In vitro reconstitution of the coenzyme A biosynthesis pathway for the study of its regulation and disruption by possible inhibitors

Thesis (PhD)--Stellenbosch University, 2021.ENGLISH ABSTRACT: The coenzyme A (CoA) biosynthesis pathway has been validated as a novel target for antimicrobial drug development against numerous disease-causing organisms such as Mycobacterium tuberculosis (Mtb) and Staphylococcus aureus. It is therefore not surprising that various enzymes of the CoA biosynthesis pathways of these organisms have been the targets of multiple target-based drug discovery and development efforts. However, many of the inhibitors that have been identified and developed to target these enzymes have not translated into potent cell growth inhibitors, while the mechanisms of action of some of the most promising CoA-directed growth inhibitors, such as homopantothenate (HoPan) and the N-substituted pantothenamides (PanAms), are not completely understood. In this study we set out to address these knowledge gaps to provide better direction for CoA-directed antimicrobial drug development. We first show that the chemical modulator HoPan, which is used to reduce CoA levels in eukaryotic disease models, does not target PanK, but rather PPCS, after metabolic activation by PanK. Next, we considered the current approach to CoA-directed drug development and make the argument that potential inhibitors should be studied in a systems context that takes into account the flux control distribution among the pathway enzymes. To demonstrate the utility of such an approach, we developed a systems-based assay to identify and characterise CoaBC-targeted inhibitors that display different mechanisms of action. The assay was validated by using known inhibitors of the PPCS activities of the S. aureus and Mtb CoaBC proteins, and then used to investigate the potential of two promising CoaBC-targeted drug development strategies: one depending on metabolic activation by S. aureus PanK (SaPanK), and the other making use of prodrugs. For the PanAms, we found that these compounds can have different modes of action depending on their interaction with SaPanK. We show that the most potent PanAm growth inhibitors target CoA- utilising processes as their main mode of action, with PanAms that only inhibit the CoA biosynthesis pathway enzymes having reduced potency. Following this, we demonstrate the importance of determining the vulnerabilities of the CoA pathway enzymes in the specific organism being targeted as a key prerequisite for a drug development campaign as previously developed CoA-directed inhibitors that failed to show growth inhibition seem to target enzymes that were found to be less vulnerable to inhibition. We used an in vitro reconstituted S. aureus CoA biosynthesis pathway to determine the potential feedback regulation mechanisms of the S. aureus CoA biosynthesis pathway as well as the vulnerabilities of the individual pathway enzymes. We discovered that DPCK, the last enzyme in the pathway, is the most vulnerable target in the pathway, followed by PanK. CoaBC and PPAT, on the other hand, are not vulnerable targets. This study ultimately sheds new light on the mode of action of existing CoA-directed inhibitors while providing important new tools for directing future CoA-directed drug development efforts.AFRIKAANSE OPSOMMING: Die koënsiem A (KoA) biosintese padweg is reeds bevestig as ’n nuwe teiken vir die ontwikkeling van antimikrobiese middels teen verskeie patogeniese organismes soos Mycobacterium tuberculosis (Mtb) en Staphylococcus aureus. Dit is dus geen verrassing dat verskeie ensieme van die KoA biosintese padweë van hierdie organismes die teikens van verskeie teiken- gebaseerde inhibeerder-ontdekking en -ontwikkelingspogings was nie. Meeste van die inhibeerders wat ontwikkel was om die KoA biosintetiese ensieme te teiken het egter nie ook selgroei sterk inhibeer nie. Terselfdertyd word die wyse van inhibisie van die mees belowende klasse van KoA padweg gerigte inhibeerders, soos homopantoteensuur (HoPan) en die N- gesubstitueerde pantoteenamiede (PanAms), nog nie heeltemal verstaan nie. In hierdie studie wou ons dus ons kennis van KoA-geteikende antimikrobiese middelontwikkeling uitbrei om hierdie kennisgapings te vul. Eerstens wys ons dat die chemiese modulator HoPan, wat gebruik work om KoA vlakke in eukariotiese siektemodelle te verlaag, nie PanK teiken soos aanvanklik gedink is nie, maar wel PPCS na dié verbinding geaktiveer word deur PanK. Volgende het ons die huidige benadering vir KoA-geteikende inhibeerderontwikkeling ondersoek en voer die argument dat potensiële inhibeerders eerder in ‘n sisteem bestudeer moet word wat die verspreiding van die koers (flux) tussen die ensieme in ag neem. Om die nut van so ‘n sisteem te demonstreer het ons ‘n sisteemgebaseerde analise ontwikkel om inhibeerders wat CoaBC teiken (met verskeie inhibisiewyses) te identifiseer en te karakteriseer. Hierdie manier van analise is gevalideer deur bestaande inhibeerders van die PPCS aktiwiteite van die S. aureus en Mtb CoaBC proteïne te toets en ook om die potensiaal van twee belowende CoaBC-gebaseerde teenmiddelontwikkelings strategieë te bepaal: een wat afhanklik is van PanK-aktivering en ’n ander wat gebruik maak van pro-middels (“prodrugs”). Vir die PanAms het ons bevind dat hierdie verbindings verskeie wyses van inhibisie kan hê afhangende van hul interaksie met die S. aureus PanK. Ons wys dat die kragtigste PanAm groei-inhibitore prosesse wat KoA verbruik teiken en dat dit hul hoofwyse van inhibisie is, terwyl PanAms wat uitsluitlik die KoA biosintetiese ensieme teiken nie voldoende selgroeiinhibisie veroorsaak nie. Hierna wys ons hoe belangrik dit is om die kwesbaarheid van KoA biosintetiese ensieme te bepaal wat in ’n spesifieke organisme geteiken word en hoe dit ’n voorvereiste vir die teenmiddel-ontwikkelingsveldtog moet wees, siende dat verskeie bestaande KoA-geteikende inhibeerders wat nie tot selgroei-inhibisie lei nie, ensieme teiken wat minder kwesbaar is. Ons het ’n in vitro hersaamgestelde S. aureus KoA biosintese padweg gebruik om die moontlike terugvoer regulasie meganismes in hierdie padweg te bepaal, sowel as die kwesbaarhede van die individuele ensieme in die padweg. Ons het ontdek dat DPCK die mees kwesbare teiken in die padweg is, gevolg deur PanK. CoaBC en PPAT is nie kwesbare teikens nie. Hierdie studie het dus nuwe lig gewerp op die wyse van inhibisie van bestaande KoA- geteikende inhibeerders en verskaf ook belangrike nuwe instrumente om toekomstige KoA- geteikende teenmiddel ontwikkelingspogings te rig.Doctora