Coenzyme A biosynthesis and Coenzyme A-dependent redox processes as targets for anti-staphylococcal drug development

[摘要] ENGLISH SUMMARY: Staphylococcus aureus, the bacterium that causes most hospital-acquired in humans is rapidly becomingmore prevalent in the community and, alarmingly, increasingly resistant to the current arsenal of availableantibacterial agents. More than ever, new treatments are urgently needed to combat this threat. In this studywe proposed an alternative strategy to current drug development methodologies that entails the identificationand 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 studyis 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 andenzymology as potential antistaphylococcal targets.The viability of coenzyme A disulfide reductase (CoADR) as a potential antistaphylococcal drug target wasevaluated. The S. aureus CoADR (SaCoADR) enzyme structures in complex with mechanism-based Michaelacceptor-containing inhibitors were examined; specifically how its interaction with these compounds relatesto the observed differences in activity between them. Consequently, the observed enzyme inhibition could beadequately explained when taking into account the chemical properties of the inhibitors in combination withtheir interactions with SaCoADR. Also, the structural data in the study provided a strong starting point forfuture inhibitor design. The reasons for the poor correlation between the in vitro inhibition of SaCoADR bythe Michael acceptor-containing CoA analogues and the whole cell inhibition of S. aureus by theircorresponding pantothenamide precursors were investigated and these results led us to the conclusion thatthe poor correlation is due to SaCoADR not being essential under normal growth conditions. However, ourresults suggest that under conditions where CoA levels are sufficiently reduced, CoADR might becomerelevant, even under normal growth conditions. This opens the door for studies on the possible synergisticeffects of CoADR inhibitors and compounds that reduce CoA levels; such combinations most likely hold themost 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 theinhibitor. 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-electronsrenders the acyl phosphate resistant to nucleophilic attack by introducing new, stable resonance forms. Thismechanism of apparent stabilisation via resonance was also applied to an unrelated system and we wereable to convert substrates of human VNN1 pantetheinase into inhibitors of the enzyme. These studiesallowed us to rationalise the tight-binding inhibition observed for PCJ-CMP. Additionally, we uncovered anew strategy whereby β-alanine-containing compounds can be rendered resistant to hydrolysis and/or acyltransfer; this strategy can likely have wide-ranging applications in the design of such small moleculeinhibitors and therapeutics.

[发布机构] Stellenbosch University