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Assay of DOHH for phycocyanin a -84 lyase and phycoerythrocyanin a -84 lyase activities. Absorption spectra of acceptor proteins, CpcA and PecA, after treatment with PCB in the presence (–) or absence (–) of DOHH, and purification by Ni 2 + affinity chromatography. (A) Assay for the attachment of PCB to CpcA in the additional presence of the lyase, CpcE. (B) Assay for the attachment of PCB to CpcA in the additional presence of the lyase, CpcF; (C) Assay for the attachment of PCB to PecA in the additional presence of the lyase, PecE. (D) Assay for PCB isomerizing to PVB and attachment to PecA in the additional presence of the lyase, PecF. All reactions were carried out in E. coli (see Materials and methods for details). doi:10.1371/journal.pone.0058318.g008
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Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) wh...
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Context 1
... enzyme the human, orthologous protein was applied in the first step of eIF-5A modification. Nickel-chelate-affinity chromatography was used to purify human DHS which is able to modify the parasitic EIF-5A precursor protein. The deoxyhypusinylated, modified EIF-5A precursor protein was enriched in two subsequent steps of size exclusion chromatography using an Amicon-Ultra 100 and an Amicon-Ultra 30 column (Fig. 5A) as described previously [10]. DHS was cut off after the first step of size exclusion chromatography. Column fractions were analysed by SDS-PAGE and checked by silver staining (Fig. 5B). The modified EIF-5A dhp occurred in the Amicon-Ultra 100 and Amicon-Ultra 30 eluates (Fig. 5A). The eluate from the Amicon-30 column contained the enriched deoxyhypusinated EIF-5A dhp . EIF-5A dhp was applied as a substrate for the DOHH activity assay. Again DOHH was cut off by two steps of size exclusion chromatography and hypusinated EIF-5A hyp was analysed further after peptide hydrolysis and derivatization with methyl chloroformate by GC/MS (Fig. 5C). Methyl chloroformate derivatizes the reactive side chains and esterifies the carboxylic groups. The authentic deoxyhypusine standard showed three prominent peaks of 87 m/z, 129 m/z and 143 m/z after hydrolysis. Hypusine was identified by its prominent peaks 88 m/z, 101 m/z and 157 m/z after hydrolysis. In a separate experiment we performed the DOHH activity assay with 100 nmol of the 5-lipoxgenase inhibitor zileuton and identified the presence of modified EIF-5A hyp and its precursor EIF-5A dhp by GC/MS analysis. The activity assay revealed a deoxyhypusine to hypusine ratio of 90.4% to 9.6% based on the peak areas while the determined deoxyhypusine to hypusine ratio in the non-treated DOHH was 47% to 53%, respectively. To compare the inhibitory effect of zileuton on human and parasitic DOHH inhibition the radioactive filter assay was applied since GC/MS analysis only allows a relative quantification. Zileuton applied in a concentration of 100 nmol inhibited parasitic DOHH approximately 9 fold while only 1.3 fold inhibition was detected for the human enzyme (Table 1). These results were furthermore substantiated by a dose-response curve with various zileuton concentrations inhibiting either recombinant DOHH from P. vivax or the human orthologue. Surprisingly, zileuton was more effective in inhibition of the parasitic enzyme in comparison to the human orthologue (Fig. 6). Moreover, zileuton resulted in a determined IC 50 value of 90 nmol for the human DOHH protein while the IC 50 value of 12,5 nmol was significantly lower for the P. vivax protein (Table 2). Since DOHH from P. vivax shares two EZ-HEAT-like repeats present in phycocyanin lyase from various species we tested the expressed DOHH protein for a possible, dual E/F type phycobilin lyase activity (Fig. 7). Recombinant plasmids expressing the acceptor proteins CpcA or PecA and the PCB catalyzing enzymes [heme oxygenase (HO1) and PCB:ferredoxin oxidoreductase (PcyA) were co-transformed together with expression plasmids encoding the non-isomerising lyase, CpcE/F or the isomerising lyase, PecE/F and the recombinant dohh plasmid respectively, into E. coli strain BL21 (DE3). Heme oxygenase (HO1) oxygenates heme to biliverdin while PcyA reduces biliverdin to phycocyanobilin (PCB). Thus both reactions lead to PCB. Controls were performed in the absence or presence of expressed DOHH protein. Addition of the apoprotein to the chromophore was determined by absorption spectroscopy. The chromophorylated CpcA or PecA were purified by Sepharose chelating chromatography. In Fig. 7A the control experiment of the residual, spontaneous addition of the CpcA to ring A of the chromophore was tested, which was minimal on assembly in E. coli . The spontaneous (non enzymatic) chromophore addition generates an absorption at 645 nm [26]. The isomerized product of the phycoerythrocyanin lyase, phycoviolobilin, is characterized by an absorption at , 565 nm for the Z-form and 505 nm for the E-form (Figure 7D). Figures 7A and C show no attachment of the chromophore to the apoprotein irrespective of the presence or absence of DOHH. In the presence of the phycocyanin a -84 lyase the chromophore was properly attached (Fig. 7B) while the presence of recombinant plasmodial DOHH had no effect. Similar results were obtained when an additional isomerizing lyase, PecE/ F encoding phycoerythrocyanin a -84 lyase/isomerase was present (Fig. 7D). Here, the DOHH protein had a slightly inhibitory effect in comparison to the control (Fig. 7C) which shows the attachment of isomerized phycoviolobilin to the apoprotein. In the next set of experiments the different subunits of the nonisomerizing lyase CpcE or CpcF, or the different subunits of the isomerizing lyase PecE or PecF in the presence or absence of the recombinant DOHH protein were tested for a possible activity of DOHH which might be similar to the subunit of E/F type phycobiliprotein lyase (Fig. 8). Fig. 8A shows the absorption spectrum for the attachment of the chromophore PCB to the apoprotein CpcA in the presence of the additional lyase subunit CpcE. In comparison to the absorption spectra obtained when both subunits of the non-isomerizing lyase CpcE/F are present (Fig. 8B) absorption is significantly lower when only one subunit either CpcE or CpcF is present (Fig. 8A,B). In case of the additional CpcE or CpcF subunit, absorption spectra were not influenced in the presence or absence of plasmodial DOHH. Moreover, no lyase activity could be detected when either PecE (Fig. 8C) or PecF (Fig. 8D) were present. Based on these experiments we conclude that DOHH from P. vivax has no phycocyanin lyase activity. In an attempt to design a homology model for P. vivax DOHH we performed a screening for a suitable template in different databases. Screening with the Protein Model Portal resulted in one hit to the 3HGC protein an acid- sensing eukaryotic ion channel protein which is proton-activated [27]. The aligned protein is an amiloride-sensitive cation channel transport protein from Gallus gallus with a sequence identity of 31% on the amino acid level. However, homology modelling resulted in a model of poor quality. A parallel screen performed with Swiss model resulted in the template of YIBA a predicted HEAT-repeat containing lyase from E. coli [28] with an amino acid sequence identity of 19% as previously described [12]. Again, the constructed model did not show the expected quality. Next the amino acid sequence of stable human 5-lipoxygenase was aligned to P. vivax DOHH. Both proteins i.e. human 5-LOX and DOHH from P. vivax are rich in a -helices [Fig. 2]. The overall amino acid identity is 21% between both proteins. Again,3 D- modeling failed because of less quality between both proteins due to the low amino acid identity. Here, we report on the cloning of the deoxyhypusine hydroxylase gene from the neglected, benign human malaria parasite P. vivax. DOHH from the benign malaria parasite encodes an ORF of 346 amino acids and thus differs in its length and in the number of structural motifs from its P. falciparum orthologue [11]. DOHH from P. vivax has a close relationship to the rodent parasites P. knowlesi (97%) and P. yoelii (78%) and no significant relationships to the orthologues from parasites belonging to the kinetoplastids i.e. trypanosomes and leishmania. In contrast to the P. falciparum dohh gene with five E-Z-HEAT like repeats, P vivax has only four E-Z- HEAT- like repeat domains. However, in both Plasmodium species two E-Z HEAT-like repeat domains recruit from phycocyanin lyases present in different cyanobacteria [29,30]. During evolution Plasmodium acquired the apicoplast which is a relic of the chloroplast from cyanobacteria [31]. The dohh gene is not located in the apicoplast genome but retained structural elements which show its recruitment from phycocyanin lyase which is an enzyme involved in photosynthesis from cyanobacteria. Therefore, it might be tempting to speculate that during evolution these two phycocyanin lyase derived E-Z HEAT-repeats in DOHH provide an extensive soluble surface that is well suited to interact with a different tetrapyrrolic chromophore rather than phycocyanobilin. An attractive candi- date could be hemoglobin being a precursor of biliverdin chromophores. Although DOHH has lost its phycocyanin lyase activity it might be a potential binding protein of hemoglobin which is essential for the parasite to maintain its amino acid requirement [32]. Experiments to investigate binding of DOHH to hemoglobin are currently under way [33]. The two other HEAT-domains which occur in P. vivax DOHH derive from a COG1413 domain present in Methanosarcina from Archae and Nostoc from Cyanobacteria . The basis of the evolutionary success of these HEAT-like repeats might be the rapid adaptation to different interacting partners [30]. At the sequence level this is reflected in the extent of sequence divergence that can be observed for the individual repeats. In consequence, beside EIF-5A as the main interacting partner for DOHH from P. vivax , different interacting partners might be involved depending on the cellular responses and environmental stimuli. In this context it is interesting to note that a recent tandem affinity based protein complementation study demonstrated a significant interaction [34] between human DOHH and LDH-A (lactate dehydrogenase isoenzyme A) (P06151) and pyruvate kinase isoenzymes M1/M2 (P52480). Lactate dehydrogenase catalyzes the interconversion of either lactate or pyruvate with concomitant interconversion of + NADH and NAD . Under facultative anaerobic or anaerobic conditions lactate dehydrogenase converts lactate to pyruvate and the reverse reaction is catalyzed during the Cori cycle. In both + reactions NAD is regenerated from NADH which is used for continuation of glycolysis. In sum, the HEAT-repeat domains identified in DOHH might provide the ...
Context 2
... In an attempt to design a homology model for P. vivax DOHH we performed a screening for a suitable template in different databases. Screening with the Protein Model Portal resulted in one hit to the 3HGC protein an acid- sensing eukaryotic ion channel protein which is proton-activated [27]. The aligned protein is an amiloride-sensitive cation channel transport protein from Gallus gallus with a sequence identity of 31% on the amino acid level. However, homology modelling resulted in a model of poor quality. A parallel screen performed with Swiss model resulted in the template of YIBA a predicted HEAT-repeat containing lyase from E. coli [28] with an amino acid sequence identity of 19% as previously described [12]. Again, the constructed model did not show the expected quality. Next the amino acid sequence of stable human 5-lipoxygenase was aligned to P. vivax DOHH. Both proteins i.e. human 5-LOX and DOHH from P. vivax are rich in a -helices [Fig. 2]. The overall amino acid identity is 21% between both proteins. Again,3 D- modeling failed because of less quality between both proteins due to the low amino acid identity. Here, we report on the cloning of the deoxyhypusine hydroxylase gene from the neglected, benign human malaria parasite P. vivax. DOHH from the benign malaria parasite encodes an ORF of 346 amino acids and thus differs in its length and in the number of structural motifs from its P. falciparum orthologue [11]. DOHH from P. vivax has a close relationship to the rodent parasites P. knowlesi (97%) and P. yoelii (78%) and no significant relationships to the orthologues from parasites belonging to the kinetoplastids i.e. trypanosomes and leishmania. In contrast to the P. falciparum dohh gene with five E-Z-HEAT like repeats, P vivax has only four E-Z- HEAT- like repeat domains. However, in both Plasmodium species two E-Z HEAT-like repeat domains recruit from phycocyanin lyases present in different cyanobacteria [29,30]. During evolution Plasmodium acquired the apicoplast which is a relic of the chloroplast from cyanobacteria [31]. The dohh gene is not located in the apicoplast genome but retained structural elements which show its recruitment from phycocyanin lyase which is an enzyme involved in photosynthesis from cyanobacteria. Therefore, it might be tempting to speculate that during evolution these two phycocyanin lyase derived E-Z HEAT-repeats in DOHH provide an extensive soluble surface that is well suited to interact with a different tetrapyrrolic chromophore rather than phycocyanobilin. An attractive candi- date could be hemoglobin being a precursor of biliverdin chromophores. Although DOHH has lost its phycocyanin lyase activity it might be a potential binding protein of hemoglobin which is essential for the parasite to maintain its amino acid requirement [32]. Experiments to investigate binding of DOHH to hemoglobin are currently under way [33]. The two other HEAT-domains which occur in P. vivax DOHH derive from a COG1413 domain present in Methanosarcina from Archae and Nostoc from Cyanobacteria . The basis of the evolutionary success of these HEAT-like repeats might be the rapid adaptation to different interacting partners [30]. At the sequence level this is reflected in the extent of sequence divergence that can be observed for the individual repeats. In consequence, beside EIF-5A as the main interacting partner for DOHH from P. vivax , different interacting partners might be involved depending on the cellular responses and environmental stimuli. In this context it is interesting to note that a recent tandem affinity based protein complementation study demonstrated a significant interaction [34] between human DOHH and LDH-A (lactate dehydrogenase isoenzyme A) (P06151) and pyruvate kinase isoenzymes M1/M2 (P52480). Lactate dehydrogenase catalyzes the interconversion of either lactate or pyruvate with concomitant interconversion of + NADH and NAD . Under facultative anaerobic or anaerobic conditions lactate dehydrogenase converts lactate to pyruvate and the reverse reaction is catalyzed during the Cori cycle. In both + reactions NAD is regenerated from NADH which is used for continuation of glycolysis. In sum, the HEAT-repeat domains identified in DOHH might provide the accessible surface for an interaction with both enzymes i.e LDH-A and pyruvate kinase to maintain energy metabolism. The occurrence of two E-Z HEAT-like repeat domains present in phycocyanin lyases from different cyanobacterial species prompted us to test the DOHH protein from P. vivax for residual phycocyanin lyase activity. Within two different sets of experiments in the additional presence of the nonisomerizing lyase CpcE/CpcF or the isomerizing lyase, pecE/F, the recombinant DOHH exhibited no phycocyanin lyase activity (Fig. 7, 8). Given that DOHH exerts its catalytic mechanism as a subunit of lyase activity, the additional presence of the isomerizing subunits CpcE or F and PecE or PecF respectively, had no effect (Fig. 8). Thus it seems likely that during evolution when photosynthesis was not necessary anymore parasitic DOHH has lost this function. DOHH from P. vivax was expressed under the control of the IPTG-inducible T7 promotor in E. coli BL21 (DE3) cells. The overexpressed protein had a molecular size of 39 kDa (Fig. 3B, E1, E2) and an isoelectric point of 5.13. P. vivax DOHH protein displayed deoxyhypusine hydroxylase activity in a novel, non radioactive assay [12] (Fig. 5) which was analyzed further either by GC/MS (Fig. 5) or one dimensional protein gelelectrophoresis and subsequent mass spectrometry (data not shown). It was demonstrated that the drug zileuton which is an inhibitor of human 5-lipoxygenase (5-LOX) inhibited parasitic DOHH significantly (Table 1). These experiments were even more supported by the dose-response curve (Fig. 6) comparing the inhibitory effect of different zileuton concentrations against human DOHH and P.vivax DOHH. The fact that DOHH from P. vivax is inhibited more selectively than the human orthologue might result from a higher affinity of the inhibitor to the active site of the parasitic enzyme. This is even more strengthened by the determined IC 50 values for DOHH from P. vivax i.e. 12,5 nmol and for its human orthologue i.e. 90 nmol, respectively (Table 2). Moreover, the E-Z-HEAT- like repeat domains might contribute to an improved binding. Human 5-lipoxygenase catalyzes the first two reactions in the production of leukotriens from arachidonic acid. Moreover, 5- LOX is a validated target for antiinflammation drug design. Many different inhibitors of 5-LOX have been reported like redox, iron ligands and nonredox inhibitors but only few maintain the in vivo activity so far. The only 5-LOX inhibitor on the market is zileuton which is used for the treatment of asthma [35]. In human whole blood, zileuton inhibited 5-LOX at a concentration of 3.3 + 0.4 m M while in vivo the weak potency and the rapid clearance are the therapeutic drawbacks. All lipoxygenases are homologous in sequence and have the same two domain structure which is an N-terminal ß-barrel domain and a C-terminal catalytic domain (lipoxygenase domain). A catalytic iron atom resides in the C-terminal domain. Moreover, the catalytic iron is ligated in an octahedral arrangement by three conserved histidines, one His/Asn/Ser, and the C-terminal isoleucine. By contrast the ferrous iron in Plasmodium DOHH is coordinated by four histidine glutamate residues. The structural similarities between 5-LOX and plasmodial DOHH might explain the selective iron complexing strategy. There are recent reports [36] about a comparative modelling of the human 5-LOX inhibitor binding structure which was used to perform a virtual screen to discover novel 5-LOX inhibitors. This strategy is pursued for the parasitic enzyme although currently a crystal structure is missing. This virtual screening will combine molecular docking and pharmacophore mapping to define structure relationships. Another interesting observation derives from a comparison of different iron-chelating compounds (Table 2) [40] tested against the P. falciparum DOOH. These data show that zileuton inhibits DOHH from P. vivax in a nanomolar range while all the other compounds unfold inhibition at a micromolar concentration. These results further support the notion of a specific inhibition by zileuton rather than the other compounds, i.e. mimosine, cyclopirox or 4-Oxo-piperidine-3-mono- carboxylate. In vivo experiments in the future will delineate whether the selectivity can be confirmed. A major advantage of zileuton is its specific inhibition of the parasitic enzyme rather than the human orthologue. In an in vitro assay zileuton inhibited the parasitic enzyme to 90% while inhibition of the human enzyme was 10% (Table 1). Obviously, zileuton [N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea] is a highly selective inhibitor of plasmodial DOHH. Since no crystallized structure of the human or parasitic enzyme exists the reaction mechanism of the inhibitor has to be elucidated. Another advantage of the drug is the anti-inflammatory property which will be tested for the treatment of cerebral malaria in the near future where host-specific immune and anti-inflammatory mechanisms may be important in response to the presence of parasites in the CNS ...
Citations
... Although a variety of specific small molecule inhibitors have been identified to block spermidine [50] and hypusine biosynthesis [51,52] in Plasmodium, a continuous strategy is still missing to develop parasite-specific efficient inhibitors to the clinical phase. A major problem with the less efficient SpdS inhibitors resulted from a sequential order of substrate binding to the protein. ...
... In the case of the hypusine pathway, the identified DHS inhibitors were spermidine mimetics similar to N 1 -guanyl-1,7-diaminoheptane GC7 with undesired side effects [53] due to a lack of target selectivity. Attempts to inhibit DOHH were based on an iron complexing strategy with known iron chelators like ciclopirox or zileuton which were used as repurposing drugs [52]. However, current crystallization experiments will hopefully foster the development of specific inhibitors against DOHH from the parasite. ...
The triamine spermidine is a key metabolite of the polyamine pathway. It plays a crucial role in many infectious diseases caused by viral or parasitic infections. Spermidine and its metabolizing enzymes, i.e., spermidine/spermine-N1-acetyltransferase, spermine oxidase, acetyl polyamine oxidase, and deoxyhypusine synthase, fulfill common functions during infection in parasitic protozoa and viruses which are obligate, intracellular parasites. The competition for this important polyamine between the infected host cell and the pathogen determines the severity of infection in disabling human parasites and pathogenic viruses. Here, we review the impact of spermidine and its metabolites in disease development of the most important, pathogenic human viruses such as SARS-CoV-2, HIV, Ebola, and in the human parasites Plasmodium and Trypanosomes. Moreover, state-of-the-art translational approaches to manipulate spermidine metabolism in the host and the pathogen are discussed to accelerate drug development against these threatful, infectious human diseases.
... GC-MS is a useful technique for the quantitative analysis of amino acids and their metabolites after chemical derivatization (Hušek et al. 2016). One of our groups analyzed biological Hyp and dHyp by GC-MS (Frommholz et al. 2009;Atemnkeng et al. 2013;von Koschitzky and Kaiser 2013;von Koschitzky et al. 2015). Biological Hyp and dHyp were identified in proteolysates by GC-MS after derivatization with chloroformate (Frommholz et al. 2009;Atemnkeng et al. 2013). ...
... One of our groups analyzed biological Hyp and dHyp by GC-MS (Frommholz et al. 2009;Atemnkeng et al. 2013;von Koschitzky and Kaiser 2013;von Koschitzky et al. 2015). Biological Hyp and dHyp were identified in proteolysates by GC-MS after derivatization with chloroformate (Frommholz et al. 2009;Atemnkeng et al. 2013). Biological Hyp and dHyp were also analyzed by GC-MS using a three-step derivatization procedure and electron ionization (EI) (von Koschitzky et al. 2015). ...
Hypusination is a unique two-step enzymatic post-translational modification of the N ε -amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography–mass spectrometry (GC–MS) method for the measurement of biological hypusine (Hyp), i.e., N ε -(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH 3 OH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CD 3 OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d 0 Me) and the deuterium-labelled methyl esters (d 3 Me) derivatives: d 0 Me-Hyp-(PFP) 5 and d 3 Me-Hyp-(PFP) 5 , respectively. Negative-ion chemical ionization generated the most intense ions with m / z 811 for d 0 Me-Hyp-(PFP) 5 and m / z 814 for the internal standard d 3 Me-Hyp-(PFP) 5 . Selected-ion monitoring of m / z 811 and m / z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1–5 µM Hyp was 91–94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black ( n = 38, age 7.8 ± 0.7 years) and white ( n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68–5.31] µM (range 0.54–9.84 µM) in the black boys and 3.87 [2.95–5.06] µM (range 1.0–11.7 µM) in the white boys ( P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20–0.29] µmol/mmol (range 0.11–0.36 µmol/mmol) in the black boys and 0.26 [0.21–0.30] µmol/mmol (range 0.10–0.45 µmol/mmol) in the white boys ( P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.
... The GC/MS method for the analysis of deoxyhypusine was developed according to an approach which was previously described [41]. A total of 200 µL of the peptide hydrolysates was transferred into a glass vial. ...
The treatment of a variety of protozoal infections, in particular those causing disabling human diseases, is still hampered by a lack of drugs or increasing resistance to registered drugs. However, in recent years, remarkable progress has been achieved to combat neglected tropical diseases by sequencing the parasites’ genomes or the validation of new targets in the parasites by novel genetic manipulation techniques, leading to loss of function. The novel amino acid hypusine is a posttranslational modification (PTM) that occurs in eukaryotic initiation factor 5A (EIF5A) at a specific lysine residue. This modification occurs by two steps catalyzed by deoxyhypusine synthase (dhs) and deoxyhypusine hydroxylase (DOHH) enzymes. dhs from Plasmodium has been validated as a druggable target by small molecules and reverse genetics. Recently, the synthesis of a series of human dhs inhibitors led to 6-bromo-N-(1H-indol-4yl)-1-benzothiophene-2-carboxamide, a potent allosteric inhibitor with an IC50 value of 0.062 µM. We investigated this allosteric dhs inhibitor in Plasmodium. In vitro P. falciparum growth assays showed weak inhibition activity, with IC50 values of 46.1 µM for the Dd2 strain and 51.5 µM for the 3D7 strain, respectively. The antimalarial activity could not be attributed to the targeting of the Pfdhs gene, as shown by chemogenomic profiling with transgenically modified P. falciparum lines. Moreover, in dose-dependent enzymatic assays with purified recombinant P. falciparum dhs protein, only 45% inhibition was observed at an inhibitor dose of 0.4 µM. These data are in agreement with a homology-modeled Pfdhs, suggesting significant structural differences in the allosteric site between the human and parasite enzymes. Virtual screening of the allosteric database identified candidate ligand binding to novel binding pockets identified in P. falciparum dhs, which might foster the development of parasite-specific inhibitors.
... Apart from the human paralogue, the enzyme has been assessed as a target for small molecules from the benign malaria parasite P. vivax and P. falciparum (Von Koschitzky and Kaiser 2014;von Koschitzky et al. 2015). Specific inhibition of recombinant P. vivax DOHH was possible by complexing the ferrous iron with zileuton (Atemnkeng et al. 2013), an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine (673). ...
... The calculated IC 50 for DOHH from the parasite was 12.5 nM determined by a relative quantification by GC/MS. In contrast, the human orthologue is only less affected since the determined IC 50 was 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme (Atemnkeng et al. 2013). Thus, zileuton represents on-target specificity of the enzyme in the parasite. ...
Cancer drug resistance, in particular in advanced stages such as metastasis and invasion is an emerging problem. Moreover, drug resistance of parasites causing poverty-related diseases is an enormous, global challenge for drug development in the future. To circumvent this problem of increasing resistance, the development of either novel small compounds or Advanced Medicinal Therapies have to be fostered. Polyamines have many fundamental cellular functions like DNA stabilization, protein translation, ion channel regulation, autophagy, apoptosis and mostly important, cell proliferation. Consequently, many antiproliferative drugs can be commonly administered either in cancer therapy or for the treatment of pathogenic parasites. Most important for cell proliferation is the triamine spermidine, since it is an important substrate in the biosynthesis of the posttranslational modification hypusine in eukaryotic initiation factor 5A (EIF5A). To date, no small compound has been identified that directly inhibits the precursor protein EIF5A. Moreover, only a few small molecule inhibitors of the two biosynthetic enzymes, i.e. deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) have been functionally characterized. However, it is evident that only some of the compounds have been applied in translational approaches, i.e. in murine models to analyze the function of this modified protein in cell proliferation. In recent years, the pharmaceutical industry shifted from small molecules beyond traditional pharmacology to new tools and methods to treat disorders involving signaling deregulation. In this review, we evaluate translational approaches on inhibition of EIF5A hypusination in pathogenic parasites and therapy-resistant tumors and discuss its feasibility for an application in Advanced Medicinal Therapies.
... Over recent years, we have elucidated the hypusine pathway in a variety of human Plasmodium parasites and performed target evaluation of the enzymes DHS and DOHH, respectively [11][12][13][14]. Although there is the common opinion that the eIF-5A-modifying enzymes are highly conserved, this is, however, not true for the plasmodial enzymes in comparison to the human paralogues. ...
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.
... This PK-PD model allows for mathematical simulations, produces testable predictions, and has implications for the study of deferiprone and ciclopirox as inhibitors of eIF5A hydroxylation in other clinical conditions known to involve hypusine formation, such as cancer [128][129][130][131] and parasitic diseases (e.g. [132,133]). ...
Trial registration:
ClinicalTrial.gov NCT02191657.
... 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. ...
... These results emphasize the necessity for inhibitors, which are highly selective for the plasmodial enzyme and effective in vivo without affecting the human paralogue. DOHH from P. vivax has already been evaluated (Atemnkeng et al. 2013) since this parasite causes severe clinical symptoms and a relapse due to dormant hypnozoites after a primary infection has been cleared. The dormant hypnozoites persist in the hepatocytes of the liver. ...
... Two different procedures of processing of the GC/ MS data were employed to analyze the samples. The first method, which was previously described (Atemnkeng et al. 2013;Njuguna et al. 2014), leads to a relative quantification of the amino acids in % within a sample by comparing the peak areas of the analytes. For obtaining insight into the absolute inhibition of P. falciparum DOHH by the compounds of interest, a method using hypusine (for synthesis as described above) and deoxyhypusine as standards as well as norvaline (Sigma Aldrich, Munich, Germany) as an internal standard was established. ...
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.
... Modification of eIF-5A by hypusine is linked to different pathways and is preferentially regulated under stress conditions (inflammation, hypoxia, abiotic stress in plants). This results in translational control of specific mRNAs in diabetes and infectious diseases (Hauber et al. 2005;Chawla et al. 2012;Atemnkeng et al. 2013;Bresciani et al. 2013), pinpointing its important role in translational medicine. The study by Tersey et al. (2013) tested the hypothesis that polyamines may contribute to inflammation and autoimmunity in type 1 diabetes. ...
... alaria remains one of the major infectious diseases in the world. Plasmodium vivax is the main cause of malaria in Asia, South America and Oceania and is also responsible for approximately 80-300 million cases of malaria per year in the world (1,2). In 2011 totally 3271 clinical malaria cases were reported by Iranian Malaria Control Department in Disease Management Center and Prevention and more than 90% of them were related to P. vivax (Center for Diseases Management and Control, Tehran, Iran, unpublished data). ...
Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran.
Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced.
A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates.
Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.
... In sum, separation of DOHH by native PAGE electrophoresis revealed two bands, i.e., one band referred to the apoprotein, which does not contain ferrous iron while the latter represents the holoenzyme with ferrous iron, which is coordinated to four histidine glutamate residues (Kerscher et al. 2010). The molecular cloning of the DOHH orthologue from the neglected human malaria parasite P. vivax (Atemnkeng et al. 2013) has confirmed the previous target assessment for P. falciparum DOHH. Purified DOHH protein shows functional activity and has only four E-Z HEAT-like repeats opposed to the five present in its orthologue from P. falciparum. ...
A first approach to discover new antimalarials has been recently performed in a combined approach with data from GlaxoSmithKline Tres Cantos Antimalarial Set, Novartis-GNF Malaria Box Data set and St. Jude Children's Research Hospital. These data are assembled in the Malaria Box. In a first phenotypic forward chemical genetic approach, 400 chemicals were employed to eradicate the parasite in the erythrocytic stages. The advantage of phenotypic screens for the identification of novel chemotypes is that no a priori assumptions are made concerning a fixed target and that active compounds inherently have cellular bioavailability. In a first screen 40 mostly heterocyclic, highly active compounds (in nmol range of growth inhibition) were identified with EC50 values ≤2 μM against chloroquine-resistant Plasmodium falciparum strains and a therapeutic window ≥10 against two mammalian cell lines. 78 % of the compounds had no violations with the Lipinski Rule of 5 and only 1 % of the compounds showed cytotoxicity when applied at concentrations of 10 μM. This pre-selective step of parasitic eradication will be used further for a test of the Malaria Box with a potential in iron chelating capacity to inhibit deoxyhypusine hydroxylase (DOHH) from P. falciparum and vivax. DOHH, a metalloprotein which consists of ferrous iron and catalyzes the second step of the posttranslational modification at a specific lysine in eukaryotic initiation factor 5A (EIF-5A) to hypusine. Hypusine is a novel, non-proteinogenic amino acid, which is essential in eukaryotes and for parasitic proliferation. DOHH seems to be a "druggable" target, since it has only 26 % amino acid identity to its human orthologue. For a High-throughput Screening (HTS) of DOOH inhibitors, rapid and robust analytical tools are a prerequisite. A proteomic platform for the detection of hypusine metabolites is currently established. Ultra performance Liquid Chromatography enables the detection of hypusine metabolites with retention times of 7.4 min for deoxyhypusine and 7.3 min for hypusine. Alternatively, the analytes can be detected by their masses with gas chromatography/mass spectrometry or one-dimensional chromatography coupled to mass spectrometry. Moreover, the identified hits will be tracked further to test their efficacy in novel "in vitro assays". Subsequently in vivo inhibition in a humanized mouse model will be tested.
