22 research outputs found

    A novel heteromeric pantothenate kinase complex in apicomplexan parasites

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    Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms. Author summary: Apicomplexans are a phylum of obligate intracellular parasites that cause diseases in humans and other animals, inflicting considerable burdens on human societies. During their intracellular stage, these parasites must scavenge vitamins from their host organisms in order to survive and proliferate. One such vitamin is pantothenate (vitamin B5), which parasites convert in a universal five-step pathway to the essential metabolite coenzyme A (CoA). The first reaction in the CoA biosynthesis pathway is catalyzed by the enzyme pantothenate kinase (PanK). The genomes of humans and many other organisms, including apicomplexans, encode multiple PanK homologues, although in all studied examples, the functional PanK enzyme exists as a homodimer. In this study, we demonstrate that the two PanK homologues encoded in the genomes of the apicomplexans Plasmodium falciparum and Toxoplasma gondii, PanK1 and PanK2, exist as functional heteromeric complexes. We provide evidence that both PanK homologues contribute to the PanK activity in these parasites, and that both PanK1 and PanK2 are essential for the proliferation of T. gondii parasites specifically for their PanK activity. Our data describe the first known instances of heteromeric PanK complexes in nature and may explain why some organisms that express multiple PanKs seemingly harbor non-functional isoforms.ETT, VMH and CS were supported by Research Training Program scholarships from the Australian Government. CS was also funded by an NHMRC Overseas Biomedical Fellowship (1016357). This work was, in part, supported by a Project Grant (APP1129843) from the National Health and Medical Research Council to KJS and a Discovery Grant (DP150102883) from the Australian Research Council to Gv

    The key glycolytic enzyme phosphofructokinase is involved in resistance to antiplasmodial glycosides

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    ABSTRACT Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparum. IMPORTANCE Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway

    The pantothenate kinase of the human malaria parasite Plasmodium falciparum

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    The intraerythrocytic stage of the malaria parasite Plasmodium falciparum has an absolute requirement for vitamin B5 (also known as pantothenate) in order to survive. The parasite takes up extracellular pantothenate and subsequently converts it into coenzyme A (CoA) via a series of five universal enzymatic steps. The first enzyme of the pathway is pantothenate kinase (PanK), which phosphorylates pantothenate and commits the molecule to CoA biosynthesis. There are two putative PanK genes (designated Pfpank1 and Pfpank2) in the parasite’s genome, both of which are expressed in the intraerythrocytic stage of the parasite. Many antiplasmodial pantothenate analogues, including pantothenol (PanOH), CJ-15,801, N-substituted pantothenamides (PanAms) and PanAm derivatives have been shown to inhibit P. falciparum growth by targeting its CoA biosynthesis and/or utilisation, although their exact mechanism of action in the parasite remains poorly characterised. In this study, a step-wise dose-escalating drug pressure regime with either PanOH or CJ-15,801 was used to generate resistant parasite lines. These parasite lines are cross-resistant to both PanOH and CJ-15,801, but exhibit different sensitivity profiles to the PanAm derivatives N5-trz-C1-Pan and N-PE-αMe-PanAm, consistent with these two groups of pantothenate analogues having different mechanisms of action. Whole-genome sequencing revealed that these parasites harbour mutations in Pfpank1. Some of these mutations significantly alter the activity of PfPanK, the parasite’s requirement for pantothenate and consequently their fitness compared to the Parent line. These results are consistent with PfPanK1 being the active PanK during this stage of the parasite’s lifecycle. When analysed in conjunction with what has been reported for other organisms, the results of functional enzymatic assays performed in this study revealed important information about the modes of action of these pantothenate analogues. PanOH and CJ-15,801 are predicted to inhibit PfPPCS (the second enzyme of CoA biosynthesis). Conversely, N5-trz-C1-Pan and N-PE-αMe-PanAm are hypothesised to be metabolised into CoA analogues, which subsequently inhibit downstream CoA-utilising enzymes. In order to characterise the activity, conformation and potential interacting partners of PfPanK1 and PfPanK2, green fluorescent protein (GFP)-fused copies of these proteins were expressed in P. falciparum parasites to enable their purification. Results of western blot and mass spectrometry analyses of immunoprecipitated PfPanK are consistent with the native protein being a complex that is comprised of PfPanK1, PfPanK2 and the adapter protein Pf14-3-3I. This marks the first description of a heterodimeric PanK in nature. In silico analysis of the amino acid sequence of the two PfPanKs and interrogation of existing phosphoproteomic studies suggest that Pf14-3-3I binds to PfPanK2. Taken together, the research presented in this study has extended our understanding of the P. falciparum PanKs and, therefore, provided further insight into the CoA biosynthesis pathway as an antimalarial drug target. Furthermore, the information generated in this study about the mechanisms of action of the pantothenate analogues will hopefully expedite the discovery of new antimalarials

    Studies with the Plasmodium falciparum hexokinase reveal that PfHT limits the rate of glucose entry into glycolysis

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    To characterise plasmodial glycolysis, we generated two transgenic Plasmodium falciparum lines, one expressing P. falciparum hexokinase (PfHK) tagged with GFP (3D7-PfHKGFP) and another overexpressing native PfHK (3D7-PfHK+). Contrary to previous reports

    A Pantetheinase-Resistant Pantothenamide with Potent, On Target, and Selective Antiplasmodial Activity

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    Pantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC50], 20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of -methyl-N-phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC50, 52 6 nM), target specificity, and low toxicity.

    Toward a Stable and Potent Coenzyme A-Targeting Antiplasmodial Agent: Structure-Activity Relationship Studies of N-Phenethyl-α-methyl-pantothenamide

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    Pantothenamides (PanAms) are potent antiplasmodials with low human toxicity currently being investigated as antimalarials with a novel mode of action. These structural analogues of pantothenate, the vitamin precursor of the essential cofactor coenzyme A, are susceptible to degradation by pantetheinase enzymes present in serum. We previously discovered that α-methylation of the β-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methyl-pantothenamide as a pantetheinase-resistant PanAm with potent, on-target, and selective antiplasmodial activity. In this study, we performed structure–activity relationship investigations to establish whether stability and potency can be improved further through alternative modification of the scissile amide bond and through substitution/modification of the phenyl ring. Additionally, for the first time, the importance of the stereochemistry of the α-methyl group was evaluated in terms of stability versus potency. Our results demonstrate that α-methylation remains the superior choice for amide modification, and that while monofluoro-substitution of the phenyl ring (that often improves ADME properties) was tolerated, N-phenethyl-α-methyl-pantothenamide remains the most potent analogue. We show that the 2S,2′R-diastereomer is far more potent than the 2R,2′R-diastereomer and that this cannot be attributed to preferential metabolic activation by pantothenate kinase, the first enzyme of the coenzyme A biosynthesis pathway. Unexpectedly, the more potent 2S,2′R-diastereomer is also more prone to pantetheinase-mediated degradation. Finally, the results of in vitro studies to assess permeability and metabolic stability of the 2S,2′R-diastereomer suggested species-dependent degradation via amide hydrolysis. Our study provides important information for the continued development of PanAm-based antimalarials.This project was funded by grants from the National Research Foundation (NRF) of South Africa and Stellenbosch University to M.deV (NRF CSUR Grant # 93732, NRF Y-rated Grant # 112089, and SU Subcommittee B funding) and E.S. (NRF CPRR Grant # 93430). C.S. was funded by an NHMRC Overseas Biomedical Fellowship (1016357). L.B. received an NRF Scare Skills doctoral bursary, and M.K. was supported by bursaries from the NRF and Stellenbosch University. We are also grateful to the Canberra Branch of the Australian Red Cross Blood Service (Australia) and the Western Cape Blood Bank (South Africa) for the provision of red blood cells

    Role of the (104) MgCl<sub>2</sub> Lateral Cut in Ziegler–Natta Catalysis: A Computational Investigation

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    Density functional theory (DFT) has been used for the study of ethylene polymerization in the Ziegler–Natta (ZN) olefin polymerization system for eight different alkoxy group containing titanium catalysts (<b>Cat-A–H</b>), Ti­(III)­Et­(OR)­(OR′) (where R = −CH<sub>3</sub>, – Et, −<i>tert</i>-butyl, −cyclohexane, R′ = −CH<sub>3</sub>, −Et, −<i>tert</i>-butyl, −cyclohexane). What is of significance is that the catalysts studied were all considered to be tethered to the (104) MgCl<sub>2</sub> surface, which has traditionally been considered a “dormant” surface in ZN catalysis systems, in contrast to the “more active” (110) MgCl<sub>2</sub> surface. Our calculations indicate that the binding of all the catalysts to the (104) surface is favorable, even after taking entropic effects into account. For purposes of comparison, ethylene polymerization has been investigated for the <b>Cat-C</b> (TiEt­(OEt)<sub>2</sub>) and the <b>Cat-H</b> (TiEt­(Cl)­(OC<sub>4</sub>H<sub>8</sub>Cl)) (OC<sub>4</sub>H<sub>8</sub>Cl = the chlorobutoxy group) cases, for both the (i) (110) and the (ii) (104) MgCl<sub>2</sub> surfaces. It has been seen that for both (i) and (ii) the energy gap between insertion and the termination barriers (Δ<i>X</i>) was nearly the same for both the <b>Cat-C</b> and <b>Cat-H</b> cases, which shows that ethylene polymerization on the (104) MgCl<sub>2</sub> surface is likely to be a prominent occurrence in Z–N catalysis, when alkoxy groups are bound to the titanium center. Additionally, for the <b>Cat-C</b> and the <b>Cat-H</b> cases, the regio- and stereoselective behavior of the propylene monomer on the titanium species present on the (110) and the (104) MgCl<sub>2</sub> surfaces has also been investigated, and the results indicate that the (104) MgCl<sub>2</sub> surface is only slightly less effective than the (110). However, the calculations also indicate that for <b>Cat-H</b> the (104) MgCl<sub>2</sub> surface significantly improves the molecular weight of polypropylene in comparison to the (110) surface, further showcasing how the (104) surface (ignored until date) might be a major player in ZN catalysis. Given that a major portion of the MgCl<sub>2</sub> support is made up of (104) lateral cuts, the current findings are of considerable relevance

    Studies with the Plasmodium falciparum hexokinase reveal the PfHT limits the rate of glucose entry into glycolysis

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    To characterise plasmodial glycolysis, we generated two transgenic Plasmodium falciparum lines, one expressing P. falciparum hexokinase (PfHK) tagged with GFP (3D7-PfHKGFP) and another overexpressing native PfHK (3D7-PfHK+). Contrary to previous reports, we propose that PfHK is cytosolic. The glucose analogue, 2-deoxy-D-glucose (2-DG) was nearly 2-fold less toxic to 3D7-PfHK+ compared with control parasites, supporting PfHK as a potential drug target. Although PfHK activity was higher in 3D7-PfHK+, they accumulated phospho-[14C]2-DG at the same rate as control parasites. Transgenic parasites overexpressing the parasite’s glucose transporter (PfHT) accumulated phospho-[ 14C]2-DG at a higher rate, consistent with glucose transport limiting glucose entry into glycolysis

    Overcoming synthetic challenges in targeting coenzyme A biosynthesis with the antimicrobial natural product CJ-15,801

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    The biosynthesis of the essential metabolic cofactor coenzyme A (CoA) has been receiving increasing attention as a new target that shows potential to counter the rising resistance to established antimicrobials. In particular, phosphopantothenoylcysteine synthetase (PPCS)—the second CoA biosynthesis enzyme that is found as part of the bifunctional CoaBC protein in bacteria, but is monofunctional in eukaryotes—has been validated as a target through extensive genetic knockdown studies in Mycobacterium tuberculosis. Moreover, it has been identified as the molecular target of the fungal natural product CJ-15,801 that shows selective activity against Staphylococcus aureus and the malaria parasite Plasmodium falciparum. As such, CJ-15,801 and 4′-phospho-CJ-15,801 (its metabolically active form) are excellent tool compounds for use in the development of new antimicrobial PPCS inhibitors. Unfortunately, further study and analysis of CJ15,801 is currently being hampered by several unique challenges posed by its synthesis. In this study we describe how these challenges were overcome by using a robust palladium-catalyzed coupling to form the key N-acyl vinylogous carbamate moiety with retention of stereochemistry, and by extensive investigation of protecting groups suited to the labile functional group combinations contained in this molecule. We also demonstrate that using TBAF for deprotection causes undesired off-target effects related to the presence of residual tertiary ammonium salts. Finally, we provide a new method for the chemoenzymatic preparation of 4′-phospho-CJ-15,801 on multi-milligram scale, after showing that chemical synthesis of the molecule is not practical. Taken together, the results of this study advances our pursuit to discover new antimicrobials that specifically target CoA biosynthesis and/or utilization.We are also grateful to the Canberra branch of the Australian Red Cross Blood Service for providing red blood cells. This work was supported by a CPRR grant (#78988) from the National Research Foundation (NRF) of South Africa and a National Institutes of Health (NIH) award (R01AI136836) to ES. RD received grant-holder and free-standing postdoctoral fellowships from the NRF and postdoctoral study support from the Oppenheimer Memorial Trust, RvdW and LB received NRF Scare Skills doctoral bursaries and KJM an NRF Innovation doctoral bursary. ETT was supported by a Research Training Program scholarship from the Australian Government
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