Differences in growth of Trypanoplasma borreli in carp serum is dependent on transferrin genotype.

Kinetoplastid parasites require transferrin (Tf), being the main source of iron, for growth and multiplication. This group of parasites developed a unique receptor-mediated system for acquiring host Tf which bears no structural homology with the host transferrin receptor. Trypanoplasma borreli, a blood parasite of common carp, probably uses a similar mechanism to sequester iron from host transferrin. In this study, we demonstrate a critical role of Tf for parasite growth. For in vitro studies we isolated and purified Tf from carp homozygous for the D or G allele of Tf. We obtained Tf-depleted serum using specific antibodies to carp Tf and studied gene expression in vivo during T. borreli infection with Real Time-quantitative PCR. We demonstrate that T. borreli cannot survive in medium supplemented with Tf-depleted serum while reconstitution with Tf restores normal growth. The critical role of Tf for parasite survival was shown in incomplete medium (medium without serum): addition of purified Tf significantly increased parasite survival. We also demonstrate that Tf polymorphism has a significant impact on T. borreli multiplication. Cultured parasites die more quickly in an environment containing D-typed Tf, as compared to medium with G-typed Tf. Gene expression during T. borreli infection in carp did not show an acute phase response. We could, however, observe an increased transcription of Tf in the head kidney, which may be associated with an immunological function of the Tf protein.

Antikinetoplastid Activity of Indolocarbazoles from Streptomyces sanyensis.

Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of Trypanosoma and Leishmania genera that affect poor and remote populations in developing countries. These parasites share similar complex life cycles and modes of infection. It has been demonstrated that the particular group of phosphorylating enzymes, protein kinases (PKs), are essential for the infective mechanisms and for parasite survival. The natural indolocarbazole staurosporine (STS, 1) has been extensively used as a PKC inhibitor and its antiparasitic effects described. In this research, we analyze the antikinetoplastid activities of three indolocarbazole (ICZs) alkaloids of the family of staurosporine STS, 2-4, and the commercial ICZs rebeccamycin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) in order to establish a plausive approach to the mode of action and to provide a preliminary qualitative structure-activity analysis. The most active compound was 7-oxostaurosporine (7OSTS, 2) that showed IC50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, and a Selectivity Index (CC50/IC50) of 52 against amastigotes of L. amazonensis compared to the J774A.1 cell line of mouse macrophages.

Small Molecule Inhibitors Targeting the Heat Shock Protein System of Human Obligate Protozoan Parasites.

Obligate protozoan parasites of the kinetoplastids and apicomplexa infect human cells to complete their life cycles. Some of the members of these groups of parasites develop in at least two systems, the human host and the insect vector. Survival under the varied physiological conditions associated with the human host and in the arthropod vectors requires the parasites to modulate their metabolic complement in order to meet the prevailing conditions. One of the key features of these parasites essential for their survival and host infectivity is timely expression of various proteins. Even more importantly is the need to keep their proteome functional by maintaining its functional capabilities in the wake of physiological changes and host immune responses. For this reason, molecular chaperones (also called heat shock proteins)-whose role is to facilitate proteostasis-play an important role in the survival of these parasites. Heat shock protein 90 (Hsp90) and Hsp70 are prominent molecular chaperones that are generally induced in response to physiological stress. Both Hsp90 and Hsp70 members are functionally regulated by nucleotides. In addition, Hsp70 and Hsp90 cooperate to facilitate folding of some key proteins implicated in cellular development. In addition, Hsp90 and Hsp70 individually interact with other accessory proteins (co-chaperones) that regulate their functions. The dependency of these proteins on nucleotide for their chaperone function presents an Achille's heel, as inhibitors that mimic ATP are amongst potential therapeutic agents targeting their function in obligate intracellular human parasites. Most of the promising small molecule inhibitors of parasitic heat shock proteins are either antibiotics or anticancer agents, whose repurposing against parasitic infections holds prospects. Both cancer cells and obligate human parasites depend upon a robust protein quality control system to ensure their survival, and hence, both employ a competent heat shock machinery to this end. Furthermore, some inhibitors that target chaperone and co-chaperone networks also offer promising prospects as antiparasitic agents. The current review highlights the progress made so far in design and application of small molecule inhibitors against obligate intracellular human parasites of the kinetoplastida and apicomplexan kingdoms.

Response of Anaerobic Protozoa to Oxygen Tension in Anaerobic System

The effect of oxygen on anaerobic protozoa was studied in anaerobic batch reactors inoculated with sludge and protozoa cultures. Among the protozoa genera, Metopus, Brachonella, Plagiopyla, Trepomonas, and Vanella were more sensitive to oxygen compared to other genera. Protozoa genera Menoidium, Rhynchomonas, Cyclidium, Spathidium, and Amoeba were found to survive under aerobic conditions, and the growth rate was slightly higher or similar to anaerobic condition. O2 tension resulted in the loss of free and endosymbiotic methanogens in anaerobic system, while methanogens were observed inside the protozoan cysts. Survival of anaerobic protozoa declined considerably when the O2 tension exceeded 1% atm. sat. and showed chemosensory behavior in response to O2 exposure. Superoxide dismutase activity was detected in survived protozoa cells under O2 tension. Facultative anaerobic protozoa with SOD activity can provide a mechanism to overcome possible occurrence of oxygen toxicity in the treatment of wastewater in anaerobic reactor

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Response of Anaerobic Protozoa to Oxygen Tension in Anaerobic System.

The effect of oxygen on anaerobic protozoa was studied in anaerobic batch reactors inoculated with sludge and protozoa cultures. Among the protozoa genera, Metopus, Brachonella, Plagiopyla, Trepomonas, and Vanella were more sensitive to oxygen compared to other genera. Protozoa genera Menoidium, Rhynchomonas, Cyclidium, Spathidium, and Amoeba were found to survive under aerobic conditions, and the growth rate was slightly higher or similar to anaerobic condition. O2 tension resulted in the loss of free and endosymbiotic methanogens in anaerobic system, while methanogens were observed inside the protozoan cysts. Survival of anaerobic protozoa declined considerably when the O2 tension exceeded 1% atm. sat. and showed chemosensory behavior in response to O2 exposure. Superoxide dismutase activity was detected in survived protozoa cells under O2 tension. Facultative anaerobic protozoa with SOD activity can provide a mechanism to overcome possible occurrence of oxygen toxicity in the treatment of wastewater in anaerobic reactor.

Revisiting tubercidin against kinetoplastid parasites: Aromatic substitutions at position 7 improve activity and reduce toxicity.

The nucleoside antibiotic tubercidin displays strong activity against different target organisms, but it is notoriously toxic to mammalian cells. The effects of tubercidin against T. brucei parasites inspired us to synthesize several C7 substituted analogs for in vitro evaluation in order to find suitable hit compounds. C7 Deazaadenosines substituted with electron-poor phenyl groups were found to have micromolar activity against T. brucei in vitro. Replacement of the phenyl for a pyridine ring gave compound 13, with submicromolar potency and much-attenuated cytotoxicity compared to tubercidin. The veterinary pathogen T. congolense was equally affected by 13in vitro. Transporter studies in T. b. brucei indicated that 13 is taken up efficiently by both the P1 and P2 adenosine transporters, making the occurrence of transporter-related resistance and cross-resistance with diamidine drugs such as diminazene aceturate and pentamidine as well as with melaminophenyl arsenicals unlikely. Evaluation of the in vitro metabolic stability of analog 13 indicated that this analog was significantly metabolized in mouse microsomal fractions, precluding further in vivo evaluation in mouse models of HAT.

Druggable Targets in Cyclic Nucleotide Signaling Pathways in Apicomplexan Parasites and Kinetoplastids against Disabling Protozoan Diseases in Humans.

Cell signaling in eukaryotes is an evolutionarily conserved mechanism to respond and adapt to various environmental changes. In general, signal sensation is mediated by a receptor which transfers the signal to a cascade of effector proteins. The cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are intracellular messengers mediating an extracellular stimulus to cyclic nucleotide-dependent kinases driving a change in cell function. In apicomplexan parasites and kinetoplastids, which are responsible for a variety of neglected, tropical diseases, unique mechanisms of cyclic nucleotide signaling are currently identified. Collectively, cyclic nucleotides seem to be essential for parasitic proliferation and differentiation. However, there is no a genomic evidence for canonical G-proteins in these parasites while small GTPases and secondary effector proteins with structural differences to host orthologues occur. Database entries encoding G-protein-coupled receptors (GPCRs) are still without functional proof. Instead, signals from the parasite trigger GPCR-mediated signaling in the host during parasite invasion and egress. The role of cyclic nucleotide signaling in the absence of G-proteins and GPCRs, with a particular focus on small GTPases in pathogenesis, is reviewed here. Due to the absence of G-proteins, apicomplexan parasites and kinetoplastids may use small GTPases or their secondary effector proteins and host canonical G-proteins during infection. Thus, the feasibility of targeting cyclic nucleotide signaling pathways in these parasites, will be an enormous challenge for the identification of selective, pharmacological inhibitors since canonical host proteins also contribute to pathogenesis.

Drug Discovery for Kinetoplastid Diseases: Future Directions.

Kinetoplastid parasites have caused human disease for millennia. Significant achievements have been made toward developing new treatments for leishmaniasis (particularly on the Indian subcontinent) and for human African trypanosomiasis (HAT). Moreover, the sustained decrease in the incidence of HAT has made the prospect of elimination a tantalizing reality. Despite the gains, no new chemical or biological entities to treat kinetoplastid diseases have been registered in more than three decades, and more work is needed to discover safe and effective therapies for patients with Chagas disease and leishmaniasis. Advances in tools for drug discovery and novel insights into the biology of the host-parasite interaction may provide opportunities for accelerated progress. Here, we summarize the output from a gathering of scientists and physicians who met to discuss the current status and future directions in drug discovery for kinetoplastid diseases.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

Toward a Drug Against All Kinetoplastids: From LeishBox to Specific and Potent Trypanothione Reductase Inhibitors.

Leishmaniasis, Chagas disease, and sleeping sickness affect millions of people worldwide and lead to the death of about 50 000 humans per year. These diseases are caused by the kinetoplastids Leishmania, Trypanosoma cruzi, and Trypanosoma brucei, respectively. These parasites share many general features, including gene conservation, high amino acid identity among proteins, the presence of subcellular structures as glycosomes and the kinetoplastid, and genome architecture, that may make drug development family specific, rather than species-specific, i.e., on the basis of the inhibition of a common, conserved parasite target. However, no optimal molecular targets or broad-spectrum drugs have been identified to date to cure these diseases. Here, the LeishBox from GlaxoSmithKline high-throughput screening, a 192-molecule set of best antileishmanial compounds, based on 1.8 million compounds, was used to identify specific inhibitors of a validated Leishmania target, trypanothione reductase (TR), while analyzing in parallel the homologous human enzyme glutathione reductase (GR). We identified three specific highly potent TR inhibitors and performed docking on the TR solved structure, thereby elucidating the putative molecular basis of TR inhibition. Since TRs from kinetoplastids are well conserved, and these compounds inhibit the growth of Leishmania, Trypanosoma cruzi, and Trypanosoma brucei, the identification of a common validated target may lead to the development of potent antikinetoplastid drugs.

Novel 8-nitroquinolin-2(1H)-ones as NTR-bioactivated antikinetoplastid molecules: Synthesis, electrochemical and SAR study.

To study the antiparasitic 8-nitroquinolin-2(1H)-one pharmacophore, a series of 31 derivatives was synthesized in 1-5 steps and evaluated in vitro against both Leishmania infantum and Trypanosoma brucei brucei. In parallel, the reduction potential of all molecules was measured by cyclic voltammetry. Structure-activity relationships first indicated that antileishmanial activity depends on an intramolecular hydrogen bond (described by X-ray diffraction) between the lactam function and the nitro group, which is responsible for an important shift of the redox potential (+0.3 V in comparison with 8-nitroquinoline). With the assistance of computational chemistry, a set of derivatives presenting a large range of redox potentials (from -1.1 to -0.45 V) was designed and provided a list of suitable molecules to be synthesized and tested. This approach highlighted that, in this series, only substrates with a redox potential above -0.6 V display activity toward L. infantum. Nevertheless, such relation between redox potentials and in vitro antiparasitic activities was not observed in T. b. brucei. Compound 22 is a new hit compound in the series, displaying both antileishmanial and antitrypanosomal activity along with a low cytotoxicity on the human HepG2 cell line. Compound 22 is selectively bioactivated by the type 1 nitroreductases (NTR1) of L. donovani and T. brucei brucei. Moreover, despite being mutagenic in the Ames test, as most of nitroaromatic derivatives, compound 22 was not genotoxic in the comet assay. Preliminary in vitro pharmacokinetic parameters were finally determined and pointed out a good in vitro microsomal stability (half-life > 40 min) and a 92% binding to human albumin.

Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids.

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

Cytotoxic and anti-kinetoplastid potential of the essential oil of Alpinia speciosa K. Schum.

Alpinia speciosa K. Schum, known as colônia (colony), is native to tropical Asia and found in parts of tropical America. Its leaves are used to wrap food, rhizomes for food preparation and seeds for health maintenance, and have been widely used by the population as a diuretic, antihypertensive, antiulcerogenic and sedative. The present study aimed to verify the leishmanicidal and trypanocidal potential, as well as the cytotoxicity, of the A. speciosa essential oil, in vitro. A. speciosa presented 1,8-cineole (28.46%), camphor (17.10%) and sabinene (9.95%) as major constituents. The cytotoxic activity of the essential oil presented a low value, while the antipromastigote and antiepimastigote activity presented values considered clinically relevant, since it had an action below 500 µg/mL. In relation to this study, it can be concluded that this is a pioneer in the potential of the A. speciosa essential oil and in the use against the parasites Trypanosoma cruzi Chagas and Leishmania brasiliensis Vianna, having its importance also rooted in this fact. Still in accordance with the results, A. speciosa was effective because it presented values of clinical relevance and low toxicity. It was also observed that the chemical constitution of the above identified compounds with remarkable antiparasitic activities.

Everybody needs sphingolipids, right! Mining for new drug targets in protozoan sphingolipid biosynthesis.

Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes. In addition, they have indispensable functions as signalling molecules controlling a myriad of cellular events. Disruption of either the de novo synthesis or the degradation pathways has been shown to have detrimental effects. The earlier identification of selective inhibitors of fungal SL biosynthesis promised potent broad-spectrum anti-fungal agents, which later encouraged testing some of those agents against protozoan parasites. In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of the Apicomplexa and Kinetoplastidae, outlining the divergence and interconnection between host and pathogen metabolism. The druggability of the SL biosynthesis is considered, alongside recent technology advances that will enable the dissection and analyses of this pathway in the parasitic protozoa. The future impact of these advances for the development of new therapeutics for both globally threatening and neglected infectious diseases is potentially profound.

Screening a Natural Product-Based Library against Kinetoplastid Parasites.

Kinetoplastid parasites cause vector-borne parasitic diseases including leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease. These Neglected Tropical Diseases (NTDs) impact on some of the world's lowest socioeconomic communities. Current treatments for these diseases cause severe toxicity and have limited efficacy, highlighting the need to identify new treatments. In this study, the Davis open access natural product-based library was screened against kinetoplastids (Leishmania donovani DD8, Trypanosoma brucei brucei and Trypanosoma cruzi) using phenotypic assays. The aim of this study was to identify hit compounds, with a focus on improved efficacy, selectivity and potential to target several kinetoplastid parasites. The IC50 values of the natural products were obtained for L. donovani DD8, T. b. brucei and T. cruzi in addition to cytotoxicity against the mammalian cell lines, HEK-293, 3T3 and THP-1 cell lines were determined to ascertain parasite selectivity. Thirty-one compounds were identified with IC50 values of ≤ 10 µM against the kinetoplastid parasites tested. Lissoclinotoxin E (1) was the only compound identified with activity across all three investigated parasites, exhibiting IC50 values < 5 µM. In this study, natural products with the potential to be new chemical starting points for drug discovery efforts for kinetoplastid diseases were identified.

Polyamine-based analogs and conjugates as antikinetoplastid agents.

Naturally occurring polyamines: putrescine, spermidine and spermine are crucial for Kinetoplastid growth and persistence. These aliphatic polycations are either biosynthesized or internalized into Kinetoplastid by active transport. Impairing the polyamine metabolism using polyamine derivatives is an interesting path in the search of new antikinetoplastid chemotherapy. In the past 30 years, research interest in this field has been constantly expanding and recent results demonstrated that the discovery of a polyamine-based antikinetoplastid drug is undoubtedly possible. In this paper, all the polyamine derivatives previously described to present an antikinetoplastid activity are reported. This review is organized around three main parts which are diamine, triamine and tetramine derivatives. Each part includes the description of the series of molecules and, their in vitro and in vivo activity when available. Structure-activity relationships of these derivatives are discussed and the most promising structures for a positive outcome are eventually highlighted.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Selenoproteins of African trypanosomes are dispensable for parasite survival in a mammalian host.

The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.