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|Biotin protein ligase inhibitors as new antibacterial agents to target Staphylococcus aureus: Studies of efficacy, mechanism of action and resistance
|Hayes, Andrew James
|School of Biological Sciences
|There is a desperate need for new antibacterials to combat the growing threat of antimicrobial resistant infections. One of the most common causes of such infections is the Gram-positive pathogen Staphylococcus aureus. S. aureus is responsible for the majority of hospital based infective deaths, with strains resistant to even last resort antibacterial agents. To combat these infections novel antibiotic agents are required. However, few novel antibacterial agents have been approved in the past 4 decades, with most products being derivatives of previously used chemical classes. As a result, resistance mechanisms are often already present in bacteria, or develop rapidly after introduction. Antibacterial agents with novel mechanism of action against S. aureus are needed to address this crisis. One potential target for the development of novel antibacterials is the essential enzyme biotin protein ligase (BPL). The BPL of S. aureus serves two major functions. Firstly, BPL catalyses attachment of the vitamin biotin onto biotin-dependent enzymes, such as acetyl-CoA (ACC) and pyruvate carboxylase (PC). Secondly BPL is a transcriptional repressor that regulates expression of proteins required for biotin biosynthesis and biotin transport. Previous work in our laboratory and others has sought to generate inhibitors that target BPL catalytic activity, however little is known about their effect on transcriptional repression. As a result, several BPL inhibitors with a variety of chemical scaffolds are described in the literature focusing on creating mimics of the reaction intermediate biotinyl-5′-AMP. Despite success in creating antibacterial inhibitors, a selective compound that completely inhibits bacterial growth has proved elusive. The most potent and selective anti-staphyloccocal compound, biotin-triazole, shows promising inhibitory activity (Ki = 90 nM) but is unable to completely inhibit bacterial growth. The first aim of this thesis is to characterise three distinct chemical classes of BPL inhibitors designed to improve whole cell antibacterial activity. Each series of inhibitors was tested to determine in vitro potency and whole cell efficacy through enzyme and antibacterial susceptibility assays. The first and second compound series are modifications of the previously described biotin-triazole pharmacophore. Series 1 describes the shortening of the triazole pharmacophore with a view to producing a refined pharmacophore with greater drug like properties. For a similar purpose series 2 was created by halogenation of the C5 position on the triazole ring. Improvements in whole cell activity were achieved in series 2, yielding our first biotin-triazole inhibitor to completely inhibit bacterial growth (MIC = 8μg/ml). The third series tested a separate sulfonyl based compound series and provided the most potent BPL inhibitor and antibacterial to date. This compound, BPL 199, exhibited a sub-nanomolar Ki (0.7 nM) and an MIC of < 0.5 μg/ml against a panel of S. aureus isolates. Importantly, BPL199 also showed no cytotoxicity against two human cell lines, HepG2 and HEK293, and was well tolerated in mouse models. The second aim of this thesis was to validate the mechanism of antibacterial action of BPL inhibitors and investigate potential mechanisms of resistance in S. aureus. To validate the mechanism of action, a BPL overexpression strain was constructed in S. aureus strain RN4220 and used to test the effect of increased BPL on compound efficacy. This system was used against the most potent compounds from all three series. The assay confirmed that BPL inhibitors exerted antibacterial effects through inhibition of the BPL enzyme for the most potent antibacterial compounds. To determine resistance mechanisms to BPL inhibitors in S. aureus several individual isolates of S. aureus NCTC 8325 were exposed to sub-optimal concentrations of BPL199 to evolve resistance. Whole genome sequencing of the strains resistant to BPL199 was then undertaken to identify potential resistance mechanisms. The mutations present occurred in a diverse range of genes including BPL and the biotin dependent enzyme pyruvate carboxylase. The one missense mutation in BPL (D200E) was further explored with both in vitro and in vivo testing. This amino acid substitution was found to not greatly affect catalytic activity, reducing the affinity for biotin by 2-fold (Km wt = 1.8 ± 0.3 μM , D200E = 3.8 ± 0.4 μM) and did not affect repression by the inhibitor (Ki wt = 4.8 ± 2.1 nM , D200E =10.9 ± 3.5 nM). However native mass spectrometry was able to show that the substitution abolished dimerization of the BPL in vitro and EMSA showed that this resulted in reduced DNA binding activity. Further in vivo testing, with a chromosomally integrated lacZ reporter assay in E. coli, demonstrated that the mutation was sufficient for dysregulation of the biotin transport and synthesis genes with a greater than 50 – fold increased biotin concentration required to facilitate repression of biotin transport. Several of the other mutations identified, such as the one in PC, were likely to induce loss of function. The individual effect of a loss of function in these genes was further explored using transposon mutagenesis knock-out strains. The effect of individual loss of function mutations in pyruvate carboxylase in S. aureus JE2 determined that this mutation alone was sufficient for a four-fold decrease in susceptibility to BPL199. In summary this thesis has looked at the development of BPL inhibitors for the purpose of antibacterial drug discovery. Using the most potent compounds the mechanism of action and resistance to BPL inhibitors was also characterised. This work will help in the future design of BPL inhibitors with the resistance mechanisms suggesting potential pathways that are important for BPL inhibitor resistance.
|Thesis (MPhil) -- University of Adelaide, School of Biological Sciences, 2017
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