In vitro models for human malaria: targeting the liver stage.

The Plasmodium liver stage represents a vulnerable therapeutic target to prevent disease progression as the parasite resides in the liver before clinical representation caused by intraerythrocytic development. However, most antimalarial drugs target the blood stage of the parasite's life cycle, and the few drugs that target the liver stage are lethal to patients with a glucose-6-phosphate dehydrogenase deficiency. Furthermore, implementation of in vitro liver models to study and develop novel therapeutics against the liver stage of human Plasmodium species remains challenging. In this review, we focus on the progression of in vitro liver models developed for human Plasmodium spp. parasites, provide a brief review on important assay requirements, and lastly present recommendations to improve models to enhance the discovery process of novel preclinical therapeutics.

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.

Galtonosides A-E: Antiproliferative and Antiplasmodial Cholestane Glycosides from Galtonia regalis.

An extract of Galtonia regalis from the Natural Products Discovery Institute showed moderate antiplasmodial activity, with an IC50 value less than 1.25 µg/mL. The two known cholestane glycosides 1 and 2 and the five new cholestane glycosides galtonosides A-E (3-7) were isolated after bioassay-directed fractionation. The structures of the new compounds were determined by interpretation of their NMR and mass spectra. Among these compounds, galtonoside B (4) displayed the most potent antiplasmodial activity, with an IC50 value of 0.214 µM against the drug-resistant Dd2 strain of Plasmodium falciparum.

Anibamine and Its Analogues: Potent Antiplasmodial Agents from Aniba citrifolia.

In our continuing search for novel natural products with antiplasmodial activity, an extract of Aniba citrifolia was found to have good activity, with an IC50 value less than 1.25 µg/mL. After bioassay-directed fractionation, the known indolizinium alkaloid anibamine (1) and the new indolizinium alkaloid anibamine B (2) were isolated as the major bioactive constituents, with antiplasmodial IC50 values of 0.170 and 0.244 µM against the drug-resistant Dd2 strain of Plasmodium falciparum. The new coumarin anibomarin A (3), the new norneolignan anibignan A (5), and six known neolignans (7-12) were also obtained. The structures of all the isolated compounds were determined based on analyses of 1D and 2D NMR spectroscopic and mass spectrometric data, and the absolute configuration of anibignan A (5) was assigned from its ECD spectrum. Evaluation of a library of 28 anibamine analogues (13-40) indicated that quaternary charged analogues had IC50 values as low as 58 nM, while uncharged analogues were inactive or significantly less active. Assessment of the potential effects of anibamine and its analogues on the intraerythrocytic stages and morphological development of P. falciparum revealed substantial activity against ring stages for compounds with two C-10 side chains, while those with only one C-10 side chain exhibited substantial activity against trophozoite stages, suggesting different mechanisms of action.

Metabolic dependency of chorismate in Plasmodium falciparum suggests an alternative source for the ubiquinone biosynthesis precursor.

The shikimate pathway, a metabolic pathway absent in humans, is responsible for the production of chorismate, a branch point metabolite. In the malaria parasite, chorismate is postulated to be a direct precursor in the synthesis of p-aminobenzoic acid (folate biosynthesis), p-hydroxybenzoic acid (ubiquinone biosynthesis), menaquinone, and aromatic amino acids. While the potential value of the shikimate pathway as a drug target is debatable, the metabolic dependency of chorismate in P. falciparum remains unclear. Current evidence suggests that the main role of chorismate is folate biosynthesis despite ubiquinone biosynthesis being active and essential in the malaria parasite. Our goal in the present work was to expand our knowledge of the ubiquinone head group biosynthesis and its potential metabolic dependency on chorismate in P. falciparum. We systematically assessed the development of both asexual and sexual stages of P. falciparum in a defined medium in the absence of an exogenous supply of chorismate end-products and present biochemical evidence suggesting that the benzoquinone ring of ubiquinones in this parasite may be synthesized through a yet unidentified route.

Antiplasmodial flavanones and a stilbene from Carpha glomerata.

Bioassay-guided fractionation of an extract of Carpha glomerata (Cyperaceae) led to the isolation of seven compounds. Compounds 1 (carphorin A), 3 (carphorin C), 4 (carphorin D), and 5 (carphabene) are new compounds, and compound 2 (8-(3″-hydroxyisoamyl)-naringenin) was isolated for the first time as a natural product. All structures were elucidated based on analyses of their HR-ESIMS and 1D and 2D NMR data. Compounds 1, 2, and 6, which have prenyl or hydroxyprenyl side chains, exhibited antiplasmodial activities with IC50 values of 5.2 ±â€¯0.6, 3.4 ±â€¯0.4, and 6.7 ±â€¯0.8 µM against the drug-resistant Dd2 strain of Plasmodium falciparum. In addition the prenylated stilbene 5 also showed good activity, with IC50 5.8 ±â€¯0.7 µM.

Antiplasmodial Chromanes and Chromenes from the Monotypic Plant Species Koeberlinia spinosa.

Nine new compounds containing either a chromane or chromene ring moiety were isolated from the monotypic plant Koeberlinia spinosa. Compounds 1-4 are chromanes with all possible E and Z isomers of the isoprenoid side chain, with compound 5 a methylated derivative of 1. Compounds 6 and 7 were assigned as diastereomeric cyclized derivatives of 2 and were probably artifacts formed during the extraction or the isolation processes. Compounds 8 and 9 were characterized as new chromenes. Structure elucidation of 1-9 was conducted via 1D and 2D NMR spectroscopic data interpretation, and absolute configurations were determined by ECD spectroscopic analysis. Compounds 2, 5, 6, and 7 had weak antiplasmodial activity, while none of the compounds exhibited antiproliferative activity. The isolation, structure elucidation, and biological evaluation of these compounds are presented.

Antiplasmodial alkaloids from bulbs of Amaryllis belladonna Steud.

A bioassay-guided fractionation and chemical investigation of Amaryllis belladonna Steud. bulbs resulted in the isolation and identification of the new crinane alkaloid 1,4-dihydroxy-3-methoxy powellan (1), along with the 3 known crinane alkaloids 2-4 and the two lycorane alkaloids 5-6. The structures were elucidated by interpretation of combined HR-ESIMS, CD and 2D NMR spectroscopic data. Among these isolated compounds the lycorane-type alkaloid acetylcaranine (5) exhibited strong antiplasmodial activity, while compounds 3 and 4 were moderately active, and compounds 1 and 6 were inactive.

Isolation of the New Antiplasmodial Butanolide, Malleastrumolide A, from Malleastrum sp. (Meliaceae) from Madagascar.

An extract of Malleastrum sp. (Meliaceae) collected in Madagascar by the Madagascar International Cooperative Biodiversity Group was found to have antimalarial activity, with an IC50 value between 2.5 and 5 µg ml-1 . After purification by liquid-liquid partition, chromatography on a Diaion open column, C18 SPE and C18 reversed phase HPLC, the new butanolide, malleastrumolide A, was isolated. The structure of malleastrumolide A was determined by mass spectrometry, NMR, and ECD. The double bond position was determined by cross-metathesis and mass spectrometry. The compound has antiproliferative activity against the A2780 ovarian cancer cell line with an IC50 value of 17.4 µm and antiplasmodial activity against the drug-resistant Dd2 strain of Plasmodium falciparum with an IC50 value of 2.74 µm.

Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease.

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.