A novel heteromeric pantothenate kinase complex in apicomplexan parasites

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

Triazole Substitution of a Labile Amide Bond Stabilizes Pantothenamides and Improves Their Antiplasmodial Potency

The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo. In this study, we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with 50% inhibitory concentrations (IC50s) of ∼50 nM, and three others have submicromolar IC50s. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA, and show that the compound inhibits the phosphorylation of [14C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase

A cross-metathesis approach to novel pantothenamide derivatives

Pantothenamides are known for their in vitro antimicrobial activity. Our group has previously reported a new stereoselective route to access derivatives modified at the geminal dimethyl moiety. This route however fails in the addition of large substituents. Here we report a new synthetic route that exploits the known allyl derivative, allowing for the installation of larger groups via cross-metathesis. The method was applied in the synthesis of a new pantothenamide with improved stability in human blood

A cross-metathesis approach to novel pantothenamide derivatives

Pantothenamides are known for their in vitro antimicrobial activity. Our group has previously reported a new stereoselective route to access derivatives modified at the geminal dimethyl moiety. This route however fails in the addition of large substituents. Here we report a new synthetic route that exploits the known allyl derivative, allowing for the installation of larger groups via cross-metathesis. The method was applied in the synthesis of a new pantothenamide with improved stability in human blood

Role of the (104) MgCl₂ Lateral Cut in Ziegler–Natta Catalysis: A Computational Investigation

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 = −CH₃, – Et, −<i>tert</i>-butyl, −cyclohexane, R′ = −CH₃, −Et, −<i>tert</i>-butyl, −cyclohexane). What is of significance is that the catalysts studied were all considered to be tethered to the (104) MgCl₂ surface, which has traditionally been considered a “dormant” surface in ZN catalysis systems, in contrast to the “more active” (110) MgCl₂ 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)₂) and the <b>Cat-H</b> (TiEt­(Cl)­(OC₄H₈Cl)) (OC₄H₈Cl = the chlorobutoxy group) cases, for both the (i) (110) and the (ii) (104) MgCl₂ 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) MgCl₂ 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) MgCl₂ surfaces has also been investigated, and the results indicate that the (104) MgCl₂ surface is only slightly less effective than the (110). However, the calculations also indicate that for <b>Cat-H</b> the (104) MgCl₂ 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 MgCl₂ support is made up of (104) lateral cuts, the current findings are of considerable relevance

Exploring Heteroaromatic Rings as a Replacement for the Labile Amide of Antiplasmodial Pantothenamides

Malaria-causing Plasmodium parasites are developing resistance to antimalarial drugs, providing the impetus for new antiplasmodials. Although pantothenamides show potent antiplasmodial activity, hydrolysis by pantetheinases/vanins present in blood rapidly inactivates them. We herein report the facile synthesis and biological activity of a small library of pantothenamide analogues in which the labile amide group is replaced with a heteroaromatic ring. Several of these analogues display nanomolar antiplasmodial activity against Plasmodium falciparum and/or Plasmodium knowlesi, and are stable in the presence of pantetheinase. Both a known triazole and a novel isoxazole derivative were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although they fail to suppress Plasmodium berghei proliferation in vivo, the pharmacokinetic and contact time data presented provide a benchmark for the compound profile likely required to achieve antiplasmodial activity in mice and should facilitate lead optimization.This work was financially supported by grants from the Australian National Health and Medical Research Council (to K.J.S. and K.A.; APP1129843) and the Canadian Institute of Health Research (to K.A.; grant no. 89784). The China Scholarship Council is thanked for providing a scholarship to J.G. The Australian Government provided scholarships for E.T.T. and V.M.H., whereas C.S. was funded by an NHMRC Overseas Biomedical Fellowship (1016357)

Pantothenate biosynthesis in Toxoplasma gondii tachyzoites is not a drug target.

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites

Optimizing the shipbuilding layout of Damex Shipbuilding and Engineering for cost efficiency

In this master thesis the shipyard layout of Damex Shipbuilding and Engineering is optimized for cost efficiency.Ship ProductionDPOMechanical, Maritime and Materials Engineerin

Sustained virologic response of 100% in HCV genotype 1b patients with cirrhosis receiving ombitasvir/paritaprevir/r and dasabuvir for 12weeks.

BACKGROUND & AIMS: Patients with chronic hepatitis C virus (HCV) infection and cirrhosis have a higher risk for liver-related complications and have historically been more difficult to cure than patients without cirrhosis. We evaluated the safety and efficacy of ombitasvir/paritaprevir/ritonavir and dasabuvir, without ribavirin, for 12weeks in patients with HCV genotype 1b infection and compensated cirrhosis. METHODS: Treatment-naïve and peginterferon/ribavirin treatment-experienced patients received 12weeks of ombitasvir/paritaprevir/ritonavir (25/150/100mg once daily) and dasabuvir (250mgtwicedaily). Key inclusion criteria were hemoglobin ⩾10g/dl, albumin ⩾2.8g/dl, platelet count ⩾25×10(9)/L, creatinine clearance ⩾30ml/min, and Child-Pugh score ⩽6. Efficacy was assessed by the percentage of patients achieving SVR (HCV RNA <25IU/ml) 12weeks post-treatment (SVR12). Efficacy and safety were assessed in all patients receiving study drug. RESULTS: Sixty patients with HCV genotype 1b infection and cirrhosis received treatment. The study population comprised 62% male, 55% treatment-experienced, 83% with IL28B non-CC genotype, 22% with platelet count <90×10(9)/L, and 17% with albumin <3.5g/dl. All 60 patients completed treatment, and SVR12 was achieved in 100% (95% CI, 94.0-100%) of patients. The most common adverse events were fatigue (22%), diarrhea (20%), and headache (18%). Only one patient (1.7%) experienced a serious adverse event. Laboratory abnormalities were infrequently observed and not clinically significant. CONCLUSIONS: The HCV regimen of ombitasvir/paritaprevir/ritonavir and dasabuvir without ribavirin for 12weeks achieved 100% SVR12 and was well tolerated in HCV genotype 1b-infected patients with cirrhosis, suggesting that this 12-week ribavirin-free regimen is sufficient in this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT02219503