Grossman, Scott
(2022)
Novel approaches to tackling antimicrobial resistance: the design, synthesis and evaluation of quorum sensing inhibitors in Pseudomonas aeruginosa.
PhD thesis, University of Nottingham.
Abstract
The bacterium Pseudomonas aeruginosa is a nosocomial pathogen, which causes infection in immunocompromised patients. Its high levels of antibiotic resistance make P. aeruginosa infections difficult to treat, and examples of pan-resistant P. aeruginosa have been observed. P. aeruginosa contains three intertwined quorum sensing (QS) systems, which aid in the onset and continuation of infection through community-wide communication. The las, rhl and pqs systems, which are controlled by their respective transcriptional regulators LasR, RhlR and PqsR, regulate the production and detection of signal molecules, as well as other virulence factors such as the toxins pyocyanin and HCN. Therefore, inhibition of QS circuits can lead to therapeutic benefits and shows promise as a method of decreasing the occurrence of drug-resistant microorganisms.
A series of tri-substituted ureas, bearing a phenyl, benzyl and thiazoline moiety were developed in order to target inhibition of the transcriptional regulator of the rhl system, RhlR. In total 24 compounds were synthesised, including resynthesizing a hit compound identified through virtual and in vitro screening of an in-house compound library. The hit compound was found to inhibit RhlR with an IC50 value of 33.5 micromolar in a whole cell, luminescence-based bioreporter assay of strain PAO1-L rhlI mCTX::rhlI-luxCDABE. Aside from this compound, only one other compound in the series showed any activity, with an IC50 value of 25.9 micromolar. However, due to an overall lack of activity across the whole series, the work was not continued any further.
A further high-throughput screening of the library identified a quinazolinone bearing a thiazole moiety as an inhibitor of PqsR, with an IC50 value of 1.6 micromolar in a whole cell, luminescence-based bioreporter assay of strain PAO1-L mCTX::PpqsA-luxCDABE. A structure-activity relationship (SAR) study enabled optimisation of this scaffold to generate two lead compounds bearing linear alkyl chains in place of the isopropyl unit, showing activities of 343 nM and 265 nM in an analogous bioreporter assay using the clinically relevant strain PA14.
X-ray crystallography studies produced crystal structures of the original hit compound, in addition to both lead compounds and a further compound in the series bearing a tert-butyl group, where each compound differed in the alkyl chain at the thiazole 2-position. Moreover, the lead compounds were shown to significantly reduce the production of the toxin pyocyanin, and both displayed an excellent therapeutic window, with minimal cytotoxicity against the A549 lung epithelial cell line up to 100 micromolar.
An attempt was made to evolve these PqsR inhibitors further, by developing them into dual inhibitors against PqsR and an epigenetic target, histone deacetylases (HDAC), which are implicated in P. aeruginosa’s ability to modulate host immunity. One target compound was successfully synthesised through addition of a 2-fluorophenone group to the terminus of the alkyl chain (Figure iii), and was shown to be a potent PqsR inhibitor (IC50 in PA14 = 122 nM), but showed no HDAC inhibitory properties. No dual PqsR/HDAC inhibitors were successfully synthesised, though possible modifications to elicit this effect include replacing the ketone with an amide, and the fluoro group with an aniline. The thiazole group could also be removed for synthetic tractability.
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