Search

Currently, widely used antimalarial drugs have a limited clinical lifespan due to parasite resistance development. With resistance continuously rising, antimalarial drug discovery requires strategies to decrease the time of delivering a new antimalarial drug while simultaneously increasing the drug's therapeutic lifespan. Lessons learnt from various chemotherapeutic resistance studies in the fields of antibiotic and cancer research offer potentially useful strategies that can be applied to antimalarial drug discovery.In this review the authors discuss current strategies to circumvent resistance in malaria and alternatives that could be employed.Scientists have been 'beating back' the malaria parasite with novel drugs for the past 49 years but the constant rise in antimalarial drug resistance is forcing the drug discovery community to explore alternative strategies. Avant-garde anti-resistance strategies from alternative fields may assist our endeavors to manage, control and prevent antimalarial drug resistance to progress beyond beating the resistant parasite back, to stopping it dead in its tracks.

Graphical abstract, Highlights • A highly specific inhibitor, Genz-644131, was identified for P. falciparum AdoMetDC. • Increased specificity for bifunctional over monofunctional PfAdoMetDC was shown. • Genz-644131 showed improved inhibition against in vitro P. falciparum parasites. • Immunoliposomes improved in vitro drug delivery of Genz-644131 by 32-fold., S-adenosyl-l-methionine decarboxylase (AdoMetDC) in the polyamine biosynthesis pathway has been identified as a suitable drug target in Plasmodium falciparum parasites, which causes the most lethal form of malaria. Derivatives of an irreversible inhibitor of this enzyme, 5′-{[(Z)-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL73811), have been developed with improved pharmacokinetic profiles and activity against related parasites, Trypanosoma brucei. Here, these derivatives were assayed for inhibition of AdoMetDC from P. falciparum parasites and the methylated derivative, 8-methyl-5′-{[(Z)-4-aminobut-2-enyl]methylamino}-5′-deoxyadenosine (Genz-644131) was shown to be the most active. The in vitro efficacy of Genz-644131 was markedly increased by nanoencapsulation in immunoliposomes, which specifically targeted intraerythrocytic P. falciparum parasites.

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.

Plasmodium falciparum like other organisms is dependent on polyamines for proliferation. Polyamine biosynthesis in these parasites is regulated by a unique bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC). Only limited biochemical and structural information is available on the bifunctional enzyme due to the low levels and impurity of an instable recombinantly expressed protein from the native gene. Here we describe the high level expression of stable monofunctional PfAdoMetDC from a codon-harmonised construct, which permitted its biochemical characterisation indicating similar catalytic properties to AdoMetDCs of orthologous parasites. In the absence of structural data, far-UV CD showed that at least on secondary structure level, PfAdoMetDC corresponds well to that of the human protein. The kinetic properties of the monofunctional enzyme were also found to be different from that of PfAdoMetDC/ODC as mainly evidenced by an increased K(m). We deduced that complex formation of PfAdoMetDC and PfODC could enable coordinated modulation of the decarboxylase activities since there is a convergence of their k(cat) and lowering of their K(m). Such coordination results in the aligned production of decarboxylated AdoMet and putrescine for the subsequent synthesis of spermidine. Furthermore, based on the results obtained in this study we propose a new AdoMetDC subclass for plasmodial AdoMetDCs.

Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.

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 3.5.3.1) is a multimeric metallo-protein that catalyses the hydrolysis of arginine to ornithine and urea by means of a binuclear spin-coupled Mn2+ cluster in the active site. A previous report indicated that P. falciparum arginase has a strong dependency between trimer formation, enzyme activity and metal co-ordination. Mutations that abolished Mn2+ binding also caused dissociation of the trimer; conversely, mutations that abolished trimer formation resulted in inactive monomers. By contrast, the monomers of mammalian (and therefore host) arginase are also active. P. falciparum arginase thus appears to be an obligate trimer and interfering with trimer formation may therefore serve as an alternative route to enzyme inhibition. In the present study, the mechanism of the metal dependency was explored by means of homology modelling and molecular dynamics. When the active site metals are removed, loss of structural integrity is observed. This is reflected by a larger equilibration rmsd for the protein when the active site metal is removed and some loss of secondary structure. Furthermore, modelling revealed the existence of a novel inter-monomer salt-bridge between Glu295 and Arg404, which was shown to be associated with the metal dependency. Mutational studies not only confirmed the importance of this salt-bridge in trimer formation, but also provided evidence for the independence of P. falciparum arginase activity on trimer formation.

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.

Polyamines are essential polycationic molecules involved in multiple cellular events, including cell differentiation, division and death. Inhibition of polyamine biosynthesis has been considered in diverse therapeutic strategies ranging from tumour suppressors to anti-parasitic agents. In the human malaria parasite, Plasmodium falciparum, inhibition of ornithine decarboxylase (ODC) results in the arrest of schizogony due to polyamine depletion. However, the exact physiological role of the polyamines in the parasite is unknown. Here, we present results of the depletion of polyamines in the malaria parasite by α-difluoromethylornithine inhibition of ODC, as observed with differential transcriptome profiling. Upon depletion of their endogenous polyamines, the up- and downregulated parasite transcripts were selected with suppression subtractive hybridisation and differences were detected using blots or DNA microarrays. A direct linkage between polyamine depletion and the differential expression of two distinct transcripts was observed, indicating the existence of a transcriptional feedback response in the P. falciparum transcriptome upon drug challenge. The data presented provide input into the role of the polyamines in the cellular biology of P. falciparum and contribute towards the validation of polyamine biosynthesis as an antimalarial target.

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.To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes.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.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 (IC5020µg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages.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.

Chemotherapy of malaria parasites is limited by established drug resistance and lack of novel treatment options. Intraerythrocytic stages of Plasmodium falciparum, the causative agent of severe malaria, are wholly dependent upon host glucose for energy. A facilitative hexose transporter (PfHT), encoded by a single-copy gene, mediates glucose uptake and is therefore an attractive potential target. The authors first established heterologous expression in Xenopus laevis to allow functional characterisation of PfHT. They then used this expression system to compare the interaction of substrates with PfHT and mammalian Gluts (hexose transporters) and identified important differences between host and parasite transporters. Certain Omethyl derivatives of glucose proved to be particularly useful discriminators between mammalian transporters and PfHT. The authors exploited this selectivity and synthesised an O-3 hexose derivative that potently inhibits PfHT expressed in oocytes. This O-3 derivative (compound 3361) also kills cultured P. falciparum with comparable potency. Compound 3361 acts with reasonable specificity against PfHT orthologues encoded by other parasites such as Plasmodium vivax, Plasmodium yoelii and Plasmodium knowlesi. Multiplication of Plasmodium berghei in a mouse model is also significantly impeded by this compound. These findings validate PfHT as a novel target.

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 α IIb β 3 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.

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.

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. Plasmodium falciparum spermidine synthase (PfSpdS) catalyzes the synthesis of spermidine and spermine. It is a major polyamine flux-determining enzyme and spermidine is a prerequisite for the post-translational activation of P. falciparum eukaryotic translation initiation factor 5A (elF5A). The most potent inhibitors of eukaryotic SpdS’s are not specific for PfSpdS. Methods ‘Dynamic’ receptor-based pharmacophore models were generated from published crystal structures of SpdS with different ligands. This approach takes into account the inherent flexibility of the active site, which reduces the entropic penalties associated with ligand binding. Four dynamic pharmacophore models were developed and two inhibitors, (1R,4R)-(N1-(3-aminopropyl)-trans-cyclohexane-1,4-diamine (compound 8) and an analogue, N-(3-aminopropyl)-cyclohexylamine (compound 9), were identified. Results A crystal structure containing compound 8 was solved and confirmed the in silico prediction that its aminopropyl chain traverses the catalytic centre in the presence of the byproduct of catalysis, 5′-methylthioadenosine. The IC50 value of compound 9 is in the same range as that of the most potent inhibitors of PfSpdS, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and 4MCHA and 100-fold lower than that of compound 8. Compound 9 was originally identified as a mammalian spermine synthase inhibitor and does not inhibit mammalian SpdS. This implied that these two compounds bind in an orientation where their aminopropyl chains face the putrescine binding site in the presence of the substrate, decarboxylated S-adenosylmethionine. The higher binding affinity and lower receptor strain energy of compound 9 compared to compound 8 in the reversed orientation explained their different IC50 values. Conclusion The specific inhibition of PfSpdS by compound 9 is enabled by its binding in the additional cavity normally occupied by spermidine when spermine is synthesized. This is the first time that a spermine synthase inhibitor is shown to inhibit PfSpdS, which provides new avenues to explore for the development of novel inhibitors of PfSpdS. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0572-z) contains supplementary material, which is available to authorized users.

The tick, Ornithodoros savignyi has been implicated in inducing paralysis and tampan toxicosis. In this study, a basic toxin (TSGP4) was identified and the presence of an acidic toxin (TSGP2) was confirmed. Both basic and acidic toxins were more lethal than previously described, with TSGP4 (34microg) and TSGP2 (24microg) causing mortality of adult mice within 30min. Pathological effects on the cardiac system, notably of salivary gland extract on an isolated rat heart perfusion system and of purified toxins on mouse electrocardiogram patterns could be observed. TSGP4 caused Mobitz type ventricular block, while TSGP2 induced ventricular tachycardia. Conversely, fractions from reversed phase high performance liquid chromatography preparations caused paralysis-like symptoms of the limbs after only 48h. The toxins also differ from previously described tick paralysis toxins in terms of molecular behavior and properties. These results indicate that tampan toxicoses and tick paralysis are unrelated pathogenic phenomena.

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of alpha-CD41 (P2) to platelets, the binding of purified alpha(IIb)beta3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin alpha(v)beta3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward alpha(IIb)beta3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.

Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the αIIbβ3 antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of αIIbβ3. These results are concurrent with targeting of αIIbβ3 and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks.

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.

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.

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

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, 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.

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 activities 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.

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 ~7 kDa and inhibited fXa by ~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.

Anthracene-polyamine conjugates inhibit the in vitro proliferation of the intraerythrocytic human malaria parasite Plasmodium falciparum , with 50% inhibitory concentrations (IC 50 s) in the nM to μM range. The compounds are taken up into the intraerythrocytic parasite, where they arrest the parasite cell cycle. Both the anthracene and polyamine components of the conjugates play a role in their antiplasmodial effect.

Progesterone-binding globulin (PBG) was purified from pooled pregnant Cape porcupine (Hystrix africaeaustralis) plasma by SP-Sephadex Chromatography, followed by Sephacryl S-200 gel filtration. The final product was homogeneous according to high performance capillary electrophoresis, SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. A molecular mass of 65,300 Da was calculated from sedimentation equilibrium centrifugation data. Scatchard analysis indicated a dissociation constant of 0.82 × 10−9M, and PBG concentration of 18.4 nmole/mg protein.

Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) is a key enzyme in the synthesis of isoprenoids in the malaria parasite, using a pathway that is absent in the human host. This enzyme is receiving attention as it has been validated as a promising drug target. However, an impediment to the characterisation of this en- zyme has been the inability to obtain sufficient quantities of the enzyme in a soluble and functional form. The expression of PfDXR from the codon harmonised coding region, under conditions of strongly controlled transcription and induction, resulted in a yield of 2 - 4 mg/L of enzyme, which is 8 to 10-fold higher than previously reported yields. The kinetic pa- rameters Km, Vmax and kcat were determined for PfDXR using an NADPH-dependent assay. Residues K295 and K297, unique to species of Plasmodium and located in the catalytic hatch region; and residues V114 and N115, essential for NADPH binding, were mutated to resemble those found in E. coli DXR. Interestingly, these mutations decreased the sub- strate affinity of PfDXR to values resembling that of E. coli DXR. PfDXR-K295N, K297S and PfDXR-V114A, N115G demonstrated a decreased ability to turnover substrate by 4-fold and 2-fold respectively in comparison to PfDXR. This study indicates a difference in the role of the catalytic hatch in capturing substrate by species of Plasmodium. The results of this study could contribute to the development of inhibitors of PfDXR.

Malaria tropica is a devastating infectious disease caused by Plasmodium falciparum. This parasite synthesizes vitamin B6de 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). PfPdx1 accommodates both the R5P and G3P substrates and intricately co-ordinates the reaction mechanism, which is composed of a series of imine bond formations, leading to the production of PLP. We demonstrate that E4P (D-erythrose 4-phosphate) inhibits PfPdx1 in a dose-dependent manner. We propose that the acyclic phospho-sugar E4P, with a C1 aldehyde group similar to acyclic R5P, could interfere with R5P imine bond formations in the PfPdx1 reaction mechanism. Molecular docking and subsequent screening identified the E4P hydrazide analogue 4PEHz (4-phospho-D-erythronhydrazide), which selectively inhibited PfPdx1 with an IC50 of 43 μM. PfPdx1 contained in the heteromeric PLP synthase complex was shown to be more sensitive to 4PEHz and was inhibited with an IC50 of 16 μM. Moreover, the compound had an IC50 value of 10 μM against cultured P. falciparum intraerythrocytic parasites. To analyse further the selectivity of 4PEHz, transgenic cell lines overexpressing PfPdx1 and PfPdx2 showed that additional copies of the protein complex conferred protection against 4PEHz, indicating that the PLP synthase is directly affected by 4PEHz in vivo. These PfPdx1 inhibitors represent novel lead scaffolds which are capable of targeting PLP biosynthesis, and we propose this as a viable strategy for the development of new therapeutics against malaria.

Total plasma and unbound progesterone and testosterone concentrations in pregnant Cape porcupines were determined by radioimmunoassay and centrifugal ultrafiltration dialysis of plasma collected at weekly intervals throughout gestation. During pregnancy, total plasma progesterone and testosterone concentrations attained values of up to ~200ng/ml and ~2000pg/ml, respectively. However, unbound progesterone and testosterone remained relatively constant at 2% and 13% of total values, respectively. The low concentrations of unbound steroids result from high concentrations of plasma binding proteins which probably protect progesterone and testosterone from metabolism during pregnancy. High testosterone concentrations may induce the production of plasma progesterone binding proteins during pregnancy to maintain high progesterone concentrations.

New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness, Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.

Polyamines are present at high levels in proliferating cells, including cancerous cells and protozoan parasites and the inhibition of their synthesis has been exploited in antiproliferative strategies. Inhibition of the malaria parasite's polyamine biosynthetic pathway causes cytostatic arrest in the trophozoite stage but does not cure in vivo infections in the murine model of malaria. This is possibly due to exogenous polyamine salvage from the host, which replenishes the intracellular polyamine pool. This implies that disruption of polyamine metabolism as an antimalarial chemotherapy strategy may require targeting both polyamine biosynthesis and transport simultaneously. In the absence of a clear understanding of the uptake mechanism of polyamines into Plasmodium falciparum parasites, polyamine transport into both the infected erythrocytes and parasites isolated from the erythrocyte were investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes (iRBC) were similar to those of transport into uninfected erythrocytes (RBC) spermidine uptake occurred via the new permeability pathways (NPP) induced by the parasite in the erythrocyte membrane. Once inside the erythrocyte cytoplasm, both putrescine and spermidine were taken up by the parasite via a temperature- and glucose-dependent, saturable process. The uptake of both these polyamines was competed for by other polyamines, and biosynthesis inhibition led to increased total uptake of both putrescine and spermidine. Polyamine uptake was pH dependent with uptake increasing with increasing pH, but did not appear to be coupled to the Na+ or K+ gradients. Membrane potential perturbations influenced polyamine uptake, consistent with the transport being an electrogenic process.

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).

Isozyme analyses have been used for the definitive identification of many plant cultivars, but not for cultivated tomatoes. Six isozyme systems, namely alcohol dehydrogenase, acid phosphatase, phosphoglucomutase, esterase, phosphoglucoisomerase and 6-phosphogluconate dehydrogenase of tomato seed extracts were resolved by isoelectric focusing on polyacrylamide gels with a narrow pH gradient. Nine alcohol dehydrogenase phenotypes were distinguished which, with three acid phosphatase phenotypes, identified twelve of the seventeen cultivars. Fewer differences were found for the other isozymes. Since this method could differentiate between breeding parents and their progeny it is concluded that further investigations are warranted.

In silico searches of new drug candidates have been considered as a cost-effective alternative to experimental drug screening. The efficiency of in silico approaches relies on important assumptions regarding the target selection, the methods employed, the quality of the drug and target molecule structures, and the computing environment (CPU-limited workstations, grids). The use of in silico methods is sometimes the only possible strategy when the infectious agent cannot be propagated safely or with sufficient reproducibility in a laboratory environment, when the target proteins cannot be heterologously expressed for structural and activity analyses or when the genetic variability requires the specific definition of invariable protein domains as potential targets. When the structure of the target is not available, in silico ligand-based drug design may be the only possible alternative strategy to find novel bioactive molecules. The strengths and limitations of in silico strategies therefore rely on the accuracy of the prior knowledge and assumptions. Here, we focus on Plasmodial proteins as a case study, since these proteins can often not be expressed in recombinant systems and are therefore difficult to characterize structurally using traditional physical approaches. This chapter will (1) briefly address the question of target selection in the context of a parasitic infection such as malaria, (2) introduce the peculiarities of malaria proteins and detail some in silico approaches to describe molecular structures of malaria drug targets, and review subsequent rational approaches for (3) receptor-based and (4) ligand-based drug design. This review also presents the evolution of the computing infrastructures that make in silico experiments possible and particularly discusses the WISDOM initiative for grid-enabled drug discovery against neglected and emergent diseases.

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.

International audience; 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.

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 3.5.3.1) is a multimeric metallo-protein that catalyses the hydrolysis of arginine to ornithine and urea by means of a binuclear spin-coupled Mn(2+) cluster in the active site. A previous report indicated that P. falciparum arginase has a strong dependency between trimer formation, enzyme activity and metal co-ordination. Mutations that abolished Mn(2+) binding also caused dissociation of the trimer; conversely, mutations that abolished trimer formation resulted in inactive monomers. By contrast, the monomers of mammalian (and therefore host) arginase are also active. P. falciparum arginase thus appears to be an obligate trimer and interfering with trimer formation may therefore serve as an alternative route to enzyme inhibition. In the present study, the mechanism of the metal dependency was explored by means of homology modelling and molecular dynamics. When the active site metals are removed, loss of structural integrity is observed. This is reflected by a larger equilibration rmsd for the protein when the active site metal is removed and some loss of secondary structure. Furthermore, modelling revealed the existence of a novel inter-monomer salt-bridge between Glu295 and Arg404, which was shown to be associated with the metal dependency. Mutational studies not only confirmed the importance of this salt-bridge in trimer formation, but also provided evidence for the independence of P. falciparum arginase activity on trimer formation.

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.

The organization and mining of malaria genomic and post-genomic data is highly motivated by the necessity to predict and characterize new biological targets and new drugs. Biological targets are sought in a biological space designed from the genomic data from Plasmodium falciparum, but using also the millions of genomic data from other species. Drug candidates are sought in a chemical space containing the millions of small molecules stored in public and private chemolibraries. Data management should therefore be as reliable and versatile as possible. In this context, we examined five aspects of the organization and mining of malaria genomic and post-genomic data: 1) the comparison of protein sequences including compositionally atypical malaria sequences, 2) the high throughput reconstruction of molecular phylogenies, 3) the representation of biological processes particularly metabolic pathways, 4) the versatile methods to integrate genomic data, biological representations and functional profiling obtained from X-omic experiments after drug treatments and 5) the determination and prediction of protein structures and their molecular docking with drug candidate structures. Progresses toward a grid-enabled chemogenomic knowledge space are discussed., Comment: 43 pages, 4 figures, to appear in Malaria Journal

Spermidine synthase is currently considered as a promising drug target in the malaria parasite, Plasmodium falciparum, due to the vital role of spermidine in the activation of the eukaryotic translation initiation factor (eIF5A) and cell proliferation. However, very limited information was available regarding the structure and mechanism of action of the protein at the start of this study. Structural and mechanistic insights of the P. falciparum spermidine synthase (PfSpdSyn) were obtained utilizing molecular dynamics simulations of a homology model based on the crystal structures of the Arabidopsis thaliana and Thermotoga maritima homologues. Our data are supported by in vitro site-directed mutagenesis of essential residues as well as by a crystal structure of the protein that became available recently. We provide, for the first time, dynamic evidence for the mechanism of the aminopropyltransferase action of PfSpdSyn. This characterization of the structural and mechanistic properties of the PfSpdSyn as well as the elucidation of the active site residues involved in substrate, product, and inhibitor interactions paves the way toward inhibitor selection or design of parasite-specific inhibitors.

Resistance of the most virulent human malaria parasite, Plasmodium falciparum, to antifolates is spreading with increasing speed, especially in Africa. Antifolate resistance is mainly caused by point mutations in the P. falciparum dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) target proteins. Homology models of the bifunctional P. falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase (PPPK-DHPS) enzyme as well as the separate domains complete with bound substrates were constructed using the crystal structures of Saccharomyces cerevisiae (PPPK-DHPS), Mycobacterium tuberculosis (DHPS), Bacillus anthracis (DHPS), and Escherichia coli (PPPK) as templates. The resulting structures were subsequently solvated and refined using molecular dynamics. The active site residues of DHPS are highly conserved in S. cerevisiae, M. tuberculosis, E. coli, S. aureus, and B. anthracis, an attribute also shared by P. falciparum DHPS. Sulfadoxine was superimposed into the equivalent position of the p-aminobenzoic acid substrate and its binding parameters were refined using minimization and molecular dynamics. Sulfadoxine appears to interact mainly with P. falciparum DHPS mainly through hydrophobic interactions. Rational explanations are provided by the model for the sulfadoxine resistance-causing effects of four of the five known mutations in P. falciparum DHPS. A possible structure for the bifunctional PPPK-DHPS was derived from the structure from the S. cerevisiae bifunctional enzyme. The active site residues of P. falciparum PPPK are also conserved when compared to S. cerevisiae, Haemophilus influenzae, and E. coli. The informative nature of these models opens up avenues for structure-based drug design approaches toward the development of alternative and more effective inhibitors of P. falciparum PPPK-DHPS.

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.

In the malaria parasite, the two main regulatory activities of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) occur in a single bifunctional protein. The AdoMetDC domain was modeled using the human and potato X-ray crystal structures as templates. Three parasite-specific inserts and the core active site region was identified using a structure-based alignment approach. The domain was modeled without the two largest inserts, to give a root mean square deviation of 1.85 A from the human template. Contact with the rest of the bifunctional complex is predicted to occur on one face of the Plasmodium falciparum AdoMetDC (PfAdoMetDC) domain. In the active site there are four substitutions compared to the human template. One of these substitutions may be responsible for the lack of inhibition by Tris, compared to mammalian AdoMetDC. The model also provides an explanation for the lack of putrescine stimulation in PfAdoMetDC compared to mammalian AdoMetDC. A network of residues that connects the putrescine-binding site with the active site in human AdoMetDC is conserved in the malarial and plant cognates. Internal basic residues are found to assume the role of putrescine, based on the model and site-directed mutagenesis: Arg11 is absolutely required for normal activity, while disrupting Lys15 and Lys215 each cause 50% inhibition of AdoMetDC activity. These novel features of malarial AdoMetDC suggest possibilities for the discovery of parasite-specific inhibitors.

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type ‘a’ and to granule type ‘b’, that shares a similar homogenous morphology with non-labeled granule type ‘d’. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.

Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains are inferred from the effect of its removal on both catalytic activities. Interference with essential protein-protein interactions mediated by parasite-specific regions therefore appears to be a viable strategy to aid the design of selective inhibitors of polyamine metabolism of P. falciparum.

An apyrase, ATP-diphosphohydrolase (EC 3.6.1.5) was purified from the soft tick, Ornithodoros savignyi . SDS–PAGE and native PAGE analysis, showed that tick apyrase has a molecular mass of 67 kDa and a basic iso-electric point. The purified enzyme conformed to properties associated with apyrases, including low substrate specificity, a dependence on bivalent metal ions and an insensitivity to normal ATPase inhibitors and sulfhydryl group reagents. It was, however, inhibited by chelating agents, mercuric chloride, dithiothreitol and fluoride. Mg 2+ had a stabilizing effect with respect to inactivation by DTT, suggesting that the enzyme is a metalloprotein. Compared to other apyrases, tick apyrase had higher kinetic rate constants (mM range). The enzyme also inhibited ADP- and collagen-, but not thrombin-induced platelet aggregation and disaggregated platelets that were aggregated by ADP. These properties indicate that apyrase fulfils an important function during tick feeding.

Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the alpha IIb/beta3 antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of alpha IIb/beta3. These results are concurrent with targeting of alpha IIb/beta3 and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks.

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 α/β-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 α-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. Proteins 2003;50:464–473. © 2003 Wiley-Liss, Inc.