Metabolomic analyses of the malaria parasite after inhibition of polyamine biosynthesis

Malaria, a disease transmitted by female mosquitoes, has plagued the world for many centuries. The disease is associated with high mortality rates, severe poverty, and economic burden. These are factors which hamper effective eradication of the disease. Drug resistant forms of the parasite have caused increasing concerns and questioned the longevity of current effective antimalarials. Efforts are therefore aimed at the identification and exploitation of essential parasite proteins as potential drug targets. The polyamine pathway of Plasmodium falciparum is an exploitable pathway which contains two distinct, chemically validated drug targets; a bifunctional PfAdoMetDC-ODC protein and PfSpdSyn. These enzymes ensure intricate regulation of polyamine production and the pathway contains various distinctive features which could be selectively targetable from the mammalian counterpart pathways. However, inhibition of polyamine production through the use of specific enzyme inhibitors has revealed various compensatory responses that negate the efficacy of these inhibitors. An account is given of the metabolomic fluctuations in the parasite during inhibition of polyamine biosynthesis. From co-inhibited P. falciparum extracts, it could be demonstrated that the characteristic growth-arrest coincided with the depletion in spermidine, the metabolic product of PfSpdSyn. The co-inhibition strategy therefore emphasised the importance of spermidine biosynthesis by PfSpdSyn. Moreover, adenosyl-related metabolite levels were not disrupted during polyamine depletion, supporting the notion that these metabolites are intricately recycled within the parasites. The identified metabolic compensatory mechanisms have further potential for exploitation, and can strategically be combined with polyamine biosynthesis inhibition to ensure parasitic attenuation. In addition, several novel inhibitors were previously computationally identified, based on a dynamic receptor-based pharmacophore model of PfSpdSyn. The in vitro inhibiting activity of these compounds was determined against PfSpdSyn. Results from the in vitro experiments supported the in silico predictions, and emphasized the supportive role of pharmacophore modelling has for the identification of novel inhibitors. The research contributed in understanding parasitic polyamine metabolite regulation, and will aid in the future optimization of therapeutic strategies, aimed at exploitation of the polyamine pathway as a potential antimalarial drug target. CopyrightDissertation (MSc)--University of Pretoria, 2009.Biochemistryunrestricte

Functional consequences of perturbing polyamine metabolism in the malaria parasite, Plasmodium falciparum

Inhibition of polyamine biosynthesis and/or the perturbation of polyamine functionality have been exploited with success against parasitic diseases such as Trypanosoma infections. However, when the classical polyamine biosynthesis inhibitor, α-difluoromethylornithine, is used against the human malaria parasite, Plasmodium falciparum, it results in only a cytostatic growth arrest. Polyamine metabolism in this parasite has unique properties not shared by any other organism. These include the bifunctional arrangement of the catalytic decarboxylases and an apparent absence of the typical polyamine interconversion pathways implying different mechanisms for the regulation of polyamine homeostasis that includes the uptake of exogenous polyamines at least in vitro. These properties make polyamine metabolism an enticing drug target in P. falciparum provided that the physiological and functional consequences of polyamine metabolism perturbation are understood. This review highlights our current understanding of the biological consequences of inhibition of the biosynthetic enzymes in the polyamine pathway in P. falciparum as revealed by several global analytical approaches. Ultimately, the evidence suggests that polyamine metabolism in P. falciparum is a validated drug target worth exploiting.This work was supported by the National Research Foundation of South Africa (NRF: Grant FA2004051300055, FA2006040400011 and FA2007050300003), the University of Pretoria and the Department of Science and Technology of South Africa for funding the South African Malaria Initiative of which LB and AIL are members. KC, TvB, SS and MW were recipients of prestigious bursaries from the NRF, South Africa. JN hold bursaries from the Carl and Emily Fuchs Foundation as well as the Ernst and Ethel Eriksen Trust.www.springerlink.co

Targeted inhibition of the Plasmodium falciparum Vitamin B6 producing enzyme Pdx1 and the biochemical and functional consequences thereof

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials.Thesis (PhD)--University of Pretoria, 2013.gm2013BiochemistryUnrestricte

Exploring inhibition of Pdx1, a component of the PLP synthase complex of the human malaria parasite Plasmodium falciparum

Malaria tropica is a devastating infectious disease caused by Plasmodium falciparum. This parasite synthesizes vitamin B6 de novo via the pyridoxal 5-phosphate (PLP) synthase enzymatic complex consisting of PfPdx1 and PfPdx2 proteins. Biosynthesis of PLP is largely performed by PfPdx1, ammonia provided by PfPdx2 subunits, is condensed together with Dribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P). PfPdx1 accommodates both the R5P and G3P substrates and intricately coordinates the reaction mechanism, which is composed of a series of imine bond formations, leading to the production of PLP. We demonstrate that D-erythrose 4-phosphate (E4P) inhibits PfPdx1 in a dose dependent manner. We propose that the acyclic phospho-sugar E4P, with a C1 aldehyde group similar to acyclic R5P, could interfere with R5P imine bond formations in the PfPdx1 reaction mechanism. Molecular docking and subsequent screening identified the E4P hydrazide analogue, 4- phospho-D-erythronhydrazide (4PEHz), which selectively inhibited PfPdx1 with an IC50 of 43 μM. PfPdx1 contained in the heteromeric PLP synthase complex was shown to be more sensitive to 4PEHz and was inhibited with an IC50 of 16 μM. Moreover, the compound had an IC50 value of 10 μM against cultured P. falciparum intraerythrocytic parasites. To further analyse the selectivity of 4PEHz, transgenic cell lines over-expressing PfPdx1 and PfPdx2 showed that additional copies of the protein complex conferred protection against 4PEHz, implicating that the PLP synthase is directly affected by 4EPHz in vivo. These PfPdx1 inhibitors represent novel lead scaffolds which are capable of targeting PLP biosynthesis, and we propose this as a viable strategy for the development of newer therapeutics against malaria.This work was funded by the grants [WR 124/2] and [WR 124/3] from the Deutsche Forschungsgemeinschaft (DFG) to CW as well as the National Research Foundation (NRF) of South Africa [65876] and NRF-DFG Scientific exchange grants to LMB, IM and CW. SBR was supported by the PhD sandwich programme of the German Academic Exchange Programme (DAAD) [A/08/99008]. CW is supported by grant [2009/54325-2] from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) within the “Jovem Pesquisador” programme.http://www.biochemj.org/bj/default.ht