Doctoral Degrees (Biochemistry)

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Browsing Doctoral Degrees (Biochemistry) by Subject "Anti-infective agents"

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    Coenzyme A biosynthesis and Coenzyme A-dependent redox processes as targets for anti-staphylococcal drug development
    (Stellenbosch : Stellenbosch University, 2015-12) Moolman, Wessel Johannes Albertus; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH SUMMARY: Staphylococcus aureus, the bacterium that causes most hospital-acquired in humans is rapidly becoming more prevalent in the community and, alarmingly, increasingly resistant to the current arsenal of available antibacterial agents. More than ever, new treatments are urgently needed to combat this threat. In this study we proposed an alternative strategy to current drug development methodologies that entails the identification and targeting of processes that are essential to the survival of pathogenic bacteria in their human host, i.e. where they need to counter the defences of the human immune system. In particular, the focus of this study is the importance of the central metabolic cofactor coenzyme A (CoA) in the defence mechanisms that S. aureus employs under such circumstances, and therefore on the targeting of CoA biosynthesis and enzymology as potential antistaphylococcal targets. The viability of coenzyme A disulfide reductase (CoADR) as a potential antistaphylococcal drug target was evaluated. The S. aureus CoADR (SaCoADR) enzyme structures in complex with mechanism-based Michael acceptor-containing inhibitors were examined; specifically how its interaction with these compounds relates to the observed differences in activity between them. Consequently, the observed enzyme inhibition could be adequately explained when taking into account the chemical properties of the inhibitors in combination with their interactions with SaCoADR. Also, the structural data in the study provided a strong starting point for future inhibitor design. The reasons for the poor correlation between the in vitro inhibition of SaCoADR by the Michael acceptor-containing CoA analogues and the whole cell inhibition of S. aureus by their corresponding pantothenamide precursors were investigated and these results led us to the conclusion that the poor correlation is due to SaCoADR not being essential under normal growth conditions. However, our results suggest that under conditions where CoA levels are sufficiently reduced, CoADR might become relevant, even under normal growth conditions. This opens the door for studies on the possible synergistic effects of CoADR inhibitors and compounds that reduce CoA levels; such combinations most likely hold the most potential for work focused on CoADR as a drug target. The mechanism of inhibition of phosphopantothenoylcysteine synthetase (PPCS) enzymes by 4’-phospho- CJ-15,801-cytidylate (PCJ-CMP) was investigated by determining the basis for the apparent stability of the inhibitor. We showed that the PPCS protein itself plays no role in the mechanism of inhibition by PCJ-CMP, but that the introduction of the double bond in the β-alanine moiety of the substrate with its extra π-electrons renders the acyl phosphate resistant to nucleophilic attack by introducing new, stable resonance forms. This mechanism of apparent stabilisation via resonance was also applied to an unrelated system and we were able to convert substrates of human VNN1 pantetheinase into inhibitors of the enzyme. These studies allowed us to rationalise the tight-binding inhibition observed for PCJ-CMP. Additionally, we uncovered a new strategy whereby β-alanine-containing compounds can be rendered resistant to hydrolysis and/or acyl transfer; this strategy can likely have wide-ranging applications in the design of such small molecule inhibitors and therapeutics.
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    A comparative analysis of CoA biosynthesis in selected organisms: a metabolite study
    (Stellenbosch : Stellenbosch University, 2016-03) Goosen, Rene; Strauss, Erick; Snoep, Jacky L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH SUMMARY: This study investigated the biochemical regulation of CoA production because it is an essential pathway that presents an important target for antimicrobial drug discovery studies. Currently, the specific life-sustaining functions of CoA are not clearly defined and a better understanding of the regulation of the CoA biosynthesis pathway would aid in the understanding of the relevance of maintaining specific CoA levels for survival. Regulation of CoA production was investigated on two levels. First, it was determined if production is up-regulated under conditions predicted to be associated with increased demand in S. aureus. Second, regulation of production of CoA by the salvage pathway in E. coli was investigated. In S. aureus it was found that CoA production is up-regulated under conditions of oxidative stress by an as yet unidentified mechanism. This led to an investigation of the regulation in the CoA biosynthesis pathway to understand how production is controlled. At present, the regulation of CoA production is thought to occur at a single, rate-limiting step identified as the first enzyme in the pathway, pantothenate kinase (PanK). Failure of inhibition of PanK to result in growth inhibition suggested that a re-evaluation of this premise is required. To this end, a systems analysis approach was taken in this study to elucidate the control of CoA production by the salvage pathway. Previously, a lack of analytical tools to measure the intermediates of CoA biosynthesis hampered investigations into regulation of the pathway and a holistic study has not been performed to elucidate the control profile. Consequently a method was also developed for the quantification of all the intermediates of the CoA salvage pathway based on derivatization with a fluorescent thiol probe and HPLC analysis. This method allowed for time course analysis of the reconstituted pathway to be performed to provide a holistic interpretation of CoA production. A kinetic model of the pathway was constructed from rate equations parameterized with a combination of experimentally determined values and values reported in the literature. Time course profiles were used to validate the model for subsequent control analyses. Both time course profiles and predictions made by the model indicated that PanK is unlikely to control the rate of CoA production under most conditions, and that it is in fact dephospho-CoA kinase (DPCK), the last enzyme in the pathway, that controls the rate under physiological conditions. This implies that DPCK is the best target for inhibition of the CoA biosynthetic pathway because it is far more likely to be in control of the rate of CoA production under physiological conditions. This finding is significant to antimicrobial drug development efforts because it suggests that the target focus should be shifted from PanK to DPCK. Therefore the findings of this study represent a major shift in our current understanding of the regulation of the rate of CoA production. It also highlights the importance of conducting a detailed systems analysis when studying metabolic pathways from both regulatory and drug development perspectives.
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    Synthesis, profiling and mode of action studies of PanSulfAms as inhibitors of coenzyme a biosynthesis and utilization
    (Stellenbosch : Stellenbosch University, 2019-12) Jana, Collins Edward; Strauss, Erick; De Villiers, Marianne; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Though tremendous progress has been made in the development of antibacterial drugs, the evolution of antimicrobial resistance is a serious problem that is affecting the successful prevention and treatment of various infections caused by microbial pathogens. Taking this into consideration, the need for new antimicrobials is of paramount importance. Since the B-vitamin pantothenate promotes the growth of microbes, analogues of this compound that may act as antimetabolites have been synthesized and tested for their inhibitory properties against bacterial growth. N-substituted pantoyltauramides (PanSulfAms) are a class of pantothenate analogues previously synthesized by various research groups in the 1940s. These analogues demonstrated promising inhibition of the proliferation of avian malaria parasites as well as showing good antibacterial activities against Streptococcus pyogenes. In this study, the chemical and structural diversity of these analogues was expanded by preparing PanSulfAms in which various amide moieties were introduced to the sulfonic acid group of taurine. These compounds were subsequently used to produce fourteen PanSulfAms that were investigated for their potential as antibacterial agents. The growth inhibitory activities of the synthesized PanSulfAms were first investigated against Escherichia coli and Staphylococcus aureus both as models of Gram-negative and Gram-positive bacteria respectively, and because these compounds have never been investigated on these microbes. We also investigated the link between the organisms’ pantothenate kinase (PanK) types and the observed growth inhibition to ascertain whether this relates to the compounds’ mode of action. In this study, we have shown for the first time that PanSulfAms have the potential to inhibit S. aureus growth, but not that of E. coli. In addition, we have also performed the first detailed comparative kinetic analysis of PanSulfAms that cause S. aureus growth inhibition, demonstrating that PanK type directs the mode of action of these compounds. Finally, the PanSulfAms were studied for their ability to inhibit CoA biosynthesis. We show that their impact is compound-specific and relates to their interaction with the various CoA biosynthetic enzymes. We demonstrate for the first time that certain phosphorylated PanSulfAms act as inhibitors of the PPCS activity of the bifunctional CoaBC protein, and that, in combination with a specific interaction with the S. aureus PanK, has strong negative impact on the rate of CoA biosynthesis that likely contributes to their overall mode of action.