In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of plant species, used traditionally to treat malaria, have been extensively investigated for their activity against Plasmodium intraerythrocytic asexual parasites in search of new antimalarial drugs. However, less effort has been directed towards examining their efficacy in blocking transmission. Here, we report the results of the in vitro screening of extracts from eight selected plant species used traditionally to treat malaria in South Africa for activity against Plasmodium falciparum NF54 early and late stage gametocytes. The species used were Khaya anthotheca, Trichilia emetica, Turraea floribunda, Leonotis leonurus, Leonotis leonurus ex Hort, Olea europaea subsp. Africana, Catha edulis and Artemisia afra. AIM OF THE STUDY: To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes. MATERIAL AND METHODS: Air-dried and ground plant leaves were extracted using acetone. Primary two point in vitro phenotypic screens against both early and late stage gametocytes were done at 10 and 20µg/ml followed by full IC50 determination of the most active extracts. Inhibition of gametocyte viability in vitro was assessed using the parasite lactate dehydrogenase (pLDH) assay. RESULTS: Of the eight crude acetone extracts from plant species screened in vitro, four had good activity with over 50-70% inhibition of early and late stage gametocytes' viability at 10 and 20µg/ml, respectively. Artemisia afra (Asteraceae), Trichilia emetica (Meliaceae) and Turraea floribunda (Meliaceae) were additionally highly active against both gametocyte stages with IC50 values of less than 10µg/ml while Leonotis leonurus ex Hort (Lamiaceae) was moderately active (IC50<20µg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages. CONCLUSION: This study shows the potential transmission blocking activity of extracts from selected South African medicinal plants and substantiates their traditional use in malaria control that broadly encompasses prevention, treatment and transmission blocking. Further studies are needed to isolate and identify the active principles from the crude extracts of A. afra, T. emetica and T. floribunda, as well as to examine their efficacy towards blocking parasite transmission to mosquitoes.

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na(+) but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential.

Functional consequences of perturbing polyamine metabolism in the malaria parasite, Plasmodium falciparum.

Inhibition of polyamine biosynthesis and/or the perturbation of polyamine functionality have been exploited with success against parasitic diseases such as Trypanosoma infections. However, when the classical polyamine biosynthesis inhibitor, alpha-difluoromethylornithine, is used against the human malaria parasite, Plasmodium falciparum, it results in only a cytostatic growth arrest. Polyamine metabolism in this parasite has unique properties not shared by any other organism. These include the bifunctional arrangement of the catalytic decarboxylases and an apparent absence of the typical polyamine interconversion pathways implying different mechanisms for the regulation of polyamine homeostasis that includes the uptake of exogenous polyamines at least in vitro. These properties make polyamine metabolism an enticing drug target in P. falciparum provided that the physiological and functional consequences of polyamine metabolism perturbation are understood. This review highlights our current understanding of the biological consequences of inhibition of the biosynthetic enzymes in the polyamine pathway in P. falciparum as revealed by several global analytical approaches. Ultimately, the evidence suggests that polyamine metabolism in P. falciparum is a validated drug target worth exploiting.

Antimalarial drug discovery: in silico structural biology and rational drug design.

Malaria remains one of the most burdensome human infectious diseases, with a high rate of resistance outbreaks and a constant need for the discovery of novel antimalarials and drug targets. For several reasons, Plasmodial proteins are difficult to characterise structurally using traditional physical approaches. However, these problems can be partially overcome using a number of in silico approaches. This review describes the peculiarities of malaria proteins and then details various in silico strategies to select and allow descriptions of the molecular structures of drug target candidates as well as subsequent rational approaches for drug design. Chiefly, homology modelling with specific focus on unique aspects of malaria proteins including low homology, large protein size and the presence of parasite-specific inserts is addressed and alternative strategies including multiple sequence and structure-based prediction methods, sampling-based approaches that aim to reveal likely global or shared features of a Plasmodial structure and the value of molecular dynamics understanding of unique features of Plasmodial proteins are discussed. Once a detailed description of the drug target is available, in silico approaches to the specific design of an inhibitory drug thereof becomes invaluable as an economic and rational alternative to chemical library screening.

Exploring functional genomics for drug target and therapeutics discovery in Plasmodia.

Functional genomics approaches are indispensable tools in the drug discovery arena and have recently attained increased attention in antibacterial drug discovery research. However, the application of functional genomics to post-genomics research of Plasmodia is still in comparatively early stages. Nonetheless, with this genus having the most species sequenced of any eukaryotic organism so far, the Plasmodia could provide unique opportunities for the study of intracellular eukaryotic pathogens. This review presents the status quo of functional genomics of the malaria parasite including descriptions of the transcriptome, proteome and interactome. We provide examples for the in silico mining of the X-ome data sets and illustrate how X-omic data from drug challenged parasites might be used in elucidating amongst others, the mode-of-action of inhibitory compounds, validate potential targets and discover novel targets/therapeutics.

Similar mechanisms regulate protein exocytosis from the salivary glands of ixodid and argasid ticks.

Numerous bioactive compounds are secreted from large dense core granules in tick salivary glands during feeding in response to an external stimulus. Investigations into the signalling pathways regulating secretion indicated that they are similar for Argasidae (fast-feeding ticks) and Ixodidae (slow-feeding ticks), but differ in their sensitivity to prostaglandin E(2). In both cases, dopamine is the external signal for inducing exocytosis. Dopamine-induced exocytosis was shown to be strongly calcium dependant. Firstly, it requires extracellular calcium via a L-type voltage-gated calcium channel located on the plasma membrane and, secondly, intracellular calcium which is released presumably in response to inositol 1,4,5-triphosphate (IP(3)). Pathways such as the activation of phospholipase C, inositol-phosphate kinases, G-proteins, GTPases and Na(+)-K(+)-ATPases have been shown to be essential.

The influence of tick behavior, biotope and host specificity on concerted evolution of the platelet aggregation inhibitor savignygrin, from the soft tick Ornithodoros savignyi.

Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding to enable control of the hemostatic system of the host. Complex interactions at the tick-host interface are an indication of the important role that the host played during tick evolution. The question is to what extent interaction with the host and the environment influences tick evolution. Previously, two isoforms (97% sequence identity) of savignygrin, an alphaIIbbeta3 antagonist, have been described. The presence of both isoforms within 20 random individuals confirmed that these isoforms must be recent gene duplicates. Analysis of the sequence differences between the isoforms shows a Kn/Ks ratio of 1, which indicates neutral selection for the isoforms. However, the biased localization of differences within the 3' end of the genes suggests that concerted evolution acts on the isoforms. Calculation of the divergence date between the isoforms (1.6-5.2 MYA) also indicates purifying selection, as ample time had passed after duplication, for inactivation of one gene copy. We conclude that concerted evolution has functioned to maintain a high copy number of the savignygrins in order for Ornithodoros savignyi to parasitize a wide host range. This contrasts with O. moubata that expresses the savignygrin homolog, disagregin, as a single copy at lower concentration levels and correlates with the confined habitat and consequently narrow host range of O. moubata. Recent "domestication" of O. savignyi due to animal husbandry practices could however, have reduced the selection constraints acting to maintain the gene copies as evidenced by the structural instability of one of the isoforms. Our results suggest that environmental factors and host associations do play an important role in the evolution of anti-hemostatic components in ticks.

Comparative properties of a three-dimensional model of Plasmodium falciparum ornithine decarboxylase.

The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal alpha/beta-barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon alpha-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structure-based approach for the design of novel malaria-specific inhibitors.

Amino acid sequence and structure modeling of savignin, a thrombin inhibitor from the tick, Ornithodoros savignyi.

The full-length gene of savignin, a potent thrombin (E.C. 3.4.21.5) inhibitor from the tick Ornithodoros savignyi has been cloned and sequenced. Both 5' and 3' UTR's, a signal peptide from the translated amino acid sequence and an unusual poly-adenylation signal (AATACA) has been identified. The translated protein sequence shows high identity (63%) with ornithodorin, the thrombin inhibitor from the tick, Ornithodoros moubata. Molecular modeling using the structure of ornithodorin as reference gave a structure with an RMSD of 0.25 A for the full-length protein, 0.11 A for the N-terminal BPTI-like domain and 0.11 A for the C-terminal BPTI-like domain, indicating that maximum deviation occurs in the mobile bridge (0.18 A) between the two domains. Docking of savignin to thrombin shows that the interaction is similar to the ornithodorin-thrombin complex. The N-terminal amino acid residues of savignin bind inside the active site cleft, while the C-terminal domain of savignin has a net negative electrostatic potential and interacts with the basic fibrinogen recognition exosite of thrombin through hydrogen bonds and hydrophobic interactions. These results correlate with kinetic data obtained, which showed that savignin is a competitive, slow, tight-binding inhibitor that requires thrombin's fibrinogen-binding exo-site for optimal inhibition.

Structure-based inhibitor screening: a family of sulfonated dye inhibitors for malaria parasite triosephosphate isomerase.

The crystal structure of malaria triosephosphate isomerase (TIM) was screened against the National Cancer Institute database of three-dimensional molecular structures. Ten top-scoring commercially available compounds were analyzed for inhibition of recombinant TIM. Two anionic dyes showed inhibition of TIM at concentrations of <100 mM. Four related sulfonated dyes were identified from the literature, docked, and screened in vitro. All showed inhibition of malaria TIM. Models indicate that these compounds bind in two suggested conformations to the active site region of the TIM enzyme. These compounds may be used in rational modification procedures for the synthesis of lead anti-TIM drugs.

In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected South African plants.

The resistance of Plasmodium spp. to currently used drugs has become a serious problem and efforts are being directed in obtaining new drugs with different structural features. One option favoured is the search for new plant derived antimalarial drugs. Bark and leaves of 20 extracts from 14 South African plant species were tested for in vitro antiplasmodial activity by means of the flow cytometric test. The most active extract of each species giving more than 70% inhibition at 50 microg/ml was selected for determination of IC(50) values. Two extracts had IC(50) values below 2 microg/ml, another seven had IC(50) values between 2 and 5 microg/ml while one had an IC(50) of 10.1 microg/ml. Chloroquine had an IC(50) of 0.043 microg/ml. Cytotoxicities of the five most active extracts at 50 microg/ml were determined with the monkey kidney cell toxicity test and the ID(50) values ranged between 35 and 100 microg/ml.

Identification of putative proteins involved in granule biogenesis of tick salivary glands.

Ticks secrete bioactive components during feeding that assist them in gaining a blood meal. Compounds secreted are stored in granules until a stimulus induces secretion during feeding. Biogenesis of tick secretory granules has not been investigated before. An adequate understanding of granule biogenesis could advance our understanding of tick salivary gland biology and could aid in the rational design of tick control methods. Putative tick salivary gland proteins 1-4 (TSGP1-4) involved in granule biogenesis were identified in this study based on their abundance in salivary gland extracts and granule preparations and their ability to aggregate under conditions of slight acidity and high calcium concentration. TSGP2 and TSGP3 have been identified as previously described toxic and nontoxic homologues, respectively, while toxicity was also associated with TSGP4.

Neuropathogenic properties of Argas (Persicargas) walkerae larval homogenates.

Several tick species have been demonstrated, described, or suspected to cause paralysis in their host during the repletion process, presumably by impairing neurotransmission. The resulting polyneuropathy gradually spreads to the upper limbs causing incoordination and ends in respiratory failure. This form of paralysis is commonly confused with Guillain-Barrè syndrome, botulism and myasthenia gravis and although the clinical symptoms of these diseases are similar, it is not clear whether the pathogenesis is also the same. During this study we investigated the mechanism of paralysis by the tick Argas (Persicargas) walkerae by determining the effect of larval homogenates on both potassium-stimulated (calcium-dependent) and veratridine-stimulated (external calcium-independent) release of [3H]glycine from crude rat brain synaptosomes. The results indicated that larval homogenates inhibited both processes. These findings are reconcilable with the results obtained for two other paralysis-causing tick species, Ixodes holocyclus and Dermacentor andersoni, which were indicated to cause paralysis by decreasing the synthesis or release of acetylcholine at the neuromuscular junction.

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi (Acari: Argasidae).

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.

Detection and micro-scale isolation of a low molecular mass paralysis toxin from the tick, Argas (Persicargas) walkerae.

This study describes the isolation of a 11 kDa paralysis toxin from crude larval extracts of Argas (Persicargas) walkerae by exploiting the cross-reactivity of a monoclonal antibody (4B12), directed against the paralysis toxin of Rhipicephalus evertsi evertsi. This low molecular mass is in contrast to previous findings of a 60-70 kDa toxin for A. (P.) walkerae, but is similar to neurotoxins isolated from venomous forms of the class Arachnida, which comprise the orders Araneae (spiders). Scorpionida (Scorpions) and Acari (ticks and mites). Since numerous antigenic bands, ranging between 11 and 115 kDa, were detected by the monoclonal antibody 4B12, the possibility of toxin-complex formation and the effect of pH on the latter were investigated by means of HPLC and ammonium sulphate precipitation. The results suggest that physiological conditions, with respect to pH and ionic strength, promote the formation of heterogeneous toxin-complexes while an acidic pH favours the formation of a more homogeneous toxin-containing complex. Furthermore, the effect of partially purified toxin on neurotransmitter release from crude rat brain synaptosomes was investigated, since tick paralysis toxins are hypothesised to inhibit neurotransmitter release from the presynaptic terminal. Both calcium-dependent, as well as calcium-independent release was inhibited by the toxin-containing sample.

Apyrase activity and platelet aggregation inhibitors in the tick Ornithodoros savignyi (Acari: Argasidae).

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activitives supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.

Cloning, nucleotide sequence and expression of the gene encoding factor Xa inhibitor from the salivary glands of the tick, Ornithodoros savignyi.

The N-terminal sequence of the competitive and slow tight-binding factor Xa inhibitor (fXaI; Ki = 0.83 +/- 0.10 nM) isolated from the salivary glands of Ornithodoros savignyi ticks (Acari: Argasidae) was employed to design a degenerate gene-specific primer (GSP) for 3'-rapid amplification of cDNA ends (3'-RACE). The primer consisted of a sequence encoding for amino acid residues 5-11. A full-length gene was next constructed from the 3'-RACE product in a two-step PCR procedure and successfully expressed by the BAC-TO-BAC baculovirus expression system. The deduced amino acid sequence of the gene showed 46% identity and 78% homology to an fXaI (TAP) from Ornithodoros moubata. Recombinant fXaI (rfXaI) consists of 60 amino acid residues, has a molecular mass of approximately 7 kDa and inhibited fXa by approximately 91%. The availability of the rfXaI will aid further investigations of its potential for therapeutic applications and as vaccine against tick infestation. The authentic nucleotide sequence of the gene encoding tick fXaI furthermore enables studies at the genetic level and probing of other tick species for similar and related genes.

Properties of proteins binding plasma progesterone in pregnant Cape porcupines (Hystrix africaeaustralis).

The properties of progesterone-binding proteins in plasma of pregnant Cape porcupines were investigated using radiolabelled progesterone and either progesterone or cortisol as competing ligands as well as native plasma and heated (60 degrees C for 30 min) plasma. The results demonstrated that plasma from pregnant porcupines contains corticosteroid-binding globulin, but that it constitutes a significant portion of plasma progesterone-binding proteins only during the early stages of pregnancy. Corticosteroid-binding globulin of porcupines appears to be as heat labile as that of guinea-pigs. Concentrations of progesterone-binding proteins in plasma increased during pregnancy to reach concentrations at the eleventh week that were 25 times higher than those of progesterone; concentrations increased significantly (r2 = 0.88) with the increase in progesterone concentration. The results indicate that plasma progesterone-binding proteins in Cape porcupines (Old World hystricomorph) are similar in composition to those in guinea-pigs (New World hystricomorph).