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

Moolman, Wessel Johannes Albertus (2015-12)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

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.

AFRIKAANSE OPSOMMING: Staphylococcus aureus, die bakterium verantwoordelik vir die grootste hoeveelheid hospitaalverworwe infeksies in mense, is vinnig besig om meer wydverspreid in die gemeenskap voor te kom terwyl dit besig is om meer weerstandbiedig te raak teen die huidige arsenaal van antimikrobiese middels. Nuwe behandelinge word nou meer as ooit benodig om hierdie bedreiging te bekamp. In hierdie studie stel ons ‘n alternatiewe strategie voor teenoor huidige antibiotiese ontwikkelingsmetodologieë wat die indentifisering en teiken van prosesse behels wat essensieël is vir die oorlewing van patogeniese bakterieë binne hul menslike gasheer; of te wel, waar hulle nodig het om die verdedigingsmeganismes van die menslike immuunsisteem teen te werk. Dienooreenkomstig is die fokus van die studie die belangrikheid van die sentrale metaboliese kofaktor koënsiem A (KoA) in die verdedigingsmeganismes wat S. aureus gebruik onder hierdie toestande en gevolglik op die evaluering van KoA-biosintese en ensiemologie as potensiële antistafilokokale teikens. Die lewensvatbaarheid van koënsiem A disulfied reduktase (KoADR) as 'n potensiële antistafilokokale teiken is geëvalueer. Die S. aureus KoADR (SaKoADR) ensiemstrukture in kompleks met meganisme-gebaseerde Michael-akseptor-bevattende inhibeerders is ondersoek; spesifiek hoe die interaksie met hierdie verbindings betrekking het tot die waargenome verskille in hul aktiwiteite. Gevolglik kon die waargenome ensieminhibisie voldoende verduidelik word met inagneming van die chemiese eienskappe van die inhibeerders in kombinasie met hul interaksie met SaKoADR. Die strukturele data in die studie verskaf 'n sterk beginpunt vir toekomstige inhibitorontwerp. Die basis vir die swak korrelasie tussen die in vitro inhibisie van SaKoADR deur die Michael-akseptorbevattende KoA-analoë en die bakterieële groeiïnhibisie van S. aureus deur hulle ooreenstemmende pantoteenamied voorlopers is ondersoek en die resultate het aangetoon dat dit te wyte is aan die feit dat SaKoADR nie noodsaaklik onder normale groeitoestande is nie. Desnieteenstaande dui die resultate daarop dat onder omstandighede waar KoA-vlakke voldoende verminder is, KoADR dalk relevant sal word, selfs onder normale groeitoestande. Dit lê die grondslag vir studies op die moontlike sinergistiese effek van KoADR-inhibeerders met verbindings wat KoA-vlakke verminder; sulke kombinasies het waarskynlik die meeste potensiaal vir werk gefokus op KoADR as 'n antistafilokokale teiken. Die meganisme van inhibisie van fosfopantotenoïelsisteïen-sintetase (FPS) ensieme deur 4'-fosfo-CJ- 15,801-sitidilaat (FCJ-SMP) is ondersoek deur die bepaling van die basis vir die oënskynlike stabiliteit van die inhibeerder. Ons het bewys dat die FPS proteïen self geen rol speel in die meganisme van inhibisie deur FCJ-SMP nie, maar dat die invoeging van die dubbelbinding in die β-alanien groep van die substraat met sy ekstra π-elektrone die asielfosfaat weerstandbiedig maak teen nukleofiele-aanval deur die vorming van nuwe, stabiele resonansvorms. Die meganisme van stabilisering deur resonansie is ook toegepas op 'n onverwante stelsel en ons was in staat om substrate van menslike VNN1 panteteïnase te omskep in inhibeerders van die ensiem. Hierdie studies het ons in staatgestel om die styf-bindende inhibisie waargeneem vir FCJ-SMP te rasionaliseer. Daarbenewens stel ons ‘n nuwe strategie bekend waardeur β- alanien-bevattende verbindings bestand gemaak kan word teen hidrolise en / of asiel-oordrag; hierdie strategie kan waarskynlik ‘n wye toepassing hê in die ontwerp van sulke klein-molekule inhibeerders en terapieë.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97697
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