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).

Lactate dehydrogenase and malate dehydrogenase: Potential antiparasitic targets for drug development studies.

Parasitic diseases remain a major public health concern for humans, claiming millions of lives annually. Although different treatments are required for these diseases, drug usage is limited due to the development of resistance and toxicity, which necessitate alternative therapies. It has been shown in the literature that parasitic lactate dehydrogenases (LDH) and malate dehydrogenases (MDH) have unique pharmacological selective and specificity properties compared to other isoforms, thus highlighting them as viable therapeutic targets involved in aerobic and anaerobic glycolytic pathways. LDH and MDH are important therapeutic targets for invasive parasites because they play a critical role in the progression and development of parasitic diseases. Any strategy to impede these enzymes would be fatal to the parasites, paving the way to develop and discover novel antiparasitic agents. This review aims to highlight the importance of parasitic LDH and MDH as therapeutic drug targets in selected obligate apicoplast parasites. To the best of our knowledge, this review presents the first comprehensive review of LDH and MDH as potential antiparasitic targets for drug development studies.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection.

BACKGROUND: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. OBJECTIVE: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. METHODS: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it's missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. RESULTS: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. CONCLUSION: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

Crystal structure and chemical inhibition of essential schistosome host-interactive virulence factor carbonic anhydrase SmCA.

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Here we identify an α-carbonic anhydrase (SmCA) that is expressed at the schistosome surface as determined by activity assays and immunofluorescence/immunogold localization. Suppressing SmCA expression by RNAi significantly impairs the ability of larval parasites to infect mice, validating SmCA as a rational drug target. Purified, recombinant SmCA possesses extremely rapid CO2 hydration kinetics (kcat: 1.2 × 106 s-1; kcat/Km: 1.3 × 108 M-1s-1). The enzyme's crystal structure was determined at 1.75 Å resolution and a collection of sulfonamides and anions were tested for their ability to impede rSmCA action. Several compounds (phenylarsonic acid, phenylbaronic acid, sulfamide) exhibited favorable Kis for SmCA versus two human isoforms. Such selective rSmCA inhibitors could form the basis of urgently needed new drugs that block essential schistosome metabolism, blunt parasite virulence and debilitate these important global pathogens.

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.

Targeting thioredoxin glutathione reductase as a potential antischistosomal drug target.

Schistosomiasis represents a world health major problem affecting more than 206 million people worldwide. Up to date, praziquantel (PZQ) is the sole chemotherapeutic agent used in clinics for the treatment of schistosomiasis. The resistance to PZQ chemotherapy that has been emerged against some schistosome phenotypes represents the most serious PZQ-related problem so far. Therefore, it is clear that there is a substantial need to develop novel and effective antischistosomal agents in order to ensure the effective drug control of schistosomiasis in the future. It is well-documented that the thiol redox homeostasis of schistosomes is entirely based on a single enzyme named thioredoxin-glutathione reductase (TGR). Thus, TGR is an essential protein for the survival of schistosomes which means that TGR is a valid and promising target for the recent antischistosomal drug-discovery approaches. This review aimed to shed light on potential lead compounds that may inhibit TGR activity and consequently could be tested as a potential antischistsomal drugs. In the current review we discussed multiple drug discovery approaches for new compounds targeting TGR and its implementation.

Improved strategy for the curation and classification of kinases, with broad applicability to other eukaryotic protein groups.

Despite the substantial amount of genomic and transcriptomic data available for a wide range of eukaryotic organisms, most genomes are still in a draft state and can have inaccurate gene predictions. To gain a sound understanding of the biology of an organism, it is crucial that inferred protein sequences are accurately identified and annotated. However, this can be challenging to achieve, particularly for organisms such as parasitic worms (helminths), as most gene prediction approaches do not account for substantial phylogenetic divergence from model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, whose genomes are well-curated. In this paper, we describe a bioinformatic strategy for the curation of gene families and subsequent annotation of encoded proteins. This strategy relies on pairwise gene curation between at least two closely related species using genomic and transcriptomic data sets, and is built on recent work on kinase complements of parasitic worms. Here, we discuss salient technical aspects of this strategy and its implications for the curation of protein families more generally.

Kinases: Molecular Stage Directors for Schistosome Development and Differentiation.

Understanding schistosome biology is still a challenging mission. The reproductive biology of this parasitic trematode is closely associated with the pathologic consequences of schistosomiasis, the devastating infectious disease caused by members of the family Schistosomatidae worldwide. Recent studies of signaling mechanisms confirmed the prominent roles of protein kinases (PKs) in directing schistosome biology, and first evidence was obtained for an additional contribution of kinases with substrates different from proteins (non-PKs). This review provides an overview of the Schistosoma mansoni kinome in the context of male-female interaction and summarizes recent studies of kinases controlling development and differentiation. Due to their importance for schistosome biology, kinases represent Achilles' heels and are therefore of high value also for translational research.

Re-characterization of hammerhead ribozymes as molecular tools for intermolecular RNA cleavage.

A new guideline for the construction of hammerhead ribozymes to achieve trans-cleavage of a single-stranded RNA molecule was developed. The sequence rule of the HHRz cleavage site was highly recommended to be "DWH" with an optimal binding arm length of 8-9nt, which diverged from the former rule of "NUX".

Evolutionary relationships among protein lysine deacetylases of parasites causing neglected diseases.

The availability of the genomic data of diverse parasites provides an opportunity to identify new drug candidates against neglected tropical diseases affecting people worldwide. Histone modifying enzymes (HMEs) are potential candidates since they play key roles in the regulation of chromatin modifications, thus globally regulating gene expression. Furthermore, aberrant epigenetic states are often associated with human diseases, leading to great interest in HMEs as therapeutic targets. Our work focused on two families of protein lysine deacetylases (HDACs and sirtuins). First, we identified potential homologues in the predicted proteomes of selected taxa by using hidden Markov model profiles. Then, we reconstructed the evolutionary relationships of protein sequences by Bayesian inference and maximum likelihood method. In addition, we constructed homology models for five parasite HDACs to provide information for experimental validation and structure-based optimization of inhibitors. Our results showed that parasite genomes code for diverse HDACs and sirtuins. The evolutionary pattern of protein deacetylases with additional experimental data points to these enzymes as common drug targets among parasites. This work has improved the functional annotation of approximately 63% HDACs and 51% sirtuins in the selected taxa providing insights for experimental design. Homology models pointed out structural conservation and differences among parasite and human homologues and highlight potential candidates for further inhibitor development. Some of these parasite proteins are undergoing RNA interference or knockout analyses to validate the function of their corresponding genes. In the future, we will investigate the main functions performed by these proteins, related phenotypes, and their potential as therapeutic targets.

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.

The Schistosome Esophagus Is a 'Hotspot' for Microexon and Lysosomal Hydrolase Gene Expression: Implications for Blood Processing.

BACKGROUND: The schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes. METHODOLOGY/PRINCIPAL FINDINGS: We have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands. CONCLUSIONS: A proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets.

The Schistosoma indicum species group in Nepal: presence of a new lineage of schistosome and use of the Indoplanorbis exustus species complex of snail hosts.

From 2007-2014, 19,360 freshwater snails from the Terai and Hilly regions of Nepal were screened for cercariae of mammalian schistosomes. Based on analysis of mitochondrial cytochrome oxidase I, 12S, 16S and 28S sequences (3,675bp) of the cercariae recovered, we provide, to our knowledge, the first report of the Schistosoma indicum species group in Nepal. Five samples of Schistosoma nasale, nine of Schistosoma spindale and 17 of Schistosoma sp. were recovered, all from the snail Indoplanorbis exustus. The last-mentioned lineage failed to group in any of our analyses with S. nasale, S. spindale or S. indicum. It diverged in cox1 sequence from them by 16%, 13% and 13%, respectively, levels of difference comparable to well-studied species pairs of Schistosoma. Analysis of cox1, 16S and internal transcribed spacer 1 sequences (1,874bp) for Nepalese specimens of I. exustus was also surprising in revealing the presence of four genetically distinct clades. They diverged from one another at levels comparable to those noted for species pairs in the sister genus Bulinus. There was no obvious pattern of use by Nepalese Schistosoma of the Indoplanorbis clades. We found high support for a close relationship between S. indicum and Schistosoma haematobium groups, but failed to retrieve support for a clean separation of the two, with a tendency for S. nasale to fall as the most basal representative. If this pattern holds, hypotheses for the origin of the Asian Indoplanorbis-transmitted S. indicum group from the Bulinus-transmitted S. haematobium group may require modification, including consideration of more contemporaneous origins of the two groups. The Indian subcontinent is under-studied with respect to schistosome diversity and our current knowledge of the S. indicum and I. exustus species groups is inadequate. Further study is warranted given the ability of indicum group species to cause veterinary problems and cercarial dermatitis, with a worrisome potential in the future to establish infections in humans.

Re-positioning protein-kinase inhibitors against schistosomiasis.

For decades, Praziquantel (PZQ) is the drug of choice against one of the most afflicting helminthic diseases worldwide, schistosomiasis. With respect to the fear of upcoming PZQ resistance, efforts are needed to find new chemotherapeutic options. Protein kinases (PKs) are essential molecules in signaling processes and indispensable to life. Aberrant PK functions take distinctive roles in human diseases and represent targets in chemotherapies. In schistosomes, conserved PKs were found to possess similar pivotal roles contributing not only to reproduction processes, but also to the pathology of schistosomiasis, which is closely associated to egg production. Exploiting the similarity of PKs of humans and schistosomes, PK inhibitors designed to treat human diseases may serve as lead compounds for new drugs against schistosomiasis.

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.

Targeting kinases in Plasmodium and Schistosoma: Same goals, different challenges.

With respect to parasite-induced infectious diseases of worldwide importance, members of the genera Plasmodium and Schistosoma are top pathogens. Nearly half a billion people suffer from malaria caused by Plasmodium spp. and schistosomiasis (bilharzia) induced by Schistosoma spp. Resistance against essentially all drugs used for malaria treatment has been reported. For schistosomiasis justified fear of upcoming resistance is discussed against the background of only one widely used drug for treatment. Research of the recent decade has demonstrated that essential steps of the biology of these and other parasites are controlled by kinases, which represent attractive targets for new-generation antiparasitic compounds. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

cis-Acting 5' hammerhead ribozyme optimization for in vitro transcription of highly structured RNAs.

RNA-mediated biological processes usually require precise definition of 5' and 3' ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5' hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.

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.

Artesunate effect on schistosome thioredoxin glutathione reductase and cytochrome c peroxidase as new molecular targets in schistosoma mansoni-infected mice.

OBJECTIVE: To investigate the possible effect of artesunate (ART) on schistosome thioredoxin glutathione reductase (TGR) and cytochrome c peroxidase (CcP) in Schistosoma mansoni-infected mice. METHODS: A total of 200 laboratory bred male Swiss albino mice were divided into 4 groups (50 mice in each group). Group I: infected untreated group (Control group) received a vehicle of 1% sodium carbonyl methylcellulose (CMC-Na); Group II: infected then treated with artesunate; Group III: infected then treated with praziquantel, and group IV: infected then treated with artesunate then praziquantel. Adult S. mansoni worms were collected by Animal Perfusion Method, tissue egg counted, TGR, and CcP mRNA Expression were estimated of in S. mansoni adult worms by semi-quantitative rt-PCR. RESULTS: Semi-quantitative rt-PCR values revealed that treatment with artesunate caused significant decrease in expression of schistosome TGR and CcP in comparison to the untreated group. In contrast, the treatment with praziquantel did not cause significant change in expression of these genes. The results showed more reduction in total worm and female worm count in combined ART-PZQ treated group than in monotherapy treated groups by either ART or PZQ. Moreover, complete disappearance (100%) of tissue eggs was recorded in ART-PZQ treated group with a respective reduction rate of 95.9% and 68.4% in ART- and PZQ-treated groups. CONCLUSION: The current study elucidated for the first time that anti-schistosomal mechanisms of artesunate is mediated via reduction in expression of schistosome TGR and CcP. Linking these findings, addition of artesunate to praziquantel could achieve complete cure outcome in treatment of schistosomiasis.