Completing the hypusine pathway in <i>Plasmodium</i>

Frommholz, David; Kusch, Peter; Blavid, Robert; Scheer, Hugo; Tu, Jing; Marcus, Katrin; Zhao, Kai‐Hong; Atemnkeng, Veronica; Marciniak, Jana; Kaiser, Annette

doi:10.1111/j.1742-4658.2009.07272.x

Cited by 15 publications

(13 citation statements)

References 29 publications

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“…A soluble protein of 42 kDa size was recovered at 250 mM imidazole concentration in eluate fractions 1 and 2 after nickel chelate affinity chromatography. The estimated size of the yielded protein matches the one of previously expressed P. falciparum DOHH protein (Frommholz et al 2009). The different steps of DOHH protein purification are illustrated in Fig.…”

Section: Resultsmentioning

confidence: 77%

“…Cloning of the dohh gene into the expression vector pET-28a and its expression in E. coli BL21 cells was described previously (Frommholz et al 2009). Following Nickel-chelate affinity chromatography and rebuffering with 50 mM NaH 2 PO 4 buffer, the protein concentration was determined and the purified DOHH protein was used in the enzyme activity assay.…”

Section: Enzymatic Synthesis Of Hypusinementioning

confidence: 99%

“…Incubation took place at 37 °C in a shaker overnight and was stopped by freezing at −80 °C. Following incubation, a two-step size exclusion chromatography with Amicon-Ultracel filters (Amicon, Schwalbach, Germany) of 30 kDa and 100 kDa pore size (Frommholz et al 2009) was performed to remove the human DHS and concentrate the modified eIF-5A (dhp) for further use.…”

Section: Enzymatic Synthesis Of Hypusinementioning

confidence: 99%

“…Hitherto, parasitic DOHH has only been identified in different Plasmodium species, i.e., P. falciparum (Frommholz et al 2009) and P. vivax (Atemnkeng et al 2013) and recently in Leishmania donovani (Chawla et al 2012). The enzymes share common structural elements like the aforementioned EZ-HEAT-like repeat motifs are also present in E/F type phycocyanin lyases from cyanobacteria.…”

Section: Introductionmentioning

confidence: 98%

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New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

et al. 2015

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Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E-Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC-MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum-mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.

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Section: Resultsmentioning

confidence: 77%

Section: Enzymatic Synthesis Of Hypusinementioning

confidence: 99%

Section: Enzymatic Synthesis Of Hypusinementioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

et al. 2015

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“…Spermidine levels of the intra-erythrocytic parasite exceed that of the other polyamines, emphasizing the role of Pf SpdS as a major polyamine flux-determining enzyme [11]. In addition, spermidine appears to have greater metabolic importance since it is a prerequisite for the post-translational activation of P. falciparum eukaryotic translation initiation factor 5A (elF5A), which is required for protein synthesis [9,14-17]. The biosynthesis of low concentrations of spermine has been attributed to a minor, secondary activity of Pf SpdS since there is no evidence for a P. falciparum equivalent to SpmS [18].…”

Section: Introductionmentioning

confidence: 99%

A novel inhibitor of Plasmodium falciparum spermidine synthase: a twist in the tail

Burger

Williams

Sprenger

et al. 2015

Malar J

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BackgroundPlasmodium falciparum is the most pathogenic of the human malaria parasite species and a major cause of death in Africa. It’s resistance to most of the current drugs accentuates the pressing need for new chemotherapies. Polyamine metabolism of the parasite is distinct from the human pathway making it an attractive target for chemotherapeutic development. Plasmodium falciparum spermidine synthase (PfSpdS) catalyzes the synthesis of spermidine and spermine. It is a major polyamine flux-determining enzyme and spermidine is a prerequisite for the post-translational activation of P. falciparum eukaryotic translation initiation factor 5A (elF5A). The most potent inhibitors of eukaryotic SpdS’s are not specific for PfSpdS.Methods‘Dynamic’ receptor-based pharmacophore models were generated from published crystal structures of SpdS with different ligands. This approach takes into account the inherent flexibility of the active site, which reduces the entropic penalties associated with ligand binding. Four dynamic pharmacophore models were developed and two inhibitors, (1R,4R)-(N1-(3-aminopropyl)-trans-cyclohexane-1,4-diamine (compound 8) and an analogue, N-(3-aminopropyl)-cyclohexylamine (compound 9), were identified.ResultsA crystal structure containing compound 8 was solved and confirmed the in silico prediction that its aminopropyl chain traverses the catalytic centre in the presence of the byproduct of catalysis, 5′-methylthioadenosine. The IC50 value of compound 9 is in the same range as that of the most potent inhibitors of PfSpdS, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and 4MCHA and 100-fold lower than that of compound 8. Compound 9 was originally identified as a mammalian spermine synthase inhibitor and does not inhibit mammalian SpdS. This implied that these two compounds bind in an orientation where their aminopropyl chains face the putrescine binding site in the presence of the substrate, decarboxylated S-adenosylmethionine. The higher binding affinity and lower receptor strain energy of compound 9 compared to compound 8 in the reversed orientation explained their different IC50 values.ConclusionThe specific inhibition of PfSpdS by compound 9 is enabled by its binding in the additional cavity normally occupied by spermidine when spermine is synthesized. This is the first time that a spermine synthase inhibitor is shown to inhibit PfSpdS, which provides new avenues to explore for the development of novel inhibitors of PfSpdS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0572-z) contains supplementary material, which is available to authorized users.

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A suggested vital function for eIF‐5A and dhs genes during murine malaria blood‐stage infection

et al. 2016

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The biological function of the post‐translational modification hypusine in the eukaryotic initiation factor 5A ( EIF ‐5A) in eukaryotes is still not understood. Hypusine is formed by two sequential enzymatic steps at a specific lysine residue in the precursor protein EIF ‐5A. One important biological function of EIF ‐5A which was recently identified is the translation of polyproline‐rich mRNA , suggesting its biological relevance in a variety of biological processes. Hypusinated eIF ‐5A controls the proliferation of cancer cells and inflammatory processes in malaria. It was shown that pharmacological inhibition of the enzymes involved in this pathway, deoxyhypusine synthase ( DHS ) and the deoxyhypusine hydroxylase ( DOHH ), arrested the growth of malaria parasites. Down‐regulation of both the malarial eIF ‐5A and dhs genes by in vitro and in vivo silencing led to decreased transcript levels and protein expression and, in turn, to low parasitemia, confirming a critical role of hypusination in eIF ‐5A for proliferation in Plasmodium . To further investigate whether eIF ‐5A and the activating enzyme DHS are essential for Plasmodium erythrocytic stages, targeted gene disruption was performed in the rodent malaria parasite Plasmodium berghei . Full disruption of both genes was not successful; instead parasites harboring the episome for eIF ‐5A and dhs genes were obtained, suggesting that these genes may perform vital functions during the pathogenic blood cell stage. Next, a knock‐in strategy was pursued for both endogenous genes eIF ‐5A and dhs from P. berghei . The latter resulted in viable recombinant parasites, strengthening the observation that they might be essential for proliferation during asexual development of the malaria parasite.

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Completing the hypusine pathway in Plasmodium

Cited by 15 publications

References 29 publications

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

A novel inhibitor of Plasmodium falciparum spermidine synthase: a twist in the tail

A suggested vital function for eIF‐5A and dhs genes during murine malaria blood‐stage infection

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