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Pseudomonas aeruginosa is one of the primary causes of opportunistic infections in humans and it is associated with both acute and chronic infections in immunocompromised individuals. This bacterium is extremely resistant to many antibiotics, making the treatments against this pathogen often unsuccessful. Azoreductases, a family of enzymes involved in the reduction of azo compounds and quinones, are found in many bacterial species including P. aeruginosa. Although the enzymatic activity of three of these enzymes has been extensively characterized, their physiological role remains unclear. In this study, the enzymatic activity as well as the effect on physiological processes such as swarming motility, biofilm formation and antibiotic resistance of known and putative azoreductase proteins from P. aeruginosa PAO1 have been investigated. Five putative azoreductase genes from P. aeruginosa PAO1 (pa0949, pa1204, pa2280, pa2580 and pa4975) were cloned and four of these (pa0949, pa1204, pa2280 and pa2580) were over expressed in E. coli strains. Recombinant proteins were purified and biochemically characterized showing the presence of FAD bound to PA1204 and PA2580 proteins. Enzymatic reaction conditions were established for each protein by determining the preferred cofactor and reductant (flavin and NAD(P)H) used by each protein. Higher reduction rates were obtained using FAD for PA1204 and PA2580, and FMN for PA0949 and PA2280, whereas NADPH was always the preferred reductant for all of the enzymes tested. Substrate specificity studies performed with azo compounds and quinones showed that PA1204, PA2280 and PA2580 recombinant proteins can reduce both classes of substrate, with higher reduction rates with quinones, whereas the recombinant PA0949 protein showed to reduce only quinone substrates. Investigation of the role of azoreductase genes on P. aeruginosa PAO1 motility, biofilm formation and antibiotic resistance was conducted using single gene transposon mutants for each of the genes paazor1, paazor2, paazor3, pa0949, pa1204, pa2280, pa2580 and pa4975. Motility analysis demonstrated greater swarming for all mutants tested compared with wild type, although both wild type and mutants showed the same growth rate. Similarly, all mutants showed higher biofilm production compared with the wild type in a short term period (24 hours), whereas no differences were observed after a longer biofilm production period (48 hours). The MICs and MBCs of several antibiotics were determined, showing that, in presence of fluoroquinolones, P. aeruginosa PAO1 azoreductase mutants exhibit higher growth inhibition (up to 127-fold) and reduced survival (up to 7 fold) compared with wild type. This suggests that azoreductase genes (or gene products) may be involved in the P. aeruginosa PAO1 resistance to antibiotic treatments, and in particular to fluoroquinolones. The findings prove that the proteins PA0949, PA1204, PA2280 and PA2580 have similar features and enzymatic functions to the already characterized paAzoR1-3 from P. aeruginosa PAO1. Therefore, these can be included in the family of azo- and quinone- oxidoreductase enzymes. The data presented here on the antibiotic resistance strongly suggest a role for azoreductase gene products in antimicrobial resistance in P. aeruginosa. These original findings provide a springboard for further investigation of azoreductases as novel targets for antimicrobial agents for this pathogen.

This thesis describes the design, synthesis, analysis, mechanistic evaluation and optimisation of small molecule probes for the specific detection of proteins, focusing on the target protein human arylamine N-acetyltransferase type 1 (HUMAN(NAT1)) and its murine homologue, mouse arylamine N-acetyltransferase type 2 (MOUSE(NAT2)). The HUMAN(NAT1) gene is reported to be one of the most highly overexpressed genes in estrogen-receptor-positive (ER+) breast tumours, leading to its potential use as both a novel diagnostic biomarker and a novel therapeutic target for this disease. Chapter 1 reviews the literature on optical methods for the specific detection of a protein target, exploring strategies both based on biosensors and on chemical probes, before introducing the arylamine N-acetyltransferases as a family of enzymes. In Chapter 2, a family of naphthoquinone inhibitors of HUMAN(NAT1) are introduced, which undergo a colour change from red to blue upon binding specifically to the enzyme. The mechanism of this colour change, a proton transfer-mediated process, is discussed via the synthesis, pharmacological and colorimetric evaluation of close analogues of the hit compound lacking a key acidic sulfonamide-NH proton. During these studies, it was found that direct O-methylation of a sulfonamide is possible under certain conditions; such a reaction has not previously been reported. Furthermore, upon heating in polar solvents the O-methylated sulfonamide was observed to undergo rearrangement, and the mechanism of this process is investigated via NMR and kinetic studies. In Chapter 3, the design, synthesis and evaluation of HUMAN(NAT1) inhibitors with improved pharmacological and colorimetric profiles over the initial hit are described. From this optimisation, structure-activity relationships and an in silico model of interactions between the inhibitors and enzyme are evaluated. Testing of these compounds in cellular environments, however, exposes some limitations of this approach, notably the lack of sensitivity of the probes when dosed at low concentrations in cellular samples. In order to overcome this limitation, in Chapter 4 fluorescent analogues of the hit compound are designed and synthesised. Initial compounds developed in this series possess promising properties, but each compound generated suffers from either a low fluorescent intensity, lack of a pH-dependent switch in fluorescence or a low fluorescence excitation wavelength, which overlaps with those of tryptophan or tyrosine residues in proteins. Insights into the mechanism of molecular fluorescence and application of some simple quantum mechanical principles, however, lead to the design of a species which possesses all the required properties. The fluorescent emission intensity of this probe correlates linearly with [MOUSE(NAT2)] in E. coli cell extracts, and can quantify as little as 0.64% MOUSE(NAT2) in the samples; furthermore, the probe is capable of unambiguously detecting HUMAN(NAT1) within a cell extract from the ER+ breast cancer cell line ZR-75-1; future work on this probe may therefore enable its clinical use in improved early diagnosis of breast tumours. This study also represents, to the best of our knowledge, the first ever example of a small molecule, non-covalent probe capable of quantifying the concentration of a target protein in cellular extracts. In Chapter 5, the series of naphthoquinone probes is further optimised in order to study the roles of HUMAN(NAT1) in a cellular environment. Firstly, structure-activity relationships are utilised to design inhibitors with improved physical properties such as aqueous solubility and cell membrane permeability, in order to test the effect of HUMAN(NAT1) inhibitors in tumour cell models, which could have implications for the future use of a HUMAN(NAT1) inhibitor as a therapeutic agent in oncology. Secondly, the effect of the cofactor folic acid on the function and activity of HUMAN(NAT1) is explored. Finally, in Chapter 6, the conclusions of this study are outlined and a hypothesis as to how the concepts developed in this thesis might be applied to alternative, more ubiquitous biological targets is discussed, paving the way for future investigations.