Plasmodium falciparum Glucose 6-Phosphate Dehydrogenase-6-Phosphogluconolactonase : Characterisation of redox-related networks as contribution to the development of novel intervention strategies

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2008

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DOI:
http://dx.doi.org/10.22029/jlupub-10113

Abstract

Plasmodium parasites are developing unacceptable levels of resistance to one drug after another and many insecticides are no longer useful against mosquitoes transmitting the disease. Years of vaccine research have produced few hopeful candidates and although scientists are redoubling the search, an effective vaccine is at best years away. Therefore there is need for identification of new drug targets and alternative antimalarial regimes. In response to this dire situation the study aimed at evaluating the pentose phosphate pathway of the malaria parasite P. falciparum in particular the bifunctional enzyme glucose-6-phosphate dehydrogenase- -phosphogluconolactonase, understanding the kynurenine pathway oftryptophan metabolism in particular the enzymes indoleamine 2,3-dioxygenase (1 and 2) and unravelling more knowledge about the thioredoxin system networks in search for a new potential drug target and new drug alternatives. The first two steps of the pentose phosphate pathway in Plasmodium falciparum are catalysed by the enzyme glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) which is a unique bifunctional enzyme exclusively found in the genus Plasmodium. In spite of the importance of the role this enzyme plays in the parasite s pentose phosphate pathway as well as in overcoming oxidative stress, the characteristics of PfGluPhoare still a mystery. For the first time PfGluPho has been successfully cloned, heterologously overexpressed and purified to homogeneity. The recombinant enzyme was found to be a hexamer which exhibits lower Km values that favour substrate turnover by the parasite enzyme when compared to the human homologue. The steady state kinetics of PfGluPho s glucose-6-phosphate dehydrogenase (PfGluPho s G6PD) demonstrates that the enzyme follows an ordered sequential mechanism with NADP+ being the leading substrate. Three novel inhibitors of PfGluPho s G6PD which are active at the lower micromolar range wereidentified and found to be non-competitive with respect to glucose-6-phosphate and NADP+. The study offers the first clear documentation of the cloning, heterologous overexpression, biochemical as well as kinetic characterisation, crystallisation and the first novel inhibitors of PfGluPho. For 30 years, the established dogma regarding tryptophan catabolism was that the first step of the kynurenine pathway, the cleavage of the 2,3 double bond of the indole ring of tryptophan was performed by two enzymes, indoleamine 2,3-dioxygenase-1 (IDO-1) and tryptophan 2,3-dioxygenase (TDO). Recently, indoleamine 2,3-dioxygenase-2 (IDO-2) a third enzyme capable of performing this reaction has been discovered. Reported here is a study ofthe kinetic activity, pH stability, oligomeric structure as well as secondary structural features of recombinant mouse IDO-2 in direct comparison with mouse IDO-1. A screen for new more potent inhibitors of IDO-1 which lack the indole core and avoid the liability arising from the use of indole derivatives which have been reported to be neuroactive gave rise to compound 55D11 (Ki 0.05 ìM) which is more potent than the already existing IDO inhibitors. Astructure activity study was done using various derivatives of compound 55D11 to determine the elements that could be modified to increase potency. The study clearly demonstrates that IDO-1 and IDO-2 differ significantly in terms of their affinity for substrates as well asstructure. The malarial parasite Plasmodium falciparum possesses a functional glutathione and thioredoxin system comprising the redox-active proteins thioredoxin (Trx), glutaredoxin (Grx), and plasmoredoxin (Plrx) which all belong to the thioredoxin superfamily and share the active site motif Cys-X-X-Cys. A better understanding of the role of these members of thethioredoxin superfamily in P. falciparum as well as other systems could be achieved if more was known about their target proteins. Using thioredoxin affinity chromatography prepared by immobilising mutants of the redoxins lacking the resolving cysteine at the active site on CNBr- activated sepharose, target proteins of P. falciparum cell extract were trapped. The covalently linked proteins were eluted with dithiothreitol and analyzed by matrix assisted laser desorption ionization time of flight (MALDI-TOF). Twenty one potential targets were identified for plasmoredoxin. Besides confirming known interacting proteins, potential targetcandidates involved in processes such as; protein biosynthesis, energy metabolism and signal transduction were identified. Further confirmations of the interaction of plasmoredoxin and the target proteins were done using BIAcore surface plasmon resonance experiments.

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