Aminopeptidases of malaria parasites:new targets for chemotherapy

Novel targets for new drug development are urgently required to combat malaria, a disease that puts half of the world's population at risk. One group of enzymes identified within the genome of the most lethal of the causative agents of malaria, Plasmodium falciparum, that may have the potential to become new targets for antimalarial drug development are the aminopeptidases. These enzymes catalyse the cleavage of the N-terminal amino acids from proteins and peptides. P. falciparum appears to encode for at least nine aminopeptidases, two neutral aminopeptidases, one aspartyl aminopeptidase, one aminopeptidase P, one prolyl aminopeptidase and four methionine aminopeptidases. Recent advances in our understanding of these genes and their protein products are outlined in this review, including their potential for antimalarial drug development.

The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum:importance of active site metal ions in the binding of substrates and inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum

The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) M. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides.

How parasites affect interactions between competitors and predators

We present a synthesis of empirical and theoretical work investigating how parasites influence competitive and predatory interactions between other species. We examine the direct and indirect effects of parasitism and discuss examples of density and parasite-induced trait-mediated effects. Recent work reveals previously unrecognized complexity in parasite-mediated interactions. In addition to parasite-modified and apparent competition leading to species exclusion or enabling coexistence, parasites and predators interact in different ways to regulate or destablize the population dynamics of their joint prey. An emerging area is the impact of parasites on intraguild predation (IGP). Parasites can increase vulnerability of infected individuals to cannibalism or predation resulting in reversed species dominance in IGP hierarchies. We discuss the potential significance of parasites for community structure and biodiversity, in particular their role in promoting species exclusion or coexistence and the impact of emerging diseases. Ongoing invasions provide examples where parasites mediate native/invader interactions and play a key role in determining the outcome of invasions. We highlight the need for more quantitative data to assess the impact of parasites on communities, and the combination of theoretical and empirical studies to examine how the effects of parasitism scale up to community-level processes.

Satellite flies (Leucophora personata, Diptera : Anthomyiidae) and other dipteran parasites of the communal bee Andrena agilissima (Hymenoptera : Andrenidae) on the island of Elba, Italy

Solitary and presocial aculueate Hymenoptera are parasitized by a range of dipteran species in the families Axithomyiidae, Bombyliidae, Conopidae, Phoridae, and Sarcophagidae that are likely to impact on their hosts. We undertook a study over several years of a univoltine and communal bee, Andrena agilissima, and its main dipteran parasites, in particular the satellite fly Leucophora personata (Diptera: Anthomyiidae). Behavioural and ecological data were collected from one nesting aggregation of the host bee on the island of Elba, Italy, from 1993 to 2003, and from a foraging site of the bee, ca 5 km from the nesting aggregation. Other Diptera associated with A. agilissmia at the field site were the bee fly Bombylius fimbriatus (Bombyliidae), the conopid fly Zodion cinereum (Conopidae), and the scuttle fly Megaselia andrenae (Phoridae). The phenology of the Diptera broadly overlapped with that of their host across the season of activity (end of April and all of May). Diurnal activity patterns differed slightly; L. personata in particular was active at the host's nesting site before A. agilissima. Female satellite flies also showed a range of behaviours in gaining entry to a host nest. We summarize published data on this and other Leucophora species that parasitize Andrena host bees. Host bees returning to their nests occasionally undertook zig-zag flight manoeuvres if followed by a satellite fly that were generally successful in evading the fly. Satellite flies that entered a nest, presumably to oviposit, were less likely to remain therein if another host bee entered the same nest, suggesting that one advantage to communal nesting for this host is a reduction in brood cell parasitism by L. personata. We provide the first clear evidence for parasitism by a Zodion of any Andrena host. Both L. personata and M. andrenae concentrated their parasitic activities in the zone of the host nesting aggregation with highest nest densities. Three of the Diptera, L. personata, B. fimbriatus, and Z. cinereum, seemed to have extremely low rates of parasitism whilst that of M. andrenae appeared low. Though they have refined parasitic behaviour that allows them to gain entry into host nests (L. personata, B. fimbriatus, and M. andrenae) or to parasitize adults (Z. cinercum), these parasites seem not to impact upon the dynamics of the host A. agilissima at the nesting aggregation, and the host possesses traits to reduce parasitism.

Microtubules as antiparasitic drug targets

Background: Parasitic diseases including malaria, leishmaniasis and schistosomiasis take a terrible toll of human life, health and productivity, especially in tropical and subtropical regions, and are also highly significant in animal health worldwide. Antiparasitic drugs are the mainstays of control of most of these diseases, but in many cases current therapies are inadequate and in some the situation is deteriorating because of drug resistance. Microtubules, as essential components of almost all eukaryotic cells, are proven drug targets in many helminth diseases and show promise as targets for the development of new antiprotozoal drugs. Objective: This article reviews the chemistry of the microtubule inhibitors in current use and under investigation as antiparasitic agents, their activities against the major parasites and their mechanisms of action. New directions in both inhibitor chemistry and biological evaluation are discussed. Conclusions: The most promising immediate avenues for discovery and design appear to lie in development of novel benzimidazoles for helminth parasites and compounds based on antimitotic herbicides for protozoal parasites. New understanding from functional genomics, structural biology and microtubular imaging will help accelerate the development of completely novel antiparasitic drugs targeting microtubules.

A keystone effect for parasites in intraguild predation?

Intraguild predation (IGP) is common in communities, yet theory suggests it should not often persist and coexistence of participating species should be rare. As parasitism can play keystone roles in interactions between competitors, and between predators and prey, here we examine the role of parasites in maintaining IGP. We used numerical exploration of population dynamic equations to determine coexistence and exclusion zones for two species engaged in IGP with shared parasitism. We demonstrate that parasitism increases the range of conditions leading to coexistence when the parasite exerts a greater deleterious effect on the 'stronger' species in terms of the combined effects of competition and predation. Such a parasite can enable an inferior competitor that is also the less predatory to persist, and may actually lead to numerical dominance of this species.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in > 2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2] Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA- M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations

Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 angstrom-long channel and at the opening of the 30 angstrom-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.

An eye on RNAi in nematode parasites

RNA interference (RNAi) has revolutionised approaches to gene function determination. From a parasitology perspective, gene function studies have the added dimension of providing validation data, increasingly deemed essential to the initial phases of drug target selection, pre-screen development. Notionally advantageous to those working on nematode parasites is the fact that Caenorhabditis elegans research spawned RNAi discovery and continues to seed our understanding of its fundamentals. Unfortunately, RNAi data for nematode parasites illustrate variable and inconsistent susceptibilities which undermine confidence and exploitation. Now well-ensconced in an era of nematode parasite genomics, we can begin to unscramble this variation.