Rational development of novel small molecule leads against the transcriptional activator protein ExsA of Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. It is present in aquatic, marine, and soil environments and can also be found in a variety of anthropological environments, including water distribution systems and hospitals. This prevalence is concerning due to P. aeruginosa’s status as an opportunistic pathogen, and the latter particularly so because it is a common cause of nosocomial infections. Burn victims and cystic fibrosis patients are particularly vulnerable to P. aeruginosa infections. The pathogen is highly virulent, and consequently it is a leading cause of death in intensive care units, especially among cases of ventilator associated pneumonia. Treating P. aeruginosa infections is complicated by its resistance to multiple drugs. Besides acquiring resistance mutations to antibiotic targets, the pathogen also possesses a suite of efflux pumps capable of exporting a wide range of antibiotics. P. aeruginosa has two distinct virulent lifestyles which correspond to acute and chronic infections. P. aeruginosa’s type three secretion system (T3SS) dominate in acute infection, and the expression of this system is regulated by the transcription factor ExsA. The latter therefore represents an attractive target for developing anti-virulence drugs against this pathogen. I have further characterised ExsA and its regulon with a proteomics experiment utilising deletion mutants which have an “ExsA always on” phenotype as well as an exsA deletion mutant. An extended ExsA regulon was revealed, including well known virulence factors such as HCN, and potential novel factors such as an uncharacterised non-ribosomal peptide synthase. Effectors of, and components for, the type six secretion system (associated with chronic infections and generally inversely regulated compared to the type three secretion system) were also identified as overexpressed in the “ExsA always on” mutants. Potential connections to other signalling systems are also examined. This work strengthened the case for ExsA as a therapeutic target, expanding its virulence inducing role beyond the T3SS. A number of other phenotypes, such as the downregulation of denitrification proteins, are also identified and validation is sought through phenotypic assays. ExsA is subsequently examined bioinformatically, and the inhibitors and ligands of related proteins (i.e. members of the AraC family of transcription factors) are examined for potential inhibitory effects. Due to dearth of potent inhibitors with well-characterised mechanism of action, some potential small molecule binding sites were predicted and subsequently utilised for in silico screening of commercial lead like libraries. Parallel to this, attempts were made to obtain the full-length crystal structure of ExsA which were ultimately unsuccessful. Several iterations of in silico docking experiments were performed, utilising a combination of published and modelled structures of the ExsA. This led to the identification of novel chemical scaffolds as potential binders against the chosen pocket of ExsA. Best hits from the in silico screening were then subjected to in vivo and biophysical analysis with mixed results. Due to the impact on Covid-19, the complete characterisation of those compounds was not feasible, though several of them appeared to be promising leads. Finally, a comprehensive effort to obtain an optimal structure of ExsA was undertaken. Whilst an experimental structure remained elusive, state of the art structural modelling was undertaken alongside all-atoms molecular dynamics simulations. Together the research presented in the thesis offers a firm foundation and several leads for further inhibitor discovery efforts against ExsA, as well as findings of biological significance concerning the full regulator effects of ExsA.
pseudomonas aeruginosa, T3SS, type three secretion system, virtual screening
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