsbA and provides insights into the development of potential DsbA inhibitors as antibacterial drug candidates.