Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

[[1]Benzothieno[3,2-b]pyridin-4-yl-amine--synthesis and investigation of activity against malaria]. / [1]Benzothieno[3,2-b]pyridin-4-yl-amine--synthese und prüfung auf wirksamkeit gegen Malaria.

The ethyl 4-chlorobenzothieno[3,2-b]pyridine-3-carboxylate (2) reacted with the hydrochlorides of the mono- and bis-phenol Mannich bases 6 to yield the amodiaquine and pyronaridine analogues 9. The chloroquine analogue 10 was formed by melting 2 with the novaldiamine base (7) in phenol. The stability of the 4-aminophenols 9 was investigated by anodic oxidation using the rotating platinum electrode by means of difference pulse voltammetry. The half wave potentials were measured giving E(1/2) approximately 1.05 V. Compound 9g displayed the highest activity against the growth of the malaria parasite Plasmodium falciparum. Testing against the chloroquine sensitive 3D7 and the chloroquine resistant Dd2 strain resulted in IC50 values of 150 nM and 210 nM, respectively. Surprisingly, the 3-carbinol 4 and the 3-chloromethyl derivative 5, synthesized from the 3-carboxylic acid ester 2, reacted with the phenol Mannich base 6a and the novaldiamine base (7), respectively, to yield the 4-pyridone 8.

Crystallization of the major cytosolic glutathione S-transferase from Onchocerca volvulus.

Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyse the conjugation of glutathione to xenobiotic and endogenous electrophilic compounds, thus facilitating their elimination from cells. The recombinant Onchocerca volvulus GST2 has been expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion technique. Two different crystal forms were grown under identical conditions. They belong to space groups P2(1)2(1)2 and P2(1), respectively. The unit-cell parameters obtained are a = 112.6, b = 84.3, c = 45.1 A for the P2(1)2(1)2 crystal form and a = 51.6, b = 82.3, c = 56.7 A, beta = 95.89 degrees for the P2(1) form. Complete data sets to 2.6 and 1.5 A, respectively, have been collected at 100 K with synchrotron radiation.

[Pyrido [3,2-b]indol-4-yl-amines--synthesis and investigation of activity against malaria]. / Pyrido[3,2-b]indol-4-yl-amine--Synthese und Prüfung auf Wirksamkeit gegen Malaria.

Pyrido[3,2-b]indol-4-yl-amines--synthesis and investigation of activity against malaria Starting with 3-aminoindole-2-carboxylic acid ester 1 the annulated pyrido[3,2-b]indoles 6 and 8 were synthesized as key substances. The 4-chloropyridine derivative 8 reacted with the phenol Mannich bases 11 and the novaldiamine base 13, respectively, to yield the amodiaquine and cycloquine analogues 12 as well as the chloroquine analogue 14. The stability of the compounds 12 and 14 were proven by the half wave potentials measured by differential pulse voltammetry. Compounds 12 and 14 were tested for in vitro antimalarial activity using a chloroquine sensitive and a chloroquine resistant Plasmodium falciparum strain. The highest activity was shown by 12g with IC50 values of 50 nM and 38 nM, respectively. The in vivo activity of 12g was tested in Plasmodium vinckei infected mice resulting in ED50 values of 22 mg/kg and 26 mg/kg after intraperitoneal and oral administration, respectively.

Crystallization and preliminary X-ray diffraction studies of the glutathione S-transferase from Plasmodium falciparum.

Glutathione S-transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cells. For high-resolution crystallographic investigations, GST from the human malarial parasite Plasmodium falciparum was overexpressed in bacterial cells and crystallized using hanging-drop vapour diffusion. X-ray intensity data to 2.8 A resolution were collected from an orthorhombic crystal form with unit-cell parameters a = 62.2, b = 88.3, c = 75.3 A. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement.

Synthesis and antifilarial evaluation of N1,Nn- xylofuranosylated diaminoalkanes.

A series of N(1),N(n)-xylofuranosylated diaminoalkanes (3-9 and 11-18) has been synthesized either by reductive amination of deoxy xylouloses (2a, 2b) with amines followed by one pot reduction with NaBH(4) or NaCNBH(3); or by 1,4-conjugate addition of amines to glycosyl olefinic esters (10a, 10b). The compounds were screened for their interference with filarial worms' glutathione metabolism, a potential target for chemotherapeutic attack. Interestingly, these compounds affected intracellular glutathione, gamma-glutamyl cysteine synthetase, glutathione reductase and glutathione-S-transferase(s) of bovine filarial worms to varying degrees. Some of the compounds though effected the motility and MTT reduction potential of filarial worms Brugia malayi, however, little microfilaricidal and macrofilaricidal were noted with compounds at 50mg/kg oral dose. Compounds 6, 16 and 17 were evaluated also for in vivo activity.

DBU-assisted cyclorelease elimination: combinatorial synthesis and gamma-glutamyl cysteine synthetase and glutathione-S-transeferase modulatory effect of C-nucleoside analogs.

A combinatorial library of 60C- nucleoside analogs was synthesized by sequential coupling of building blocks followed by cyclative cleavage with DBU in an efficient manner. Only DMSO soluble compounds were tested for their modulatory effect against filarial gamma-glutamyl cysteine synthetase (gamma-GCase) and glutathione-S-transeferases (GSTs). Several compounds were found to be weak inhibitors of filarial gamma-GCase, whereas, most of them stimulated filarial GSTs.

Thioredoxin and glutathione system of malaria parasite Plasmodium falciparum.

Plasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new targets for the chemotherapeutic intervention of parasite development in the human host. It is established that P. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria.

Structural analysis and antibody response to the extracellular glutathione S-transferases from Onchocerca volvulus.

Onchocerca volvulus is a human pathogenic filarial parasite which, like other parasitic nematodes, is capable of surviving in an immunologically competent host by employing a variety of immune evasion strategies and defense mechanisms including the detoxification and repair mechanisms of the glutathione S-transferases (GSTs). In this study we analyzed the glycosylation pattern and the immunological properties of extracellular O. volvulus GST1a and -1b (OvGST1a and -1b). The enzymes differ in only 10 amino acids, and both are glycoproteins that have cleavable signal peptides and unusual N-terminal extensions. These characteristics have not been described for other GSTs so far. Mass spectrometry analyses indicate that both enzymes carry high-mannose type oligosaccharides on at least four glycosylation sites. Glycosylation sites 1 to 3 of OvGST1a (OvGST1b sites 2 to 4) are occupied by truncated N-glycans (Man(2)GlcNAc2 to Man(5)GlcNAc(2)), and N glycosylation site 4 of OvGST1a (OvGST1b site 5) carries Man(5)GlcNAc2 to Man(9)GlcNAc(2). To analyze the capacity of these secretory GSTs to stimulate host immune responses, we studied the antibody responses of onchocerciasis patients against the native affinity-purified OvGST1a and -1b. By enzyme-linked immunosorbent assay we showed that OvGST1a and -1b are immunodominant antigens, with less than 7% nonresponder patients. A direct comparison of the antibody responses to the glycosylated and deglycosylated forms demonstrates the high immunogenicity of the N-glycans. Analyses of the antibody responses to the unusual N-terminal extension show an enhanced recognition of this portion by patients as opposed to recognition of the recombinant protein without extension.

Gene structure of the extracellular glutathione S-transferase from Onchocerca volvulus and its overexpression and promoter analysis in transgenic Caenorhabditis elegans.

Two highly similar genes encoding unique extracellular, glycosylated glutathione S-transferases (GSTs) of the human-pathogenic nematode, Onchocerca volvulus (Ov-GST1a and Ov-GST1b), have been isolated and characterised. The genes are approximately 3 kb in length and consist of seven exons interrupted by introns of approximately 100 bp in length, with the exception of intron II, which is approximately 1.6 kb in length. Interestingly, exon I and II encode a signal peptide and an N-terminal extension before sequence homology to other GSTs begins. The 5' flanking region was sequenced and analysed for transcription factor binding sites. Consistent with the lack of a TATA box, analysis of the mRNAs by primer extension showed multiple transcription start sites spread over a 60 bp region. To examine the activity and specificity of the Ov-GST1a gene promoter, we have exploited Caenorhabditis elegans as a heterologous transformation system. To analyse whether transgenic C. elegans are able to carry out processing and post-transcriptional modifications of the Ov-GST1a correctly, the protein was ectopically overexpressed in C. elegans. The parasite-derived Ov-GST1a gene product was correctly processed in transgenic C. elegans and posttranslational modifications, such as signal peptide cleavage and N-glycosylation, were performed successfully. This further demonstrates the potential of C. elegans as a host for expression of candidate vaccine antigens from O. volvulus and affirms the role of C. elegans as a model for parasitic nematodes.

The Plasmodium falciparum bifunctional ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, enables a well balanced polyamine synthesis without domain-domain interaction.

In the human malaria parasite Plasmodium falciparum (Pf), polyamines are synthesized by a bifunctional enzyme that possesses both ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase (AdoMetDC) activities. The mature enzyme consists of the heterotetrameric N-terminal AdoMetDC and the C-terminal dimeric ODC, which results in the formation of a heterotetrameric complex. For the native bifunctional protein a half-life longer than 2 h was determined, which is in contrast to the extreme short half-life of its mammalian monofunctional counterparts. The biological advantage of the plasmodial bifunctional ODC/AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein. An interesting feature in the regulation of the bifunctional protein is that putrescine inhibits PfODC activity approximately 10-fold more efficiently than the mammalian ODC activity, and in contrast to the mammalian AdoMetDC the activity of the PfAdoMetDC domain is not stimulated by the diamine. To analyze post-translational processing, polymerization, and domain-domain interactions, several mutant proteins were generated that have single mutations in either the PfODC or PfAdoMetDC domains. The exchange of amino acids essential for the activity of one domain had no effect on the enzyme activity of the other domain. Even prevention of the post-translational cleavage of the AdoMetDC domain or ODC dimerization and thus the interference with the folding of the protein hardly affected the activity of the partner domain. In addition, inhibition of the activity of the PfODC domain had no effect on the activity of the PfAdoMetDC domain and vice versa. These results demonstrate that no domain-domain interactions occur between the two enzymes of the bifunctional PfODC/AdoMetDC and that both enzymatic activities are operating as independent catalytic sites that do not affect each other.

Targeting polyamines of parasitic protozoa in chemotherapy.

All parasitic protozoa contain polyamines and in recent years they, and their associated enzymes, have attracted attention as drug targets because they might reveal novel antiparasite therapies. How justified is this approach to drug discovery? In this review, Sylke Müller, Graham Coombs and Rolf Walter summarize the current status of research into drugs that exploit polyamine metabolism of trypanosomatid and malaria parasites, and propose priorities for research into such drugs. This review was inspired by an Expert Meeting entitled 'Polyamine Metabolism of Parasitic Protozoa as a Drug Target'.

A stress-responsive glyoxalase I from the parasitic nematode Onchocerca volvulus.

Glyoxal, methylglyoxal and other physiological alpha-oxoaldehydes are formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates. They are detoxified enzymically by the glyoxalase system. To investigate the physiological function of glyoxalase I in parasitic organisms, the cDNA for glyoxalase I from the filarial nematode Onchocerca volvulus (designated Ov-GloI) has been cloned and characterized. The isolated cDNA contains an open reading frame of 579 bp encoding a protein with a calculated molecular mass of 21930 Da. Owing to the high degree of sequence identity (60%) with human glyoxalase I, for which the X-ray structure is available, it has been possible to build a three-dimensional model of Ov-GloI. The modelled core of Ov-GloI is conserved compared with the human glyoxalase I; however, there are critical differences in the residues lining the hydrophobic substrate-binding pocket of Ov-GloI. A 22 kDa protein was obtained by heterologous expression in Escherichia coli. A homogeneous enzyme preparation was obtained by affinity purification and functional characterization of the recombinant enzyme included the determination of kinetic constants for methylglyoxal and phenylglyoxal as well as inhibition studies. Gel filtration demonstrated a dimeric structure. To assess the role of Ov-GloI as a potential vaccine candidate or serodiagnostic tool, the serological reactivity of the recombinant Ov-GloI was analysed with sera from microfilaria carriers and specific IgG1 antibodies were detected. The effects of oxidative insult, namely plumbagin and xanthine/xanthine oxidase, on the gene transcript level of Ov-GloI were investigated. By using a semi-quantitative PCR ELISA it was shown that Ov-GloI is expressed at elevated levels under conditions of oxidative stress.

The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system.

The thioredoxin system consists of the NADPH dependent disulphide oxidoreductase thioredoxin reductase (TrxR) which catalyses the reduction of the small protein thioredoxin. This system is involved in a variety of biological reactions including the reduction of deoxyribonucleotides, transcription factors and hydrogen peroxide. In recent years the TrxR of the malaria parasite Plasmodium falciparum was isolated and characterised using model substrates like 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and Escherichia coli thioredoxin. Here we report on the isolation of a cDNA encoding for P. falciparum thioredoxin (PfTrx) and the expression and characterisation of the recombinant protein, the natural substrate of PfTrxR. The deduced amino acid sequence of PfTrx encodes for a polypeptide of 11715 Da and possesses the typical thioredoxin active site motif CysGlyProCys. Both cysteine residues are essential for catalytic activity of the protein, as shown by mutational analyses. Steady state kinetic analyses with PfTrxR and PfTrx in several coupled assay systems resulted in K(m)-values for PfTrx in the range of 0.8--2.1 microM which is about 250-fold lower than for the model substrate E. coli thioredoxin. Since the turnover of both substrates is similar, the catalytic efficiency of PfTrxR to reduce the isolated PfTrx is at least 250-fold higher than to reduce E. coli thioredoxin. PfTrx contains a cysteine residue in position 43 in addition to the active-site cysteine residues, which is partially responsible for dimer formation of the protein as demonstrated by changing this amino acid into an alanine residue. Using DTNB we showed that all three cysteine residues present in PfTrx are accessible to modification by this compound. Surprisingly the first cysteine residue of the active site motif (Cys30) is less accessible than the second cysteine (Cys33), which is highly prone to the modification. These results suggest a difference in the structure and reaction mechanism of PfTrx compared to other known thioredoxins.

Call to consolidate achievements for onchocerciasis and lymphatic filariasis control.

The Bernhard Nocht Institute for Tropical Medicine and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases held an international conference to review recent achievements in research and control of onchocerciasis and lymphatic filariasis on 19-23 September 2001 in Hamburg, Germany.

The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs.

The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.

Intersubunit interactions in Plasmodium falciparum thioredoxin reductase.

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M(r) and the low M(r) proteins. The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.