Plasmodium falciparum pyruvate kinase as a novel target for antimalarial drug-screening.

BACKGROUND: Global travellers are increasingly at risk of contracting malaria. The increasing occurrence of drug-resistance in many endemic areas emphasizes the need for novel drug targets for antimalarial-screening. In this study, the use of pyruvate kinase as a drug-target is evaluated. The functional validation of a gene encoding pyruvate kinase (designated PK1) has previously been reported. However, alternative copies of this enzyme encoded by Plasmodium falciparum could also circumvent the role of PK1. A survey of genome data revealed a putative ORF seemingly coding for another pyruvate kinase (designated PK2). METHODS: The expression of PK1 and PK2 in in vitro cultures were investigated by RT-PCR. Biocomputational analysis was carried out to identify structural differences between the P. falciparum pyruvate kinases and the corresponding enzymes from its human host. RESULTS: Both PK1 and PK2 were indeed actively transcribed during the intraerythrocytic stages, suggesting the involvement of both enzymes during infection. A comparison of amino acid residues at the effector binding sites of PK1 and PK2, to those of the human pyruvate kinases revealed some significant differences that could serve as targets for selective inhibitors to be designed against parasitic pyruvate kinases. CONCLUSION: Experimental evidence for the expression of both PK1 and PK2 during the blood stages of malaria infection was provided. Interestingly, phylogenetic analysis revealed that the "PK2" type of enzyme appears to be confined to Apicomplexans, an important observation with respect to the assessment of PK2 as a drug-target.

A relevant in vitro eukaryotic live-cell system for the evaluation of plasmodial protein localization.

Understanding the functional genomics and proteomics of plasmodia underpins the development of new approaches to antimalarial chemotherapy. Although genome databanks (e.g. PlasmoDB) and biocomputing tools (e.g. PlasMit, PlasmoAP, PATS) are useful in providing a global albeit predictive view of the myriad of about 5000 genes, only 40% are annotated, with few cases of endorsed subcellular localizations of the corresponding proteins in animal models. Progress in plasmodial protein trafficking has been hampered by the lack of a simple yet reliable method for studying subcellular localization of plasmodial proteins. In this study, we have used a combination of fluorescent markers, organelle-specific probes, phase contrast microscopy, and confocal microscopy to locate a selection of signal peptides from 10 plasmodial proteins in CHO-K1 cells. These eukaryotic cells serve as an in vitro living system for studying the cellular destinations of four mitochondrial-targeted TCA cycle proteins (citrate synthase, CS; isocitrate dehydrogenase, ICDH; branched chain alpha-keto-acid dehydrogenase E1alpha subunit, BCKDH; succinate dehydrogenase flavoprotein-subunit, SDH), two nuclear-targeted proteins (histone deacetylase, HDAC; RNA polymerase, RPOL), two apicoplast-targeted proteins (pyruvate kinase 2, PK2; glutamate dehydrogenase, GDH), and two cytoplasmic resident proteins (malate dehydrogenase, MDH; glycerol kinase, GK). The respective localizations of these malarial proteins have complied with the selected molecular targets, viz. mitochondrial, nuclear and cytoplasmic. Interestingly, MDH that is widely known to be resident in eukaryotic mitochondria was found to be cytoplasmic, probably due to the absence of molecular target sequences. Since the localization of plasmodial proteins is central to the authentication of their pathophysiological roles, this experimental system will serve as a useful a priori approach.