Expanding the scope of bacterial pantothenate kinase inhibitors for application in antibacterial drug development
| dc.contributor.advisor | Strauss, Erick | en_ZA |
| dc.contributor.author | Brigg, Siobhan Ernan | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. | en_ZA |
| dc.date.accessioned | 2021-03-08T14:22:47Z | en_ZA |
| dc.date.accessioned | 2021-04-22T10:12:54Z | en_ZA |
| dc.date.available | 2022-09-30T03:00:12Z | en_ZA |
| dc.date.issued | 2021-03 | en_ZA |
| dc.description | Thesis (PhD)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Pantothenate kinase (PanK) is the first enzyme of the universal coenzyme A (CoA) biosynthesis Pathway. There are three main types of PanK: type I and type III PanKs are found in prokaryotes while type II is predominantly found in eukaryotes, with one exception being Staphylococcus aureus, a pathogen which commonly causes drug resistant infections. Many other drug resistant pathogens possess a type III PanK; these include including Acinetobacter baumannii (Ab), Pseudomonas aeruginosa (Pa) and Helicobacter pylori (Hp). The CoA biosynthesis enzymes, and PanK in particular, have been highlighted as potential drug targets for the development of novel antimicrobials. For S. aureus, inhibitors of its type II PanK (SaPanK-II) — known as pantothenamides (PanAms) — have been developed, but inhibition of SaPanK-II’s Pan phosphorylation activity has been shown to not be the only mechanism by which these compounds cause growth inhibition. PanAms are also substrates for SaPanK-II, and through transformation by it and two other enzymes in the CoA biosynthesis pathway, they are converted to CoA-antimetabolites which have been shown to inhibit CoA-dependent processes, including fatty acid biosynthesis. In this thesis, we explored the relative contribution of SaPanK-II inhibition vs it acting as a metabolic activator in the mode of action of the PanAms. This was done by investigating the extent to which CoA production is interrupted in the presence of these PanAms, and by investigating the rate at which the CoA antimetabolites are produced. In both cases, the work was done by performing extensive HPLC-based analysis of the reconstituted S. aureus CoA pathway, and by using a systems-based analysis to understand the results. The findings show that various PanAms interact differently with SaPanK-II, and this impacts on the rates of production of the corresponding CoA metabolite, and the extent to which CoA production is inhibited. Overall, the results of this study indicate that the mode of action of each PanAm appears to be weighted differently towards SaPanK-II inhibition and CoA-antimetabolite production. Unfortunately, PanAms suffer from two major drawbacks. First, their complex, ambiguous mode of action make their development and optimisation difficult, and second, they are inactivated by the ubiquitous pantetheinases found in human serum. For these reasons, another a further study reported in this thesis was to evaluate different approaches to developing novel SaPanK-II inhibitors that are not based on the PanAm scaffold. We evaluated the value of drug repurposing and optimisation of ibuprofen, since it has been shown to be an antistaphylococcal agent and there was some evidence that this activity may be related to SaPanK-II inhibition. Unfortunately, this was found not to be the case; however, the pH-dependent antistaphylococcal activity of ibuprofen was established and derivatives were designed to improve activity at physiological pH. Hybrid ibuprofen- PanAms, where the ibuprofen carboxylate was exchanged for the N-substituent of the PanAms led us to a compound which showed low level SaPanK-II inhibition (EC50 ~100 μM). This compound could potentially be optimised as a novel SaPanK-II inhibitor but in its current form it suffers from solubility issues. We also investigated the use of high throughput virtual screening (HTVS). However, it was found that these methods need to be carefully designed for them to yield useful results. Truncated derivatives of the most potent SaPanK-II inhibitors were designed, synthesised and tested for SaPanK-II inhibition to determine which functionalities need to be maintained when designing novel inhibitors. It was found that truncation from the pantoyl moiety of PanAms gave compounds which were inactive as inhibitors of SaPanK-II. Finally, we investigated the development of an in vitro high throughput screening (HTS) assay for the identification of SaPanK-II inhibitors based on fluorescence polarisation. Unfortunately, we were not successful in developing a fluorescent probe with the required characteristics to be useful for such an assay. However, the synthesis of a carbostyril-based fluorescent probe was been investigated and can be implemented in a future project. Compared to the rational design approaches, HTS of large compound libraries is a more attractive option for finding novel scaffolds for the development of SaPanK-II inhibitors as it bypasses the trial-and-error approach of rational design. In a final project, we investigated the potential for PanK-IIIs as selective drug targets for the development of novel narrow spectrum antimicrobials. Several PanK-IIIs were compared by performing multiple sequence alignment and phylogenetic analysis, and an active site analysis based on residue–ligand interactions and active site shape and form. This information was used to determine the extent of the variations between the different PanK-III enzymes. To establish whether this analysis had predictive value in proposing inhibitor scaffolds for other PanK-IIIs, substrate and inhibitor screening assays were performed with various pantothenic acid analogues. The results show that more data is needed to improve the predictive ability of the analysis, as different PanK-IIIs showed significant divergence in their ability to accept different substrates, and their sensitivity to substrate-like inhibitors. Overall, the results reveal the potential for the development of specific PanK-III inhibitors as narrow-spectrum antimicrobials. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Pantoteensuurkinase (PanK) is die eerste ensiem in die universele koënsiem A (KoA) biosintese padweg. Daar bestaan drie hoof tipes PanK: tipe I en tipe III PanKs word in prokariote gevind, terwyl tipe II hoofsaaklik in eukariote gevind word, met die uitsondering van Staphylococcus aureus, ’n patogeen wat algemeen verantwoodelik is vir antibiotika-weerstandige infeksies. Vele ander weerstandige patogene besit ’n tipe III PanK, insluitende Acinetobacter baumannii (Ab), Pseudomonas aeruginosa (Pa) en Helicobacter pylori (Hp). Boon-op is die KoA biosintetiese ensieme, en PanK in besonder, uitgewys as potensiële teikens vir die ontwikkeling van nuwe antimikrobiese middels. In die geval van S. aureus is inhibeerders van sy tipe II PanK (SaPanK-II) — bekend as pantoteenamiede (PanAms) — reeds ontwikkel, maar daar is getoon dat die inhibisie van SaPanK-II se pantoteensuur fosforileringsaktiwiteit nie die enigste meganisme is waardeur hierdie verbindings selgroei inhibeer nie. PanAms is ook substrate van SaPanK-II en die transformasie daarvan deur PanK en twee ander ensieme in die KoA biosintese padweg lei tot die vorming van KoA-antimetaboliete wat reeds bewys is om op te tree as inhibeerders van KoA-afhanklike prosesse soos vetsuur biosintese. In hierdie tesis verken ons die relatiewe bydraes van SaPanK-II inhibisie en sy rol as metaboliese aktiveerder in die PanAms se wyse van inhibisie. Dit is vermag deur die omvang in die onderbreking in die tempo van KoA produksie in die teenwoordigheid van hierdie PanAms te ondersoek, en ook deur die tempo waarteen die KoA-antimetaboliete gevorm word te bepaal. In beide gevalle is dit gedoen deur die resultate van uitgebreide HPLC-gebaseerde analises van die hersaamgestelde S. aureus KoA padweg te ontleed m.b.v sisteemsgebaseerde analises. Hierdie bevindinge het gewys dat verskeie PanAms verskillende interaksies met SaPanK-II het en dat hierdie ’n impak het op die tempo waarby die ooreenstemmende KoA-antimetaboliet gevorm word, asook die omvang waarteen KoA produksie geïnhibeer word. Die algehele resultate van hierdie studie dui aan dat die wyse van inhibisie van elk van die PanAms blyk om tot verskillende mates afhanklik te wees van SaPanK-II inhibisie en KoA-antimetaboliet produksie. Ongelukkig word die aktiwiteit van die PanAms beïnvloed deur twee groot nadele. Eerstens maak hul komplekse, meerledige wyse van inhibisie die ontwikkeling en optimalisering van hierdie inhibeerders moeilik, en tweedens word hulle geïnaktiveer deur alomteenwoordige panteteïenase ensiem wat in menslike serum voorkom. As gevolg van hierdie redes word daar in hierdie tesis verslag gedoen van ’n bykomende studie wat verskillende benaderings evalueer om nuwe inhibeerders van SaPanK-II te ontwikkel wat nie struktureel op die PanAm raamwerk gebaseer is nie. Ons evalueer ook die waarde van die hertoewysing en optimisering van ibuprofen, aangesien daar reeds gewys is dat hierdie verbinding ’n antistafilokokkale middel is, e nook omdat daar bewyse is dat hierdie aktiwiteit dalk ’n gevolg is van SaPanK-II inhibisie. Ongelukkig het ons gevind dat dit nie die geval is nie; ons het wel die pH-afhanklike antistafilokokkale aktiwiteit van ibufrofen bepaal en afgeleides van hierdie verbinding is ontwerp om sy aktiwiteit by ’n fisologiese pH te verbeter. Ibuprofen-PanAm hibriede molekules, waarin die karboksilaat van ibuprofen verruil is vir die N-gesubstitueerde stert van die PanAms, is ontwikkel en het gelei tot die ontdekking van ’n verbinding met ’n lae vlak van SaPanK-II inhibisie (EC50 ~100 μM). Hierdie verbinding kan moontlik geoptimaliseer word as ’n nuwe SaPanK-II inhibeerder, maar ly tans aan oplosbaarheidsprobleme. Ons het ook die gebruik van virtuele hoë deurvoer-sifting ondersoek. Ons het egter gevind dat hierdie metodes noukeurig ontwerp moet word om enige bruikbare resultate te lewer. Verkorte afgeleides van die kragtigste SaPanK-II inhibeerders was ontwerp, gesintetiseer en getoets vir hulle SaPanK-II inhibisie vermoëns om te bepaal watter funktionele groepe behoue moet bly wanneer nuwe inhibeerders ontwerp word. Ons het gevind dat verkorting van die pantoïel-groep van die PanAms gelei het tot verbindings wat onaktief was as inhibeerders van SaPanK-II. Laastens het ons die ontwikkeling van hoë deurvoer-siftingstoets ondersoek vir die identifisering van SaPanK-II inhibeerders deur gebruik te maak van fluoressensie polarisasie. Ongelukkig kon ons nie ‘n suksesvolle fluoressensie indikator ontwikkel met die noodsaaklike kenmerke om in die bogenoemde toets gebruik te word nie. Ons het egter die sintese van ’n karbosteriel-gebaseerde fluoressensie indikator ondersoek wat moontlik in ’n toekomstige studie geïmplimenteer kan word. Vergeleke met die rasionele ontwerpsbenadering is die hoë deurvoer-sifting van groot verbindingsbiblioteke ’n aantrekliker opsie rakende die ontdekking van nuwe strukturele raamwerke vir die ontwikkeling van SaPanK-II inhibeerders aangesien dit die raak-en-mis benadering van rasionele ontwerp systap. In die finale projek het ons die potensiaal van PanK-III ensieme ondersoek as selektiewe teikens vir die ontwikkeling van nou-spektrum antimikrobiese middels. Verskeie PanK-III ensieme was vergelyk deur meervoudige volgorde belynings en filogenetiese analise, en ’n aktiewe setel-analise gebaseer op residu-ligand interaksies en die vorm van die aktiewe setel. Hierdie inligting was gebruik om die omvang van die verskille tussen verskillende PanK-III ensieme te bepaal. Om verder te bepaal of hierdie analise die vermoë het om voorspellings te maak oor voordelige strukturele raamwerke vir ander PanK-III ensieme, het ons substraat- en inhibeerder siftingstoetse gedoen met verskeie pantoteensuur-analoë. Die resultate wys dat bykomende data benodig word om die voorspellingsvermoë van die analise te verbeter, aangesien verskeie PanK-III ensieme getoon het dat daar beduidende afwykings is in hul vermoëns om verskillende substrate te aanvaar, asook hulle sensitiwiteit vir verskillende substraatagtige inhibeerders. Oor die algemeen het hiedie resultate die potensiaal vir die ontwikkeling van spesifieke PanK-III inhibeerders as nou-spektrum antimikrobiese middels onthul. | af_ZA |
| dc.description.version | Doctorate | en_ZA |
| dc.embargo.terms | 2022-09-30 | en_ZA |
| dc.format.extent | xv, 177 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/110297 | en_ZA |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject.lcsh | Protein kinases -- Inhibitors | en_ZA |
| dc.subject.lcsh | Antibacterial agents | en_ZA |
| dc.subject.lcsh | Pantothenic acid | en_ZA |
| dc.subject.lcsh | Drug development | en_ZA |
| dc.subject.lcsh | Drug resistance -- Infections | en_ZA |
| dc.subject.lcsh | Anti-infective agents | en_ZA |
| dc.title | Expanding the scope of bacterial pantothenate kinase inhibitors for application in antibacterial drug development | en_ZA |
| dc.type | Thesis | en_ZA |