Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Aptamer-Based Imaging of Polyisoprenoids in the Malaria Parasite.

Dolichols are isoprenoid end-products of the mevalonate and 2C-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a cis-prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques.

Cephalotane-type C20 diterpenoids from Cephalotaxus fortunei var. alpina.

Seventeen new cephalotane-type diterpenoids, fortalides A-Q (1-17), along with five known analogues, were isolated from the seeds of Cephalotaxus fortunei var. alpina. Their structures were determined by extensive spectroscopic methods, as well as electronic circular dichroism (ECD) and X-ray crystallographic data analyses. Some isolates exhibited unusual structural features that were first found in cephalotane-type diterpenoids, such as the occurrence of the 7-oxabicyclo[4.1.1]octane moiety in 14 and 15 and the cis-arrangement of 3-OH and Me-19 in 9. Besides, the antiplasmodial activity of these compounds was evaluated in this study.

Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum.

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.

Limonoids from Cipadessa baccifera.

Eighteen new limonoids, including eight methyl angolensates (1-8) and 10 cipadesins (9-18), were isolated from the leaves of Cipadessa baccifera. Their structures were characterized by means of spectroscopic data analyses, single-crystal X-ray diffraction, and quantum chemistry computational methods. The C-6 configurations in those compounds possessing a C-6 hydroxy group were all assigned as S regardless of the magnitude of J5,6, and the C-2' configuration in those bearing a 2-methylbutyryl residue was defined by single-crystal X-ray diffraction and NMR data. Compounds 1, 5, 6, 7, 11, and 12 showed moderate antimalarial activities with IC50 values ranging from 12 to 28 µM.

Molecular Modeling Applied to Nucleic Acid-Based Molecule Development.

Molecular modeling by means of docking and molecular dynamics (MD) has become an integral part of early drug discovery projects, enabling the screening and enrichment of large libraries of small molecules. In the past decades, special emphasis was drawn to nucleic acid (NA)-based molecules in the fields of therapy, diagnosis, and drug delivery. Research has increased dramatically with the advent of the SELEX (systematic evolution of ligands by exponential enrichment) technique, which results in single-stranded DNA or RNA sequences that bind with high affinity and specificity to their targets. Herein, we discuss the role and contribution of docking and MD to the development and optimization of new nucleic acid-based molecules. This review focuses on the different approaches currently available for molecular modeling applied to NA interaction with proteins. We discuss topics ranging from structure prediction to docking and MD, highlighting their main advantages and limitations and the influence of flexibility on their calculations.

Oxidative stress control by apicomplexan parasites.

Apicomplexan parasites cause infectious diseases that are either a severe public health problem or an economic burden. In this paper we will shed light on how oxidative stress can influence the host-pathogen relationship by focusing on three major diseases: babesiosis, coccidiosis, and toxoplasmosis.

Vitamin B6-dependent enzymes in the human malaria parasite Plasmodium falciparum: a druggable target?

Malaria is a deadly infectious disease which affects millions of people each year in tropical regions. There is no effective vaccine available and the treatment is based on drugs which are currently facing an emergence of drug resistance and in this sense the search for new drug targets is indispensable. It is well established that vitamin biosynthetic pathways, such as the vitamin B6 de novo synthesis present in Plasmodium, are excellent drug targets. The active form of vitamin B6, pyridoxal 5-phosphate, is, besides its antioxidative properties, a cofactor for a variety of essential enzymes present in the malaria parasite which includes the ornithine decarboxylase (ODC, synthesis of polyamines), the aspartate aminotransferase (AspAT, involved in the protein biosynthesis), and the serine hydroxymethyltransferase (SHMT, a key enzyme within the folate metabolism).

Aptamers: novel molecules as diagnostic markers in bacterial and viral infections?

Worldwide the entire human population is at risk of infectious diseases of which a high degree is caused by pathogenic protozoans, worms, bacteria, and virus infections. Moreover the current medications against pathogenic agents are losing their efficacy due to increasing and even further spreading drug resistance. Therefore, there is an urgent need to discover novel diagnostic as well as therapeutic tools against infectious agents. In view of that, the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) represents a powerful technology to target selectively pathogenic factors as well as entire bacteria or viruses. SELEX uses a large combinatorial oligonucleic acid library (DNA or RNA) which is processed a by high-flux in vitro screen of iterative cycles. The selected ligands, termed aptamers, are characterized by high specificity and affinity to their target molecule, which are already exploited in diagnostic and therapeutic applications. In this minireview we will discuss the current status of the SELEX technique applied on bacterial and viral pathogens.

In vitro activity of extracts and isolated polyphenols from West African medicinal plants against Plasmodium falciparum.

The aim of the study was to screen 11 selected traditional medicinal plants from West Africa for their in vitro antiplasmodial activity in order to determine the activity of single and of combination of plant extracts and to examine the activity of isolated pure compounds. Ethanolic and aqueous extracts of the 11 selected plants and pure compounds from Phyllanthus muellerianus and Anogeissus leiocarpus were tested in vitro against Plasmodium falciparum 3D7. Proliferation inhibitory effects were monitored after 48 h. Among the plants and pure compounds investigated in this study, geraniin from P. muellerianus, ellagic, gentisic, and gallic acids from A. leiocarpus, and extracts from A. leiocarpus, P. muellerianus and combination of A. leiocarpus with P. muellerianus affected the proliferation of P. falciparum most potently. Significant inhibitory activity was observed in combination of A. leiocarpus with P. muellerianus (IC(50) = 10.8 µg/ml), in combination of A. leiocarpus with Khaya senegalensis (IC(50) = 12.5 µg/ml), ellagic acid (IC(50) = 2.88 µM), and geraniin (IC(50) = 11.74 µM). In general growth inhibition was concentration-dependent revealing IC(50) values ranging between 10.8 and -40.1 µg/ml and 2.88 and 11.74 µM for plant extracts and pure substances respectively. Comparison with literature sources of in vivo and in vitro toxicity data revealed that thresholds are up to two times higher than the determined IC(50) values. Thus, the present study suggests that geraniin from P. muellerianus; ellagic acid, gallic acid, and gentisic acid from A. leiocarpus; and combination of extracts from A. leiocarpus with either P. muellerianus or K. senegalensis could be a potential option for malaria treatment.