Browsing by Author "Brigg, Siobhan Ernan"

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    Expanding the scope of bacterial pantothenate kinase inhibitors for application in antibacterial drug development
    (Stellenbosch : Stellenbosch University, 2021-03) Brigg, Siobhan Ernan; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.
    ENGLISH ABSTRACT: Pantothenate kinase (PanK) is the first enzyme of the universal coenzyme A (CoA) biosynthesis Pathway. There are three main types of PanK: type I and type III PanKs are found in prokaryotes while type II is predominantly found in eukaryotes, with one exception being Staphylococcus aureus, a pathogen which commonly causes drug resistant infections. Many other drug resistant pathogens possess a type III PanK; these include including Acinetobacter baumannii (Ab), Pseudomonas aeruginosa (Pa) and Helicobacter pylori (Hp). The CoA biosynthesis enzymes, and PanK in particular, have been highlighted as potential drug targets for the development of novel antimicrobials. For S. aureus, inhibitors of its type II PanK (SaPanK-II) — known as pantothenamides (PanAms) — have been developed, but inhibition of SaPanK-II’s Pan phosphorylation activity has been shown to not be the only mechanism by which these compounds cause growth inhibition. PanAms are also substrates for SaPanK-II, and through transformation by it and two other enzymes in the CoA biosynthesis pathway, they are converted to CoA-antimetabolites which have been shown to inhibit CoA-dependent processes, including fatty acid biosynthesis. In this thesis, we explored the relative contribution of SaPanK-II inhibition vs it acting as a metabolic activator in the mode of action of the PanAms. This was done by investigating the extent to which CoA production is interrupted in the presence of these PanAms, and by investigating the rate at which the CoA antimetabolites are produced. In both cases, the work was done by performing extensive HPLC-based analysis of the reconstituted S. aureus CoA pathway, and by using a systems-based analysis to understand the results. The findings show that various PanAms interact differently with SaPanK-II, and this impacts on the rates of production of the corresponding CoA metabolite, and the extent to which CoA production is inhibited. Overall, the results of this study indicate that the mode of action of each PanAm appears to be weighted differently towards SaPanK-II inhibition and CoA-antimetabolite production. Unfortunately, PanAms suffer from two major drawbacks. First, their complex, ambiguous mode of action make their development and optimisation difficult, and second, they are inactivated by the ubiquitous pantetheinases found in human serum. For these reasons, another a further study reported in this thesis was to evaluate different approaches to developing novel SaPanK-II inhibitors that are not based on the PanAm scaffold. We evaluated the value of drug repurposing and optimisation of ibuprofen, since it has been shown to be an antistaphylococcal agent and there was some evidence that this activity may be related to SaPanK-II inhibition. Unfortunately, this was found not to be the case; however, the pH-dependent antistaphylococcal activity of ibuprofen was established and derivatives were designed to improve activity at physiological pH. Hybrid ibuprofen- PanAms, where the ibuprofen carboxylate was exchanged for the N-substituent of the PanAms led us to a compound which showed low level SaPanK-II inhibition (EC50 ~100 μM). This compound could potentially be optimised as a novel SaPanK-II inhibitor but in its current form it suffers from solubility issues. We also investigated the use of high throughput virtual screening (HTVS). However, it was found that these methods need to be carefully designed for them to yield useful results. Truncated derivatives of the most potent SaPanK-II inhibitors were designed, synthesised and tested for SaPanK-II inhibition to determine which functionalities need to be maintained when designing novel inhibitors. It was found that truncation from the pantoyl moiety of PanAms gave compounds which were inactive as inhibitors of SaPanK-II. Finally, we investigated the development of an in vitro high throughput screening (HTS) assay for the identification of SaPanK-II inhibitors based on fluorescence polarisation. Unfortunately, we were not successful in developing a fluorescent probe with the required characteristics to be useful for such an assay. However, the synthesis of a carbostyril-based fluorescent probe was been investigated and can be implemented in a future project. Compared to the rational design approaches, HTS of large compound libraries is a more attractive option for finding novel scaffolds for the development of SaPanK-II inhibitors as it bypasses the trial-and-error approach of rational design. In a final project, we investigated the potential for PanK-IIIs as selective drug targets for the development of novel narrow spectrum antimicrobials. Several PanK-IIIs were compared by performing multiple sequence alignment and phylogenetic analysis, and an active site analysis based on residue–ligand interactions and active site shape and form. This information was used to determine the extent of the variations between the different PanK-III enzymes. To establish whether this analysis had predictive value in proposing inhibitor scaffolds for other PanK-IIIs, substrate and inhibitor screening assays were performed with various pantothenic acid analogues. The results show that more data is needed to improve the predictive ability of the analysis, as different PanK-IIIs showed significant divergence in their ability to accept different substrates, and their sensitivity to substrate-like inhibitors. Overall, the results reveal the potential for the development of specific PanK-III inhibitors as narrow-spectrum antimicrobials.
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    Lead optimisation of an indole based HIV-1 non-nucleoside reverse transcriptase inhibitor
    (Stellenbosch : Stellenbosch University, 2017-12) Brigg, Siobhan Ernan; Pelly, Stephen, C.; Blackie, Margaret A. L.; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.
    ENGLISH ABSTRACT: HIV-1 remains the worst pandemic faced by mankind since its discovery as the causative agent of AIDS in the early 1980s. An enormous amount of research has been done to find a cure, but to date there has been no success and resistance is widespread among the available treatment. This project focused on the development of novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) using a rational design approach. The lead compound, ethyl 5-chloro-3-(methoxy(phenyl)methyl)-1H-indole-2-carboxylate, was shown to have low nano-molar potency against HIV-1 (IC50 = 16 nM), however it had two main shortcomings which needed to be addressed; poor resistance profile and poor acid stability. Previous research had shown the resistance profile could be improved by introducing meta substitution on the phenyl moiety which interacts with Tyr181 of the NNRTI binding pocket (NNIBP). We were successful in synthesising several meta substituted phenyl derivatives of the lead compound and these were shown to be equally as potent as the lead compound. Their activity against resistant strains is yet to be determined as we are awaiting the results from biological testing. The presence of an acid labile methyl ether functionality on the molecule which was susceptible to an acid catalysed indole mediated SN1 substitution in aqueous acidic medium meant that the lead compound could never be considered as a candidate for an orally available drug. The methyl ether moiety was exchanged for a sulfide moiety and several of these derivatives were successfully synthesised. Acid stability tests showed that we were successful in our endeavour to improve the acid stability, offering an advantage over the lead compound despite a slight reduction in potency. However to completely eliminate the possibility of substitution, we replaced the methyl ether moiety for an ethyl group, successfully synthesising ethyl 5-chloro-3-(1-phenylpropyl)-1H-indole-2-carboxylate and 5-chloro-3-(1-phenylpropyl)-1H-indole-2-carboxamide and we are currently awaiting the results from biological testing to determine whether this derivative is active against HIV-1. The functionality in the 2-position of the indole was also investigated through the synthesis of 5-chloro-3-(methoxy(phenyl)methyl)-1H-indole and 5-chloro-3-((methylthio)(phenyl)methyl)-1H-indole. These derivatives lacking a group in the 2-position of the indole showed significant reduction in potency. Replacement of the ethyl ester for an isobutyl ester to give isobutyl 5-chloro-3-((3,5-dimethylphenyl)(methylthio)methyl)-1H-indole-2-carboxylate, showed some maintenance of potency, however the larger side chain was not well accommodated in the NNIBP. The presence of a chiral centre on the lead compound, and all derivatives synthesised in the project, resulted in our final aim; we set out to develop a method for resolving these enantiomers. Unfortunately, although we employed a variety of different strategies, including the use of chiral auxiliaries and the classical resolution method of attempting to make diastereomeric salts, we were not successful in achieving this aim.