High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) has been employed to study the molecular anatomical structure of rodent malaria vector Anopheles stephensi mosquitoes. A dedicated sample preparation method was developed which suits both, the special tissue properties of the sample and the requirements of high-resolution MALDI imaging. Embedding in 5% carboxymethylcellulose (CMC) was used to maintain the tissue integrity of the whole mosquitoes, being very soft, fragile, and difficult to handle. Individual lipid compounds, specifically representing certain cell types, tissue areas, or organs, were detected and imaged in 20 μm-thick whole-body tissue sections at a spatial resolution of 12 μm per image pixel. Mass spectrometric data and information quality were based on a mass resolution of 70,000 (at m/z 200) and a mass accuracy of better than 2 ppm in positive-ion mode on an orbital trapping mass spectrometer. A total of 67 imaged lipids were assigned by database search and, in a number of cases, identified via additional MS/MS fragmentation studies directly from tissue. This is the first MSI study at 12 μm spatial resolution of the malaria vector Anopheles. The study provides insights into the molecular anatomy of Anopheles stephensi and the distribution and localization of major classes of glycerophospholipids and sphingolipids. These data can be a basis for future experiments, investigating, e.g., the metabolism of Plasmodium-infected and -uninfected Anopheles mosquitoes.
: The most important parasitic diseases, malaria, leishmaniasis, trypanosomiasis, and schistosomiasis, are a great burden to mankind, threatening the life of millions of people worldwide and mostly affecting the poorest. Because drug resistance is increasing and vaccines are rarely available, novel chemotherapeutic compounds are necessary in order to treat these devastating diseases. Insects serve as vectors of many human parasitic diseases and have been shown to express a huge variety of antimicrobial peptides (AMPs). Therefore, research activity on insect-derived AMPs has been increasing in the last 40 years. This chapter summarizes the current state of research on the possible role of AMPs as potential chemotherapeutic compounds against human parasitic diseases. As a representative antimicrobial peptide with antiparasitic activity, the structure of insect defensin A is shown [PDB accession code: 1ICA]. The molecule is surrounded by schematic representations of the human pathogenic parasites Plasmodium, Leishmania and Trypanosoma.
Plasmodium falciparum is responsible for the most severe form of human malaria. P. vivax, in contrast, is the most widespread malaria parasite with an enormous impact on health and economy, since the infection is characterized by high rates of relapses. Due to the mild course of malaria tertiana and complicated in vitro culturing conditions of P. vivax, most of the research on malaria parasites has focused on P. falciparum so far. The redox metabolism of P. falciparum is a promising target for novel antimalarial drugs, since maintaining a redox equilibrium is of fundamental importance for the parasite. P. falciparum contains a cytosolic glutathione and thioredoxin system, as well as redox systems in the apicoplast and the mitochondrion. In contrast to P. falciparum, little is known about the redox processes in P. vivax so far. This review summarizes the current knowledge of the redox metabolism in malaria parasites and provides a detailed in silico comparison of the known and mostly well characterized redox enzymes from P. falciparum and the largely unknown redox proteins from P. vivax. Known antimalarials at least partially mediating their antiparasitic activity by influencing the redox balance of Plasmodium, including dehydroepiandrosterone, Mannich bases, methylene blue, and naphthoquinones, are discussed. Furthermore, we present novel inhibitors identified via screening of a compound library from the Leibniz Institute for Natural Product Research and Infection Biology, Jena that are active against the redox-related enzymes thioredoxin reductase, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase 6- phosphoglucono- lactonase from P. falciparum.
A virtual screening campaign is presented that led to small molecule inhibitors of thioredoxin reductase of Mycobacterium tuberculosis (MtTrxR) that target the protein-protein interaction site for the substrate thioredoxin (Trx). MtTrxR is a promising drug target because it dominates the Trx-dependent hydroperoxide metabolism and the reduction of ribonucleotides, thus facilitating survival and proliferation of M. tuberculosis. Moreover, MtTrxR sufficiently differs from its human homologs to suggest the possibility of selective inhibition if the MtTrxR-Trx interaction site is targeted. To this end, high-throughput docking of 6.5 million virtual compounds to the thioredoxin binding site of MtTrxR combined with constraints as filtering steps was applied. A total of 170 high-scoring compounds yielded 18 compounds that inhibited MtTrxR with IC50 values up to the low micromolar range, thus revealing that the protein-protein interaction site of MtTrxR is indeed druggable. Most importantly, selectivity toward MtTrxR in comparison to human TrxR (HsTrxR) is also demonstrated.
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