Browsing by Author "Moolman, Wessel Johannes Albertus"

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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.