Browsing by Author "Barnard, Leanne"

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    Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues
    (Public Library of Science, 2018-04-03) Tjhin, Erick T.; Spry, Christina; Sewell, Alan L.; Hoegl, Annabelle; Barnard, Leanne; Sexton, Anna E.; Siddiqui, Ghizal; Howieson, Vanessa M.; Maier, Alexander G.; Creek, Darren J.; Strauss, Erick; Marquez, Rodolfo; Auclair, Karina; Saliba, Kevin J.; Phillips, Margaret A.
    The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.
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    Pantothenamides as antibacterials: mode of action tudies and Improvement of their potency by structural modification
    (Stellenbosch : Stellenbosch University, 2015-12) Barnard, Leanne; Strauss, Erick; Van Otterlo, Willem A. L.; Stellenbosch University. Faculty of Engineering. Dept. of Chemistry and Polymer Science.
    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.