Pantothenamides as antibacterials: mode of action tudies and Improvement of their potency by structural modification

Thesis (PhD)--Stellenbosch University, 2015.ENGLISH ABSTRACT: The emergence of multidrug-resistant organisms is one of the main driving forces for the continuous development of new antimicrobial chemotherapies. Previous research established that Coenzyme A (CoA), biosynthesized from pantothenic acid, promotes the growth of various disease-causing pathogens, including Staphylococcus aureus and Plasmodium falciparum. Selective inhibition of CoA biosynthesis in pathogens might be accomplished with selected small molecule inhibitors due to the high level of structural and mechanistic divergence between the prokaryotic and eukaryotic enzymes. Consequently, the CoA biosynthetic pathway is seen as a prospective target for such chemotherapies and therefore specific analogues of pantothenic acid have been used in the search for new antimicrobials in various studies. One particular class of analogues, named N-substituted pantothenamides, has shown potential as inhibitors of CoA biosynthesis and utilization in S. aureus. However, our poor understanding of their mechanism of action has hampered their development as clinically relevant agents. Consequently, in this study we set out to elucidate the mode of action of pantothenamides by designing a compound that can only act as an inhibitor of S. aureus pantothenate kinase (SaPanKII) (the first enzyme in the CoA biosynthesis pathway) and not as a substrate. We were able to confirm that the mode of action of bacterial pantothenamide inhibition is determined by the PanK type of the targeted organism. Specifically, we show that in S. aureus growth inhibition is as a result of at least two factors working in combination: 1) by the formation of inactive acyl carrier proteins (ACPs) and CoA antimetabolites and 2) by the reduction of CoA levels through the inhibition of SaPanK-II. Although pantothenamides act as potent inhibitors of S. aureus in vitro, this promising antimicrobial activity is lost when such tests are performed in vivo due to enzymatic degradation of the pantothenamides by pantetheinase enzymes. This also translates to their inhibition of the malariacausing parasite, P. falciparum, since pantetheinase enzymes are present in plasma and serum. Therefore, the second part of this study focused on the design and synthesis of new potent inhibitors that are resistant to pantetheinase-mediated degradation. N-Heptyl pantothenamide (N7- Pan) and N-phenethyl pantothenamide (N-PE-PanAm) were used as scaffolds, since these pantothenamides were previously shown to have excellent potential as inhibitors of S. aureus and P. falciparum proliferation, respectively. Structural modifications were made to the pantothenamides to protect the scissile amide bond from hydrolysis. Specifically, these modifications were chosen to increase the steric bulk around the amide bond, by replacing it with a bioisostere moiety that should withstand pantetheinase degradation, or by preventing the compound from being recognized as a substrate. Ten N7-Pan analogues were successfully synthesized and fully characterized as inhibitors of SaPanK-II and S. aureus, while nine N-PEPanAm analogues were successfully synthesized and partially characterized as inhibitors of P. falciparum. Our results show that while modifications do result in imparting pantetheinase resistance, they also can impact negatively on target recognition.AFRIKAANSE OPSOMMING: Die verskyning van weerstandbiedende organismes is een van die belangrikste dryfkragte vir die voordurende ontwikkeling van nuwe antimikrobiese middels. Vorige navorsing het vasgestel dat koënsiem A (KoA), wat gebiosintesitieer word vanaf pantoteensuur, die groei van verskeie siekteveroorsakende patogene, insluitend Staphylococcus aureus en Plasmodium falciparum, bevorder. Weens die strukturele en meganistiese verskille tussen die prokariotiese en eukariotiese ensieme in die KoA-padweg is dit moontlik om die patogeniese ensieme selektief te inhibeer met spesifieke klein molekuul-inhibitore. Die KoA biosintese padweg word dus beskou as 'n voornemende teiken vir sulke inhibitore, en gevolglik was spesifieke analoë van pantoteensuur gebruik in die soektog na nuwe antimikrobiese middels in verskeie studies. Een spesifieke klas van hierdie analoë, naamlik die N-gesubstitueerde pantoteenamiede, is potensieel goeie inhibitore van KoA biosintese en KoA gebruik in S. aureus. Ongelukkig, weens ons swak begrip van hul meganisme van aksie, word hul ontwikkeling as klienies relevante middels beperk. Die fokus van die eerste deel van hierdie studie was om die aksiemodus van werking van pantoteenamiede te bepaal deur ‘n verbinding te ontwerp wat slegs kan optree as 'n inhibitor van S. aureus pantoteensuurkinase (SaPanK-II) (die eerste ensiem in die KoA biosintese padweg). Die resultate wys dat die meganisme van aksiemodus van die pantoteenamiede in bakterië bepaal word deur die tipe PanK wat die organisme van belang bevat. Ons toon spesifiek dat in S. aureus groei-inhibisie veroorsaak word deur ‘n kombinasie van twee faktore: 1) die vorming van onaktiewe asieldraerproteïene en KoA antimetaboliete en 2 ) die vermindering van die KoA vlakke deur die direkte inhibering van die SaPanK-II ensiem. Alhoewel pantoteenamiede optree as kragtige inhibitore van S. aureus in vitro, word hierdie belowende antimikrobiese aktiwiteit verloor in vivo weens ensiematiese afbraak deur pantetiënase ensieme teenwoordig in plasma en serum. Hierdie effek is ook waargeneem in studies met P. falciparum. Die tweede deel van hierdie studie het dus gefokus op die ontwerp en sintese van inhibitore wat bestand is teen hidrolise deur pantetiënase-ensieme. Die ontwerp van hierdie inhibitore is gebaseer op die N-heptiel pantoteenamied (N7-Pan) en N-fenetiel pantoteenamied (NPE- PanAm) raamwerk, aangesien verskeie studies reeds bewys het dat hierdie pantoteenamiede uitstekende inhibitore van onderskeidelik S. aureus en P. falciparum is. Strukturele veranderinge was gemaak om die geteikende amiedbinding in die pantoteenamiede teen hidrolise te beskerm. Hierdie veranderinge sluit in: 1) toevoeging van steriese hindernis rondom die geteikende amiedbinding; 2) vervanging met 'n bioisosteer-groep wat hidrolise deur pantetiënase-ensieme sal weerstaan; of 3) strukturele veranderings wat verhoed dat die verbinding erken word as 'n substraat vir pantetiënase-ensieme. Tien N7-Pan-analoë is suksesvol gesintetiseer en ten volle gekarakteriseer as inhibitore van SaPanK-II en S. aureus, terwyl nege N-PE-PanAm-analoë suksesvol gesintetiseer en gedeeltelik gekarakteriseer is as inhibitore van P. falciparum. Ons resultate wys dat alhoewel die strukturele veranderinge tot toenemende weerstand teen pantetiënase-ensieme lei, hierdie veranderinge ook 'n negatiewe invloed op teiken-herkenning het