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PubMed2002

Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells. Characterization, comparison with the parasite protease, and homology modeling.

Serine proteases play crucial roles in erythrocyte invasion by merozoites of the malaria parasite. Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is synthesized during maturation of the intraerythrocytic parasite and accumulates in a set of merozoite secretory organelles, suggesting that it may play a role in host cell invasion or post-invasion events. We describe the production, purification, and characterization of recombinant PfSUB-1 and comparison with the authentic protease detectable in parasite extracts. The recombinant protease requires high levels of calcium for optimum activity and has an alkaline pH optimum. Using a series of decapeptide and protein substrates, PfSUB-1 was found to have a relaxed substrate specificity with regard to the P1 position but is unable to efficiently cleave substrates with a P1 leucine residue. Similarly, replacement of a P4 valine with alanine severely reduced cleavage efficiency, whereas its replacement with lysine abolished cleavage. In all respects investigated, the recombinant protease was indistinguishable from parasite-derived enzyme. Three-dimensional homology modeling of the PfSUB-1 catalytic domain based on an alignment with closely related bacterial subtilisins and an orthologue from the rodent malaria Plasmodium yoelii suggests that the protease has at least three potential calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine within the enzyme S1 pocket. A predicted highly polar S1 pocket and a hydrophobic S4 subsite are in broad agreement with the experimentally determined substrate specificity.

Amino Acid Sequence↗
PubMed2002

Function of region I and II adhesive motifs of Plasmodium falciparum circumsporozoite protein in sporozoite motility and infectivity.

The circumsporozoite protein of Plasmodium falciparum contains two conserved motifs (regions I and II) that have been proposed to interact with mosquito and vertebrate host molecules in the process of sporozoite invasion of salivary glands and hepatocytes, respectively. To study the function of this protein we have replaced the endogenous circumsporozoite protein gene of Plasmodium berghei with that of P. falciparum and with versions lacking either region I or region II. We show here that P. falciparum circumsporozoite protein functions in rodent parasite and that P. berghei sporozoites carrying the P. falciparum CS gene develop normally, are motile, invade mosquito salivary glands, and infect the vertebrate host. Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. These data shed new light on the role of the CS protein in sporozoite motility and infectivity.

Amino Acid Motifs↗
PubMed2002

Structure of the C-terminal domains of merozoite surface protein-1 from Plasmodium knowlesi reveals a novel histidine binding site.

The protozoan parasite Plasmodium causes malaria, with hundreds of millions of cases recorded annually. Protection against malaria infection can be conferred by antibodies against merozoite surface protein (MSP)-1, making it an attractive vaccine candidate. Here we present the structure of the C-terminal domains of MSP-1 (known as MSP-1(19)) from Plasmodium knowlesi. The structure reveals two tightly packed epidermal growth factor-like domains oriented head to tail. In domain 1, the molecule displays a histidine binding site formed primarily by a highly conserved tryptophan. The protein carries a pronounced overall negative charge primarily due to the large number of acidic groups in domain 2. To map protein binding surfaces on MSP-1(19), we have analyzed the crystal contacts in five different crystal environments, revealing that domain 1 is highly preferred in protein-protein interactions. A comparison of MSP-1(19) structures from P. knowlesi, P. cynomolgi, and P. falciparum shows that, although the overall protein folds are similar, the molecules show significant differences in charge distribution. We propose the histidine binding site in domain 1 as a target for inhibitors of protein binding to MSP-1, which might prevent invasion of the merozoite into red blood cells.

Amino Acid Sequence↗
PubMed2003

Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum.

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.

Amino Acid Sequence↗
PubMed2003

Multiple splice variants encode a novel adenylyl cyclase of possible plastid origin expressed in the sexual stage of the malaria parasite Plasmodium falciparum.

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.

Adenylyl Cyclases↗
PubMed2003

Identification, characterization, and inhibition of Plasmodium falciparum beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ).

The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a Km of 199 microM and kcat/Km of 80.4 m-1 s-1 for the substrate analog beta-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (Km = 86 microM, kcat/Km = 220 m-1 s-1). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli beta-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria.

Amino Acid Sequence↗
PubMed2003

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein.

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.

Animals↗
PubMed2003

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Amino Acid Sequence↗
PubMed2003

A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites.

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.

Animals↗
PubMed2003

Plasmodium falciparum-activated chloride channels are defective in erythrocytes from cystic fibrosis patients.

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.

Animals↗
PubMed2005

Dissection of merozoite surface protein 3, a representative of a family of Plasmodium falciparum surface proteins, reveals an oligomeric and highly elongated molecule.

Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the full-length molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of alpha-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The full-length MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 mum. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f(0) > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.

Amino Acid Sequence↗
PubMed2006

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein.

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.

Amino Acid Sequence↗
PubMed2006

Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog.

Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.

Amino Acid Sequence↗
PubMed2006

Structural and functional characterization of Falcipain-2, a hemoglobinase from the malarial parasite Plasmodium falciparum.

Malaria is caused by protozoan erythrocytic parasites of the Plasmodium genus, with Plasmodium falciparum being the most dangerous and widespread disease-causing species. Falcipain-2 (FP-2) of P. falciparum is a papain-family (C1A) cysteine protease that plays an important role in the parasite life cycle by degrading erythrocyte proteins, most notably hemoglobin. Inhibition of FP-2 and its paralogues prevents parasite maturation, suggesting these proteins may be valuable targets for the design of novel antimalarial drugs, but lack of structural knowledge has impeded progress toward the rational discovery of potent, selective, and efficacious inhibitors. As a first step toward this goal, we present here the crystal structure of mature FP-2 at 3.1 A resolution, revealing novel structural features of the FP-2 subfamily proteases including a dynamic beta-hairpin hemoglobin binding motif, a flexible N-terminal alpha-helical extension, and a unique active-site cleft. We also demonstrate by biochemical methods that mature FP-2 can proteolytically process its own precursor in trans at neutral to weakly alkaline pH, that the binding of hemoglobin to FP-2 is strictly pH-dependent, and that FP-2 preferentially binds methemoglobin over hemoglobin. Because the specificity and proteolytic activity of FP-2 toward its multiple targets appears to be pH-dependent, we suggest that environmental pH may play an important role in orchestrating FP-2 function over the different life stages of the parasite. Moreover, it appears that selectivity of FP-2 for methemoglobin may represent an evolutionary adaptation to oxidative stress conditions within the host cell.

Amino Acid Motifs↗
PubMed2004

Maurer's cleft organization in the cytoplasm of plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections.

Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum. The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the cytoplasm of the host cell to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single-section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts. Verification of this model would be greatly assisted by a better understanding of the morphology, dimensions and origin of the Maurer's clefts. Here, we have generated and analyzed three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 microm thickness. Our results indicate that Maurer's clefts are heterogeneous in structure and size. We have found Maurer's clefts consisting of a single disk-shaped cisternae localized beneath the plasma membrane. In other examples, Maurer' clefts formed an extended membranous network that bridged most of the distance between the parasite and the plasma membrane of the host erythrocyte. Maurer's cleft membrane networks were composed of both branched membrane tubules and stacked disk-shaped membrane cisternae that eventually formed whorls. Maurer's clefts were visible in other cells as a loose membrane reticulum composed of scattered tubular and disk-shaped membrane profiles. We have not seen clearly discernable isolated vesicles in the analyzed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.

Animals↗
PubMed1988

Comparative evaluation of the protective effect of immune spleen cells and immune RNA against Plasmodium berghei.

Earlier studies from this laboratory indicated that passive transfer of viable or frozen-thawed cells from spleens and lymph nodes of immune mice resulted in a significant protective immunity against Plasmodium berghei in syngeneic recipients. To assess whether immune RNA played a role in conferring such protection, experiments were designed wherein immune RNA was isolated from immune monkeys, rats and mice and transferred to normal mice. The effect of transfer was assessed by challenging RNA-primed animals with Plasmodium berghei. Results indicated that immune RNA failed to confer resistance against P. berghei both in syngeneic and in heterologous systems.

Animals↗
PubMed1992

A new model for testing gametocytocidal effects of some antimalarial drugs on Plasmodium falciparum in vitro.

A technique is described for obtaining pure gametocyte cultures of Plasmodium falciparum, using pyrimethamine at the minimum concentration for inhibition of asexual parasites. Routine cultures producing sexual stages were exposed to pyrimethamine on days 5 and 6. These cultures grew synchronously and contained gametocytes of stages II, III and V on day 7, 9 and 15 of the cultures respectively. The pyrimethamine-treated gametocytes were more infective to mosquitoes than were untreated controls. This model for the culture of pure gametocytes was used to observe the activity of chloroquine, halofantrine, pyrimethamine and quinine on the gametocyte stage III of Plasmodium falciparum strain NF54 in vitro. NF54 was shown to be sensitive to chloroquine, quinine and pyrimethamine, but the results showed that halofantrine was the most effective drug in reducing the number of gametocytes. A concentration of 3 x 10(-9) M halofantrine was lethal to both asexual parasites and gametocytes. The gametocytocidal EC90 of chloroquine (1 x 10(-6) M) and that of quinine (9 x 10(-7) M) were equal to the minimum inhibitory concentration of asexual stages of isolates of P. falciparum considered as highly resistant to these drugs. A high concentration of pyrimethamine (1 x 10(-4) M) had, in contrast, little effect on gametocytes.

Animals↗
PubMed1997

Effects of folic and folinic acids in the activities of cycloguanil and WR99210 against Plasmodium falciparum in erythrocytic culture.

The in-vitro effects of folinic acid on the antimalarial activities of the triazine antifolates, cycloguanil and WR99210, were compared with those of their parent biguanides, proguanil and PS-15, a dihydrofolate-reductase inhibitor, pyrimethamine, and a pyrimidine antagonist, atovaquone. It was found that the minimum inhibitory concentrations of cycloguanil and WR99210 were not affected by physiological concentrations of folic or folinic acids in human serum. Experiments with Plasmodium falciparum growing in erythrocytic culture showed that the antimalarial effect of cycloguanil is readily antagonised by folinic acid, whereas WR99210 is much more refractory. Plasmodium falciparum exposed to cycloguanil (2.5 microM, 6 h) and WR99210 (1 microM, 6 h), showed depressed levels of thymidine 5'-triphosphate (dTTP) in the absence and presence of folinic acid (25 microM and 10 microM, respectively). The decrease in dTTP may be attributed to inhibition of dihydrofolate reductase. However, as the addition of folinic acid did not restore dTTP levels in the parasites, the drugs may have an additional or different mechanism of toxicity.

Animals↗