Malaria is one of the world s most deadly infectious diseases, mainly caused by P. falciparum (Pf) and P. vivax (Pv). Increasing resistance of Plasmodium strains against commonly used drugs endangers an effective treatment. Therefore, new antimalarial drugs need to be developed. During their life cycle, Plasmodium parasites are continuously exposed to oxidative stress, the major antioxidative systems highly depend on NADPH. For Plasmodium, the oxidative pentose phosphate pathway is the main source of NADPH, generated by two enzymes: the bifunctional glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) and the 6-phosphogluconate dehydrogenase (6PGD), both considered as promising drug targets. The aim of this thesis was to (further) characterize these enzymes and to investigate the potential of several small molecules as future antimalarial drug components.In PfGluPho, naturally occurring mutations have been discovered; however, in contrast to mutations in the gene for hG6PD, these mutations and the studied naturally occurring phosphorylations do not lead to major changes in the properties of the enzyme. In addition to P. falciparum, recombinant production and characterization of P. vivax G6PD are described. Notably, PvG6PD has lower activity and higher KM values for substrate and cofactor than PfGluPho, indicating that it has some functional disadvantages. Full-length PvGluPho ought to be characterized to investigate potential advantages of the fusion. However, since the recombinant production of PvGluPho is extremely challenging, the characterization of PvG6PD makes this important enzyme in the meantime accessible to drug discovery activities.Furthermore, recombinant production and kinetic characterization of Pf6PGD are described. We were able to solve the X-ray structures of native Pf6PGD as well as in complex with its substrate or its cofactor at resolutions of 2.8 Å, 1.9 Å, and 2.9 Å, respectively. With its dimeric conformation, each subunit consisting of a cofactor and a substrate binding domain, as well as a C-terminal tail threading through the adjacent subunit, the overall structure of Pf6PGD is similar to 6PGDs from other species. We could show that a flexible loop bordering the substrate binding site rearranges upon binding 6PG, thereby likely regulating the binding conformation of NADP+. Furthermore, the interaction between the Plasmodium-specific residue W104 and the conserved residue W265 was shown to play a role in the interaction between the cofactor and the substrate binding domain of the enzyme.Moreover, the impact of the post-translational modifications S-glutathionylation and S-nitrosation on the enzymes was tested. None of the enzymes were prone to S-glutathionylation, but all of them were prone to S-nitrosation. The three enzymes catalyzing the G6PD reaction were only moderately inhibited upon S-nitrosation, while the Pf6PGD activity was reversibly inhibited by up to 65%. This might protect the enzyme from irreversible nitrosative damage.All tested compounds had comparable IC50 values and mode of inhibition on PfGluPho and PvG6PD. This supports the hypothesis that it might be possible to develop a drug that effectively treats malaria caused by both species. The most promising compound was SBI-0797750 with an IC50 in the very low nanomolar range. Moreover, inhibition of hG6PD was below 50% at 99 µM as the highest concentration tested, showing the high selectivity for the plasmodial enzymes. In mode of inhibition studies, SBI-0797750 was determined to compete with the substrate for the binding site in PfGluPho. Furthermore, it effectively inhibits the growth of asexual blood stage parasites. Further characterization of SBI-0797750 with regard to its pharmacokinetic behavior is needed. An initial set of Pf6PGD inhibitors with IC50 values in the very low micromolar range was identified by screening the MMV Malaria Box. This is a promising starting point for structure-based optimization approaches.
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