NADPH-producing Enzymes from the Pentose Phosphate Pathway of Plasmodium and Leishmania as Targets for New Anti-infective Agents

Abstract

Malaria and the so-called neglected tropical diseases (NTDs) such as leishmaniasis, Chagas disease, and schistosomiasis remain a major public health problem that disproportionately affects the most vulnerable populations in low-income countries. These diseases cause a substantial burden on affected communities, resulting in around 800,000 deaths per year, and millions more suffering from long-term disabilities, and socioeconomic consequences. Current chemotherapeutic agents to treat malaria and NTDs, face challenges related to safety, efficacy, and drug resistance. As a result, there is an urgent need for novel anti-infective drugs with new mechanisms of action⁠. A promising approach is targeting the major enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) that produce NADPH, the primary electron source for antioxidant defense in intracellular parasites. Previous studies have demonstrated the potential of these enzymes as drug targets in Plasmodium falciparum (Pf). A high-throughput screening against PfG6PD, also called GluPho, identified two promising compounds, ML276 and ML304, from the National Institutes of Health collection. Further optimization of ML304 resulted in SBI-0797750 as the most promising candidate in terms of potency and efficacy. One aim of this work was to further characterize the mode of inhibition (MOI) and action of SBI-0797750. Dose-response studies revealed an approximately 25-fold more potent compound with low nanomolar activity against recombinant PfGluPho, Plasmodium (P.) vivax G6PD, and asexual blood stage parasites. SBI-0797750 is highly selective over the human counterpart and, like ML304, competes with G6P for its binding site. The mode of action is related to a disruption of the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages. In addition, the studies have shown that SBI-0797750 does not affect the integrity of erythrocytes from either G6PD-normal or G6PD-deficient patients, whose erythrocytes are also highly dependent on NADPH production by their own G6PD. Based on the results from Plasmodium, this work aimed to transfer the concept to G6PDs and 6PGDs from NTD-causing pathogens, such as Leishmania and Schistosoma. To this end, the respective enzymes were first recombinantly produced and their oligomerization behavior and kinetic properties were determined. The next step was to determine their three-dimensional structure and identify novel inhibitors. In this work, the first three-dimensional crystal structure of Leishmania donovani G6PD (LdG6PD), both native and complexed with one or both substrates, was solved. In contrast to previous kinetoplastid G6PD structures, the unique N-terminal domain is fully visible. The N-domain is not essential for enzyme activity, but this study indicates that it is involved in tetramerization, albeit quite different to the previously observed tetramer of Trypansoma cruzi G6PD, which lacks the N-terminal domain. Structural and kinetic studies of the substrate binding mode further indicated G6P-dependent domain motions involving the N-domains. This work also led to the solution of the first crystal structure of Leishmania donovani 6PGD (Ld6PGD). Most interestingly, a previously unknown conformation of NADPH was observed in the respective structure. In addition, auranofin, and most likely gold(I)-containing compounds in general, were identified as an interesting class of compounds against Leishmania 6PGDs. Although previous studies have shown that auranofin, which was used over decades against rheumatoid arthritis, is also highly effective against parasites, trypanothione reductase and its orthologues were previously thought to be the sole target of auranofin. Interestingly, Leishmania and Plasmodium 6PGD were also inhibited in the low micromolar range, while the human 6PGD remained fully active. MOI studies revealed that the gold(I) moiety of auranofin is responsible for the observed inhibition, competing with 6-phosphogluconate for its binding site, followed by a rapid irreversible inhibition. The findings of this thesis contribute to the understanding of parasitic G6PDs and 6PGDs and their potential as drug targets. The novel structural and biochemical insights into Kinetoplastida G6PDs and 6PGDs, exhibiting major differences to the human enzymes, will provide an excellent basis for further structure-based inhibitor studies and increase the prospects of developing selective G6PD / 6PGD inhibitors.