Drug Repurposing and De Novo Drug Discovery of Protein Kinase Inhibitors as New Drugs against Schistosomiasis.

Schistosomiasis is a neglected tropical disease affecting more than 200 million people worldwide. Chemotherapy relies on one single drug, praziquantel, which is safe but ineffective at killing larval stages of this parasite. Furthermore, concerns have been expressed about the rise in resistance against this drug. In the absence of an antischistosomal vaccine, it is, therefore, necessary to develop new drugs against the different species of schistosomes. Protein kinases are important molecules involved in key cellular processes such as signaling, growth, and differentiation. The kinome of schistosomes has been studied and the suitability of schistosomal protein kinases as targets demonstrated by RNA interference studies. Although protein kinase inhibitors are mostly used in cancer therapy, e.g., for the treatment of chronic myeloid leukemia or melanoma, they are now being increasingly explored for the treatment of non-oncological conditions, including schistosomiasis. Here, we discuss the various approaches including screening of natural and synthetic compounds, de novo drug development, and drug repurposing in the context of the search for protein kinase inhibitors against schistosomiasis. We discuss the status quo of the development of kinase inhibitors against schistosomal serine/threonine kinases such as polo-like kinases (PLKs) and mitogen-activated protein kinases (MAP kinases), as well as protein tyrosine kinases (PTKs).

Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain).

BACKGROUND: Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1). RESULTS: Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion. CONCLUSIONS: SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Lysine Deacetylase Inhibitors in Parasites: Past, Present, and Future Perspectives.

Current therapies for human parasite infections rely on a few drugs, most of which have severe side effects, and their helpfulness is being seriously compromised by the drug resistance problem. Globally, this is pushing discovery research of antiparasitic drugs toward new agents endowed with new mechanisms of action. By using a "drug repurposing" strategy, histone deacetylase inhibitors (HDACi), which are presently clinically approved for cancer use, are now under investigation for various parasite infections. Because parasitic Zn2+- and NAD+-dependent HDACs play crucial roles in the modulation of parasite gene expression and many of them are pro-survival for several parasites under various conditions, they are now emerging as novel potential antiparasitic targets. This Perspective summarizes the state of knowledge of HDACi (both class I/II HDACi and sirtuin inhibitors) targeted to the main human parasitic diseases (schistosomiasis, malaria, trypanosomiasis, leishmaniasis, and toxoplasmosis) and provides visions into the main issues that challenge their development as antiparasitic agents.

HDAC8: a multifaceted target for therapeutic interventions.

Histone deacetylase 8 (HDAC8) is a class I histone deacetylase implicated as a therapeutic target in various diseases, including cancer, X-linked intellectual disability, and parasitic infections. It is a structurally well-characterized enzyme that also deacetylates nonhistone proteins. In cancer, HDAC8 is a major 'epigenetic player' that is linked to deregulated expression or interaction with transcription factors critical to tumorigenesis. In the parasite Schistosoma mansoni and in viral infections, HDAC8 is a novel target to subdue infection. The current challenge remains in the development of potent selective inhibitors that would specifically target HDAC8 with fewer adverse effects compared with pan-HDAC inhibitors. Here, we review HDAC8 as a drug target and discuss inhibitors with respect to their structural features and therapeutic interventions.

The evolution of drugs on schistosoma treatment: looking to the past to improve the future.

Schistosomiasis is a devastating worldwide widespread tropical disease that currently affects more than 230 million people, making it an issue of great socioeconomic and public health importance. Unfortunatelly there is a single drug for the treatment of all forms of schistosomiasis, praziquantel, which was introduced in therapy in 1980. The article goes by antimony compounds, emetine, hydantoin, nitrofurans, lucanthone, hycanthone, oxamniquine derivatives and organophosphates until it finally gets to praziquantel derivatives. The intent of this review is to provide a panorama of drugs that were and are being used in human chemotherapy looking to the past to improve rational design drugs in the future. Not only clinical used compounds will be shown but also synthesized and tested compounds in vitro and in vivo in animal models which haven't yet to be used in humans. Prospects for drug discovery and vaccines to be used in the treatment and prevention of schistosomiasis, clinical trials, concerns about the resistance/decreased effectiveness of the treatment, and patent database will also be discussed. At the end of the review the reader will notice that much has been done but much still needs to be done yet.

[Progress in researches of molecular mechanism of schistosome cercariae infection].

Schistosome cercariae must penetrate skin as an initial step to successfully infect the final host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the invasion process. Nowadays, the researches of molecular mechanism of schistosome infection mainly focus on the cercarial secretions including serine protease and cysteine protease. Previous researches already showed that Schistosoma mansoni penetrates the skin mainly depend on cercarial elastease secreted by cercariae while Schistosoma japonicum penetrates the skin chiefly by cathepsin B2. The illustration of molecular mechanism of schistosome cecariae infection will accelerate the identification of novel vaccines and drug targets.

Protein kinases as potential targets for novel anti-schistosomal strategies.

Schistosome parasites are the causative pathogens of schistosomiasis (bilharzia), a disease of worldwide significance. In terms of patient numbers, schistosomiasis ranks second to malaria as a parasitosis affecting more than 200 million people of the tropics and subtropics. Since the 1970s Praziquantel (PZQ) is the drug of choice and nearly exclusively used for treatment. However, drug resistance is an increasing threat, particularly with respect to large-scale PZQ administration programs. Last decade's research indicated that resistance against PZQ can be induced under laboratory conditions, and field studies provided first indications for the possibility of reduced PZQ efficacy. Furthermore, clear evidence for the molecular armamentarium of schistosomes with multidrug transporters was found, one of which was responding to PZQ challenge. Also the development of a vaccine still represents an elusive goal, although effort and time have been invested in this subject. In light of these facts it is commonly accepted that new drugs are urgently needed. Research on signal transduction processes in Schistosoma mansoni has provided an unexpected and novel perspective towards this end. Molecular, biochemical, and physiological studies elucidating principles of schistosome development have demonstrated the essential role of protein kinases (PKs). In humans, PKs are known to be involved in cancer development. Since a variety of approved anticancer drugs targeting PKs exist, first studies have been performed to investigate whether these drugs are able to also inhibit schistosome PKs. Indeed, promising results have been obtained indicating the potential of PKs as privileged targets for new concepts in fighting schistosomes.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets.

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Cathepsins B1 and B2 of Trichobilharzia SPP., bird schistosomes causing cercarial dermatitis.

Trichobilharzia regenti and T. szidati are schistosomes that infect birds. although T. regenti/T. szidati can only complete their life cycle in specific bird hosts (waterfowl), their larvae-cercariae are able to penetrate, transform and then migrate as schistosomula in nonspecific hosts (e.g., mouse, man). Peptidases are among the key molecules produced by these schistosomes that enable parasite invasion and survival within the host and include cysteine peptidases such as cathepsins B1 and B2. These enzymes are indispensable bio-catalysts in a number of basal biological processes and host-parasite interactions, e.g., tissue invasion/migration, nutrition and immune evasion. Similar biochemical and functional characteristics were observed for cathepsins B1 and B2 in bird schistosomes (T. regenti, T. szidati) and also for their homologs in human schistosomes (Schistosoma mansoni, S. japonicum). Therefore, data obtained in the research of bird schistosomes can also be exploited for the control of human schistosomes such as the search for targets of novel chemotherapeutic drugs and vaccines.

Characterization of schistosome tegumental alkaline phosphatase (SmAP).

Schistosomes are parasitic platyhelminths that currently infect over 200 million people globally. The parasites can live for years in a putatively hostile environment - the blood of vertebrates. We have hypothesized that the unusual schistosome tegument (outer-covering) plays a role in protecting parasites in the blood; by impeding host immunological signaling pathways we suggest that tegumental molecules help create an immunologically privileged environment for schistosomes. In this work, we clone and characterize a schistosome alkaline phosphatase (SmAP), a predicted ∼60 kDa glycoprotein that has high sequence conservation with members of the alkaline phosphatase protein family. The SmAP gene is most highly expressed in intravascular parasite life stages. Using immunofluorescence and immuno-electron microscopy, we confirm that SmAP is expressed at the host/parasite interface and in internal tissues. The ability of living parasites to cleave exogenous adenosine monophosphate (AMP) and generate adenosine is very largely abolished when SmAP gene expression is suppressed following RNAi treatment targeting the gene. These results lend support to the hypothesis that schistosome surface enzymes such as SmAP could dampen host immune responses against the parasites by generating immunosuppressants such as adenosine to promote their survival. This notion does not rule out other potential functions for the adenosine generated e.g. in parasite nutrition.

Piggy-backing the concept of cancer drugs for schistosomiasis treatment: a tangible perspective?

The fear that schistosomes will become resistant to praziquantel (PZQ) motivates the search for alternatives to treat schistosomiasis. Recent studies of signaling proteins in schistosomes uncovered a way of achieving this goal relatively quickly. It was shown that protein kinases (PKs) control important biological processes in schistosomes. Concurrently, the involvement of mutant forms of PKs was demonstrated in the etiology of cancer. Therefore, different anticancer drugs have been developed to inhibit deregulated PKs. These can also inhibit schistosome PKs, thus blocking parasite development. Recent studies characterizing schistosome PKs are summarized and we discuss the concept of PK inhibitors, including approved cancer drugs, as novel candidate anti-schistosome agents. This is also likely to be of significance for other worm infections.

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti.

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

Protein tyrosine kinases as new potential targets against human schistosomiasis.

In spite of the numerous efforts made to control their transmission, parasite schistosomes still represent a serious public health concern and a major economic problem in many developing countries. Praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis and the only one that is available for mass chemotherapy. However, its widespread use and its inefficacy on juvenile parasites raise fears that schistosomes will develop drug resistance, and make the development of alternative drugs highly desirable. Protein tyrosine kinases (PTKs) are key molecules that control cell differentiation and proliferation and they already represent important targets for molecular cancer therapy. The recent characterization in Schistosoma mansoni of several cytosolic and receptor PTKs, with properties similar but also divergent from their vertebrate counterparts, opens new perspectives for the development of novel strategies in chemotherapy of schistosomiasis, which could be based on the use of parasite-specific tyrosine phosphorylation inhibitors.

Electrophilic aromatic substituted luciferins as bioluminescent probes for glutathione S-transferase assays.

New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.

Worms take the 'phyto' out of 'phytochelatins'.

Phytochelatin synthase is the enzyme responsible for the synthesis of heavy-metal-binding peptides (phytochelatins) from glutathione and related thiols. It has recently been determined that it is not only restricted to plants and some fungi, as was once thought, but also has an essential role in heavy-metal detoxification in the model nematode Caenorhabditis elegans. These findings and others that demonstrate phytochelatin synthase-coding sequences in the genomes of several other invertebrates, including pathogenic nematodes, schistosomes and roundworms, herald a new era in phytochelatin research, in which these novel post-translationally synthesized peptides will not only be investigated in the context of phytoremediation but also from a clinical parasitological standpoint.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis.

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology

[Proteases in helminthic parasites]. / Les protéases chez les helminthes.

Proteases catalyse the cleavage of internal peptide bonds within peptides and proteins. They are classified into four major classes and are involved in a broad range of eukaryotic processes. Proteases have also been found to play a number of critical roles in the virulence of pathogenic agents, particularly of nematode parasites. Parasitic proteases are involved in different aspects of host-parasite interactions. They facilitate the invasion of host tissues and allow nutrition as well as the survival of the parasite in its host. Proteases also participate in the parasite's evasion from the host's immune response. The functional diversity and complexity of these enzymes are described in this review, with a particular focus on the principally identified proteases of four helminths: Schistosoma sp., Fasciola sp., Taenia sp. and Haemonchus sp. Some of these proteases, especially the cysteine proteases secreted by the parasitic trematode Fasciola hepatica, have been successfully tested in experimental immunodiagnosis. Proteases identified in different parasites are currently under study for a use as recombinant vaccines. In this respect, proteases are proposed as major potential targets for immunotherapy and chemotherapy against parasitic diseases.