Oligomeric protein interference validates druggability of aspartate interconversion in Plasmodium falciparum.

The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum.

Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of Plasmodium falciparum in vitro.

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest.

Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections.

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin).

Antiplasmodial Limonoids from Trichilia rubescens (Meliaceae).

Two new limonoids, named rubescins D-E (1-2) along with eight known compounds, including five havanensin type limonoids, TS1 (3), TS3 (4), rubescins A-C (5-7) and three known phytosterols, β-sitosterol, stigmasterol and its 3β-O-glucopyranoside derivative were isolated from the roots and stem bark of Trichilia rubescens, collected from Cameroon. The structures of the new compounds were determined by detailed analyses of 1D and 2D NMR spectra, in combination with high-resolution mass spectrometry data and by comparison with related data from literature. Anti-plasmodial activities of some of the isolated limonoids 1, 2, 4, 6 and 7 were evaluated against erythrocytic stages of strain 3D7 Plasmodium falciparum.

A novel inhibitor of Plasmodium falciparum spermidine synthase: a twist in the tail.

Background: Plasmodium falciparum is the most pathogenic of the human malaria parasite species and a major cause of death in Africa. It's resistance to most of the current drugs accentuates the pressing need for new chemotherapies. Polyamine metabolism of the parasite is distinct from the human pathway making it an attractive target for chemotherapeutic development.

Stereoselective preparation of pyridoxal 1,2,3,4-tetrahydro-β-carboline derivatives and the influence of their absolute and relative configuration on the proliferation of the malaria parasite Plasmodium falciparum.

We have selectively synthesized by Pictet-Spengler condensation of tryptophan and pyridoxal the four stereoisomers of a pyridoxal β-carboline derivative that was designed to inhibit the proliferation of Plasmodium falciparum. Biological investigation of the four compounds revealed that they all inhibit the growth of P. falciparum.

Staphylococcus aureus thiaminase II: oligomerization warrants proteolytic protection against serine proteases.

Staphylococcus aureus TenA (SaTenA) is a thiaminase type II enzyme that catalyzes the deamination of aminopyrimidine, as well as the cleavage of thiamine into 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 5-(2-hydroxyethyl)-4-methylthiazole (THZ), within thiamine (vitamin B1) metabolism. Further, by analogy with studies of Bacillus subtilis TenA, SaTenA may act as a regulator controlling the secretion of extracellular proteases such as the subtilisin type of enzymes in bacteria. Thiamine biosynthesis has been identified as a potential drug target of the multi-resistant pathogen S.

Chemical and genetic validation of thiamine utilization as an antimalarial drug target.

Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite.

UV-triggered affinity capture identifies interactions between the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) and antimalarial agents in live parasitized cells.

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites.

Folate metabolism in human malaria parasites--75 years on.

Malaria still poses one of the most serious threats to human health worldwide and the prevailing lack of effective, clinically licensed, vaccines means that prophylaxis and treatment depend heavily on a small number of compounds whose efficacies are progressively compromised at varying rates by the inevitable emergence of drug-resistant parasite populations. Of these antimalarials, those inhibiting steps in folate metabolism, along with chloroquine, are the oldest synthetic compounds, with origins dating back three-quarters of a century. Despite widespread parasite resistance, the antifolates still play an important role in malaria control, and our understanding of the underlying mechanisms of folate metabolism and genesis of drug resistance has increased considerably over the last twenty years.

Exploring inhibition of Pdx1, a component of the PLP synthase complex of the human malaria parasite Plasmodium falciparum.

Malaria tropica is a devastating infectious disease caused by Plasmodium falciparum. This parasite synthesizes vitamin B6 de novo via the PLP (pyridoxal 5'-phosphate) synthase enzymatic complex consisting of PfPdx1 and PfPdx2 proteins. Biosynthesis of PLP is largely performed by PfPdx1, ammonia provided by PfPdx2 subunits is condensed together with R5P (D-ribose 5-phosphate) and G3P (DL-glyceraldehyde 3-phosphate).

Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ((1)O(2)). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of (1)O(2) by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye.

Crystallization and preliminary X-ray diffraction of malate dehydrogenase from Plasmodium falciparum.

The expression, purification, crystallization and preliminary X-ray diffraction characterization of malate dehydrogenase (MDH) from the malarial parasite Plasmodium falciparum (PfMDH) are reported. In order to gain a deeper understanding of the function and role of PfMDH, the protein was purified to homogeneity. The purified protein crystallized in space group P1, with unit-cell parameters a = 72, b = 157, c = 159 Å, α = 105, β = 101, γ = 95°.

Aspartate aminotransferase: bridging carbohydrate and energy metabolism in Plasmodium falciparum.

In this mini-review we briefly examine and summarize evidence on the role of the plasmodial aspartate aminotransferase (AspAT) of the malarial parasite. Recent data have provided information on the products of the purine salvage pathway as well as the glycolytic and oxidative phosphorylation pathways, suggesting that the reaction catalyzed by AspAT is an essential step in all these biochemical processes. While the biological role of the oxidative phosphorylation cycle still remains to be demonstrated, the presence of a single protein that is functional in multiple pathways (i.

The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference.

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5'-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite.

Antimalarial drugs: modes of action and mechanisms of parasite resistance.

Malaria represents one of the most serious threats to human health worldwide, and preventing and curing this parasitic disease still depends predominantly on the administration of a small number of drugs whose efficacy is continually threatened and eroded by the emergence of drug-resistant parasite populations. This has an enormous impact on the mortality and morbidity resulting from malaria infection, especially in sub-Saharan Africa, where the lethal human parasite species Plasmodium falciparum accounts for approximately 90% of deaths recorded globally. Successful treatment of uncomplicated malaria is now highly dependent on artemisinin-based combination therapies.

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum.

Background: The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes.

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum.

Aspartate aminotransferases (AspATs; EC 2.6.1.

Purification, crystallization and preliminary X-ray analysis of the aspartate aminotransferase of Plasmodium falciparum.

Aspartate aminotransferases (EC 2.6.1.

Secretion of an acid phosphatase provides a possible mechanism to acquire host nutrients by Plasmodium falciparum.

As an intracellular proliferating parasite, Plasmodium falciparum exploits the human host to acquire nutrients. However, nutrients such as nucleotides and cofactors are mostly phosphorylated in the host cell cytosol and thus have to be dephosphorylated in order to be taken up by the parasite. Here we report the functional characterization of a unique secreted phosphatase in P.

Vitamin B metabolism in Plasmodium falciparum as a source of drug targets.

The malaria parasite Plasmodium falciparum depends primarily on nutrient sources from its human host. Most compounds, such as glucose, purines, amino acids, as well as cofactors and vitamins, are abundantly available in the host cell, and can be readily salvaged by the parasite. However, in some cases the parasite can also synthesize cofactors de novo in reactions that appear to be essential.

The vitamin B1 metabolism of Staphylococcus aureus is controlled at enzymatic and transcriptional levels.

Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism.

The activity of Plasmodium falciparum arginase is mediated by a novel inter-monomer salt-bridge between Glu295-Arg404.

A recent study implicated a role for Plasmodium falciparum arginase in the systemic depletion of arginine levels, which in turn has been associated with human cerebral malaria pathogenesis. Arginase (EC 3.5.

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer.

Background: Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

Methods: Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

Poisoning pyridoxal 5-phosphate-dependent enzymes: a new strategy to target the malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal 5-phosphate (PLP), a crucial cofactor, during erythrocytic schizogony. However, the parasite possesses additionally a pyridoxine/pyridoxal kinase (PdxK) to activate B6 vitamers salvaged from the host. We describe a strategy whereby synthetic pyridoxyl-amino acid adducts are channelled into the parasite.