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

ABSTRACT: Plasmodium 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.'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.A 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.The 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.

SUBMITTER: Burger PB

PROVIDER: S-EPMC4342090 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Burger Pieter B PB Williams Marni M Sprenger Janina J Reeksting Shaun B SB Botha Mariëtte M Müller Ingrid B IB Joubert Fourie F Birkholtz Lyn-Marie LM Louw Abraham I AI

Malaria journal 20150205

<h4>Background</h4>Plasmodium 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 polyami ...[more]

PMID: 25651815

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Project description:BackgroundPlasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.ResultsMorphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.ConclusionsThis study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.

Project description:BackgroundHypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs.MethodsTransgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling.ResultsClonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N 1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes.DiscussionLoss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds.

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

Publications

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

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