Sequencing data obtained from the Plasmodium, Anopheles gambiae and human genome projects provide a new basis for drug and vaccine development. One of the most characteristic features in the process of drug development against parasitic protozoa is target identification in a biological pathway. The next step must be a structure-based rational drug design if the target is not only present in the parasite. In mouse models of malaria, such drugs should be tested for efficacy of the new therapies. Here, we present data that pinpoint the existence of two enzymes of the polyamine pathway involved in spermidine metabolism in P. falciparum, i.e. deoxyhypusine synthase (DHS; EC 1.1.1.249) and homospermidine synthase (HSS; EC 2.5.1.45). Recent data obtained from the malaria genome databases showed that at least a putative gene encoding DHS is present in the parasite. Sequencing data from the P. falciparum genome project prove that the eukaryotic initiation factor eIF5A (the substrate for DHS) exists in P. falciparum. Here, we present the amino acid sequence of eIF5A from P. vivax, which causes tertiary malaria. EIF5A from P. vivax shows 82% nucleic acid and 97% amino acid identity to its homologue from P. falciparum. GC/MS data and inhibitor studies with agmatine prove that the triamine homospermidine occurs in the parasite. These data suggest a separate locus encoding HSS in P. falciparum. The hss gene recruits from the dhs gene in eukaryotes. Here, we present genomic DNA fragments obtained by amplification with primers of a conserved region (amino acid positions 550-1,043) between the putative P. falciparum DHS gene ( dhs) and the HSS gene ( hss) from the plant Senecio vulgaris (Asteraceae). The amplification product from different P. falciparum strains reveals differences in sequence identity, compared with the putative dhs gene from P. falciparum strain 3D7. Expression of the full-length clone and determination of HSS-specific activity will finally prove whether a separate region encoding HSS exists.
Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC 50 ) values determined for both strains revealed dierent inhibition pro®les. The IC 50 value for the chloroquine-sensitive NF54 strain was 97 lM and 501 lM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a signi®cant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental eect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition pro®les with 1,7-diaminoheptane resulted in an IC 50 value of 466 lM for the NF54 strain and 319 lM for the R strain. Spermidine pools changed signi®cantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC 50 value of 431 lM and an IC 50 value of 340 lM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the ®rst evidence of homo-spermidine in P. falciparum. The use of speci®c inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.
Abstract. Targeting polyamines of parasitic protozoa in chemotherapy has attracted attention because polyamines might reveal novel drug targets for antiparasite therapies (Müller et al. 2001). The biological function of the triamine spermidine in parasitic protozoa has not been studied in great detail although the results obtained mainly imply three different functions, i.e., cell proliferation, cell differentiation, and biosynthesis of macromolecules. Sequence information from the malaria genome project databases and inhibitor studies provide evidence that the current status of spermidine research has to be extended since enzymes of spermidine metabolism are present in the parasite (Kaiser et al. 2001). Isolation and characterisation of these enzymes, i.e., deoxyhypusine synthase (EC 1.1.1.249) (DHS) and homospermidine synthase (EC 2.5.1.44) (HSS) might lead to valuable new targets in drug therapy. Currently research on spermidine metabolism is based on the deposition of the deoxyhypusine synthase nucleic acid sequence in GenBank while the activity of homospermidine synthase was deduced from inhibitor studies. Spermidine biosynthesis is catalyzed by spermidine synthase (EC 2.5.1.16) which transfers an aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine. Spermidine is also an important precursor in the biosynthesis of the unusual amino acid hypusine and the uncommon triamine homospermidine in eukaryotes, in particular in pyrrolizidine alkaloid-producing plants (Ober and Hartmann 2000). Hypusine is formed by a two-step enzymatic mechanism starting with the transfer of an aminobutyl moiety from spermidine to the ε-amino group of one of the lysine residues in the precursor protein of eukaryotic initiation factor eIF5A by DHS (Lee and Park 2000). The second step of hypusinylation is completed by deoxyhypusine hydroxylase (EC 1.14.9929) (Abbruzzese et al. 1985). Homospermidine formation in eukaryotes parallels deoxyhypusine formation in the way that in an NAD + -dependent reaction an aminobutyl moiety is transferred from spermidine. In the case of homospermidine synthase, however the acceptor is putrescine. Thus the triamine homospermidine consists of two symmetric aminobutyl moieties while there is one aminobutyl and one aminopropyl moiety present in spermidine. Here, we review the metabolism of the triamine spermidine with particular focus on the biosynthesis of hypusine and homospermidine in parasitic protozoa, i.e., Plasmodium, Trypanosoma and Leishmania, compared to that in prokaryotes i.e., Escherichia coli, a phytopathogenic virus and pyrrolizidine alkaloid-producing plants (Asteraceae) and fungi.
In the present study, we have tested the effect of different polyamine inhibitors of the spermidine metabolizing enzymes deoxyhypusine synthase and homospermidine synthase in different chloroquine resistant Plasmodium falciparum strains, in the mosquito Anopheles stephensi (Diptera: Culicidae) and in a Trypanosoma evansi clone I from strain STIB 806 K China. Recent experiments have shown that agmatine is a growth inhibitor of the malaria parasite P. falciparum (Kaiser et al. 2001) in vitro. A comparison of agmatine efficacy with the new antimalarials artemisinin, triclosan and conventional chloroquine showed similar or even better results on the basis of growth inhibition and the reduction of developmental forms. However, no effect of triclosan or agmatine was observed at the ribonucleic acid level. In a second set of experiments, we tested the effect of 1,7-diaminoheptane and agmatine on oocyst formation in A. stephensi after infection with Plasmodium yoelii. Agmatine had an antisporozoite effect since 1,000 microM led to a 59.5% inhibition of oocysts. A much weaker inhibitor of oocyst formation was 1,7-diaminoheptane. The most effective in in vitro inhibition of T. evansi was dicyclohexylamine, an inhibitor of spermidine biosynthesis with an IC(50 ) value of 47.44 microM and the deoxyhypusine inhibitor 1,7-diaminoheptane with an IC(50) value of 47.80 microM. However, both drugs were ineffective in in vivo experiments in a Trypanosoma mouse model. Two different spermidine analogues, 1,8-diaminooctane and 1,3-diaminopropane with IC(50) values of 171 microM and 181.37 microM, respectively, were moderate inhibitors in vitro and ineffective in vivo.
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