QSAR Study of N-Myristoyltransferase Inhibitors of Antimalarial Agents.

Malaria is a disease caused by protozoan parasites of the genus Plasmodium that affects millions of people worldwide. In recent years there have been parasite resistances to several drugs, including the first-line antimalarial treatment. With the aim of proposing new drugs candidates for the treatment of disease, Quantitative Structure⁻Activity Relationship (QSAR) methodology was applied to 83 N-myristoyltransferase inhibitors, synthesized by Leatherbarrow et al. The QSAR models were developed using 63 compounds, the training set, and externally validated using 20 compounds, the test set. Ten different alignments for the two test sets were tested and the models were generated by the technique that combines genetic algorithms and partial least squares. The best model shows r² = 0.757, q²adjusted = 0.634, R²pred = 0.746, R²m = 0.716, ∆R²m = 0.133, R²p = 0.609, and R²r = 0.110. This work suggested a good correlation with the experimental results and allows the design of new potent N-myristoyltransferase inhibitors.

Ubiquitin Proteasome System as a Potential Drug Target for Malaria.

Parasites of Plasmodium genus are responsible for causing malaria in humans. Resistant strains to all available antimalarials can be found in several locations around the globe, including parasites resistant to the latest generation of combination drugs, such as piperaquine + artemisinin. Plasmodium develops between two completely different hosts such as a vertebrate one and the mosquito vector, thus it has the ability to adapt to very extreme and different environments. Through the complex life cycle in the hosts, Plasmodium invades and replicates in totally different cells thus making the study of the biology of the parasite and the identification of targets for drug development affecting all stages very difficult. It was shown that host molecules, such as melatonin and derivatives, have a role in the progression and regulation of the parasite cell cycle; In fact, when the parasite is exposed to melatonin there is an increase in transcription levels of genes encoding for proteins related to the Ubiquitin Proteasome (UPS) System. This system is essential for the survival of the parasite, and drugs such as bortezomib, MLN-273, ZL3B, epoxomicins and salinosporamides are capable of eliminating the parasite by inhibiting the degradation of proteins via the proteasome system. In addition, the Plasmodium UPS shows low similarity to the ubiquitin proteasome system in Humans; the identification of unique targets to be used for therapeutic molecules development increases the importance of UPS studies in malaria challenging. Drugs that cause oxidative stress, such as artemisinin, show a strong synergistic effect with proteasome inhibitors, increasing the possibilities of combined therapies, which are more effective with lower concentration of drugs. Thus, the study of the mechanism of action of the UPS and the identification of potential targets for new drugs development are promising alternative strategies to fight the drug-resistance problem in malaria parasites.

Natural cysteine protease inhibitors in protozoa: Fifteen years of the chagasin family.

Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archaea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.

Surface-expressed enolases of Plasmodium and other pathogens.

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

Surface-expressed enolases of Plasmodium and other pathogens

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

Lack of sequence variation of the mitochondrial cytochrome b gene from a malaria parasite, Plasmodium mexicanum.

Very slight sequence differences in the mitochondrial cytochrome b gene, even single nucleotide substitutions, have been proposed as indicative of different species of avian malaria parasites. However, few studies have examined within-species variation in that gene for Plasmodium or related genera. We examined sequences for the entire cytochrome b gene from Plasmodium mexicanum , a parasite of lizards, for sites where microsatellite markers revealed substantial genetic diversity. For sites where the parasite is geographically genetically differentiated, and may have been isolated for thousands of years, there was no sequence variation (1,153 nucleotides) for >160 infections studied. The low degree of variation found in the cytochrome b gene for two human malaria parasites world-wide, as well as the lack of variation for P. mexicanum , contrast with the substantial variation found in surveys of bird malaria parasites, even in restricted geographic regions.

Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.

[Metacaspases and their role in the life cycle of human protozoan parasites]. / Las metacaspasas y su rol en la vida y muerte de los parásitos protozoarios humanos.

Metacaspases are caspase-related cysteine-proteases that are present in organisms devoid of caspases such as plants, yeast, and protozoan parasites. Since caspases are important effector molecules in mammalian apoptosis, the possible role of metacaspases in programmed cell death has been evaluated in the organisms where they are expressed. In some species of the human protozoan parasites Trypanosoma spp. and Leishmania spp., metacaspases have been involved in programmed cell death, although a role of metacaspases in recycling processes in T. brucei has also been suggested. Metacaspases are also expressed in Plasmodium spp., however their role in these organisms is still unknown. More than one metacaspase gene is present in some of these parasites, which suggests that these proteins are physiologically redundant or have different functions depending on their localization and protein interactions. The catalytic activity of metacaspases is different from that of caspases-again noting that metacaspase genes are absent in mammals. These characteristics make metacaspases and their activating mechanisms interesting subjects of research in the development of new drug targets for the treatment of trypanosomiasis, leishmaniasis, and malaria. A summary of the literature on metacaspases is provided, and Latin American researchers are encouraged to more actively explore the metacaspase potential.

¿La hemozoína, presente y futuro? / ¿Hemozoin present and future

La malaria es una enfermedad causada por parásitos del género Plasmodium estos parásitos tienen un ciclo intraeritocítico en el hospedador vertevrado. En el glóbulo rojo, el parásito ingiere la hemoglobina, obteniendo aminácidos y formando hemozoína. La hemozoína es un un material microcristalino oscuro, de color marrón amarillento, insoluble en agua, no tóxico, producido en la vacuola parasitófora del Plasmodium; este compuesto producido por el Plasmodium carece de la toxicidad que tiene el grupo hemo para el parásito. Asimismo se ha evidenciado que la hemozoína es una sustancia inmuno moduladora que tiene diversos efectos, como mediar la activación y migración de neutrófilos, incrementar la producción de óxido nítrico, inducir la activación de mataloproteínas 9, inducir la secreción de diferentes mediadores proinflamatorios, alterar las funciones de los monocitos y macrófagos humanos, tales como el estallido oxidativo, eliminación de bacterias, presentación de antígenos y la habilidad de diferenciarse a células dendríticas funcionales; por lo que la hemozína tiene efectos duales, tanto activadores como supresores de la respuesta inmune. Asimismo, la hemozoína es unblanco terapéutico potente, ya que los fármacos que inhiban su formación provocan toxicidad al parasíto e incluso la muerte del mismo.

Malaria is a disease caused by parasites of the genus Plasmodium. These parasites have intraerythrocytic cycle in the vertbrate host. In the red cell, the parasite ingests hemoglobin, obtaining amino acids and formin hemozoin. The microcrystaline material hemozoin is a dark, yellowish brown, insoluble in water, nontoxic, produced in the Plasmodium parasitophorous vacuole, this compound produced by Plasmodiun lacks the toxicity that has heme to the parasite. It has also been shown that hemozoin is an immune modulating substance that has different ffects, mediating the neutrophils activation and migration, increased nitric oxide production, induce activation of metallproteinase-9, induce the secretion of various proinflammatory mediators, alter the funcions of human monocytes and macrophages such as oxidative burst, removing bacteria, antigen presentation and the ability to differentiate into functional dendritic cells, so the hemozoin has dual effects, both activators and suppressors of the immune response. Also, the hemozoin is a potent therapeutic target, since rugs that inhibit their formation causes toxicity to the parasite and even death itself.

Detection of a malaria parasite (Plasmodium mexicanum) in ectoparasites (mites and ticks), and possible significance for transmission.

Two species of sandflies (Lutzomyia) are competent vectors of Plasmodium mexicanum, a malaria parasite of lizards. The very patchy distribution of sites with high P. mexicanum prevalence in the lizards, and often low or even nil sandfly density at such sites, provoked an evaluation of 2 common lizard ectoparasites, the tick Ixodes pacificus and the mite Geckobiella occidentalis, as potential passive vectors. Plasmodium sp.-specific polymerase chain primers were used to amplify a long segment of the mitochondrial cytochrome b gene that is unlikely to survive intact if the parasite cells are killed within a blood-feeding arthropod. The segment was strongly amplified from sandflies (the positive control for the method) from 1 to 96 hr postfeeding on an infected lizard. For ticks, the gene fragment was poorly amplified at 0 hr postfeed, and not amplified after 2 hr. In contrast, strong amplification of the parasite DNA was observed from mites from 0 to 20 hr postfeed, and weak amplification even at 96 hr.

The eukaryotic Pso2p/Snm1p family revisited: in silico analyses of Pso2p A, B and Plasmodium groups.

The eukaryotic family of Pso2/Snm1 exo/endonuclease proteins has important functions in repair of DNA damages induced by chemical interstrand cross-linking agents and ionizing radiation. These exo/endonucleases are also necessary for V(D)J recombination and genomic caretaking. However, despite the growing biochemical data about this family, little is known about the number of orthologous/paralogous Pso2p/Snm1p sequences in eukaryotes and how they are phylogenetically organized. In this work we have characterized new Pso2p/Snm1p sequences from the finished and unfinished eukaryotic genomes and performed an in-depth phylogenetic analysis. The results indicate that four phylogenetically related groups compose the Pso2p/Snm1p family: (i) the Artemis/Artemis-like group, (ii) the Pso2p A group, (iii) the Pso2p B group and (iv) the Pso2p Plasmodium group. Using the available biochemical and genomic information about Pso2p/Snm1p family, we concentrate our research in the study of Pso2p A, B and Plasmodium groups. The phylogenetic results showed that A and B groups can be organized in specific subgroups with different functions in DNA metabolism. Moreover, we subjected selected Pso2p A, B and Plasmodium proteins to hydrophobic cluster analysis (HCA) in order to map and to compare conserved regions within these sequences. Four conserved regions could be detected by HCA, which are distributed along the metallo-beta-lactamase and beta-CASP motifs. Interestingly, both Pso2p A and B proteins are structurally similar, while Pso2p Plasmodium proteins have a unique domain organization. The possible functions of A, B and Plasmodium groups are discussed.

Fosforilación en células eucarióticas: papel de fosfatasas y quinasas en la biología, patogenia y control de protozoosis tisulares y sanguíneas / Phosphorylation in eukaryotic cells: role of phosphatases and kinases in biology, pathogenesis and control of intracellular and bloodstream protozoa

Cells respond to environmental or cellular changes, rapidly switching protein activities from one state to another. In eukaryotes, a way to achieve these changes is through protein phosphorylation cycles, involving independent protein kinase and protein phosphatase activities. Current evidences show that phosphatases and kinases are also involved in the molecular basis of immune response and in diseases such as diabetes obesity and Alzheimer. In protozoan parasites like Trypanosoma and Leishmania, several kinases and phosphatases have been identified, many of them have been cloned but in several cases their biological role remains undetermined. In this review, the state-of-the art is summarized and the role of phosphatases and kinases in biological phenomena such as remodeling, invasion and pathogenic capacity of protozoan parasites is described. The real chance to use these components of signal transduction pathways as target for chemotherapeutic intervention is also discussed

Malaria parasites: enzymes involved in red blood cell invasion.

Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a preformed serine-protease active site but no detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erythrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented.