Schistosomiasis is the second most prevalent parasitic disease in the world. Currently, the treatment of this disease relies on a single drug, praziquantel, and due to the identification of resistant parasites, the development of new drugs is urged. The demand for the development of robust high‐throughput parasite screening techniques is increasing as drug discovery research in schistosomiasis gains significance. Here, we review the most common methods used for compound screening in the parasites life stages and also summarize some of the methods that have been recently developed. In addition, we reviewed the methods most commonly implemented to search for promising targets and how they have been used to validate new targets against the parasite Schistosoma mansoni. We also review some promising targets in this parasite and show the main approaches and the major advances that have been achieved by those studies. Moreover, we share our experiences in schistosomiasis drug discovery attained with our S. mansoni drug screening platform establishment.
- Schistosoma mansoni
- drug screening
- histone‐modifying enzymes
- protein kinases
- RNA interference
Schistosomiasis is a chronic parasitic disease caused by flatworms of the genus
2. Drug screening techniques in schistosomiasis
Several approaches are used to search new drugs for infectious diseases. Among them, we can highlight: selective, empirical, biochemical, and genomic approaches. In the selective approach, compounds targeting molecules important for parasite survival and/or development (a “chokepoint”) are tested. The empirical method consists of a blind random test of a large number of compounds, without any previous knowledge. The biochemical approach verifies whether the compounds are capable to change parasite metabolism. Finally, the genomic approach aims to search for new drug targets based on parasite and/or host genome analysis [8, 9].
Drug screening in
A wide range of
Recently, new methods based on high‐throughput (HTS) and whole‐organism screens in helminths have emerged. The HTS method is based on screening a parasite target against a large number of compounds in parallel (minimum of 10,000) and may be performed manually or automatically using robotic systems [13–15]. In contrast, the whole‐organism screen intends to test a small number of compounds against the pathogen and the drug effect is usually individually analyzed . These new methods monitor the parasite by video, impedance, enzymatic activity, colorimetry, and fluorimetry among others [17–23].
An example of fluorimetric method is the quantification of lactate excreted by the parasite in the culture medium. The parasite tegument presents two glucose transporters, SGTP1 and SGTP4, which acquire glucose present in the host bloodstream . Lactate is the final product after glycolysis, and it is excreted through aquaporin SmAQP, an aquaglyceroporin homologue . The amount of lactate excreted by the parasite can be measured by fluorimetric assays with probes that bind to lactate and emit fluorescence. The measurement of lactate produced by cells has frequently been used for the analysis of cells or whole‐organism viability [26, 27]. This method was used by Howe and collaborators  and was proved feasible in
Fluorescence viability analysis in schistosomula was also performed by Peak and collaborators  combining the use of propidium iodide (PI) (544 nm excitation/620 nm emission) and fluorescein diacetate (FDA) (485 nm excitation/520 nm emission). The PI intercalates into DNA if the membrane of cells is permeable in damaged or dead parasites, but in viable schistosomula, PI is incapable to cross the membrane. Breach of the membrane permeability allows PI to stain nucleic acids. On the other hand, FDA is able to penetrate the membrane of live schistosomula, and due to the parasite esterase activity, it is converted into a highly fluorescent and charged fluorescein, and FDA cannot readily exit living cells. This test requires a fluorescence inverted microscope to evaluate each spectrum. However, even without a fluorescence readout, staining with PI is a quantitative, simple and low‐cost method that has been used for a long time for viability evaluation in
The blue dye resazurin has been widely used in drug testing in
Movement‐based assays are widely used for anthelmintic drug screening in adult worms and might be the first phenotype tested in most screenings, whereas movement measures are not explored as much for testing viability of the
Recently, Rinaldi et al.  have adapted a cell viability method, which is based on impedance, called xCELLigence, to evaluate the movement of cercariae, schistosomula and adult worms, and measure egg hatching of
A very promising method that provides a viability assay in a high‐throughput fashion and with semiquantitative measurements of movement of helminth worms was developed by Marcellino and collaborators . This method has demonstrated to be efficient and sensitive for drug screening using
Another promising method based on video capture was developed by Lee and collaborators . This assay is an automated method to analyze images of schistosomula or other parasites in 96, 384, or 1536 well plate format and qualify innumerous phenotypes. A machine‐trained algorithm was able to quantitatively describe the following characteristics: size, shape, movement, texture, and color. With this method, it is possible to perform a high‐throughput whole‐organism drug screening. The web server called quantal dose‐response calculator (QDREC), which was described by Asarnow and collaborators , is based on this methodology. QDREC compares drug‐treated parasites with untreated parasites and automatically determines dose‐response. This method is the only automated method to date that provides EC50 (half maximal effective concentration) values based on phenotypic analysis. QDREC was validated using schistosomula and proved to be a high‐throughput and reliable method.
3. Case studies
In order to perform
Drug combinations for infectious diseases therapy represent an alternative to retard drug resistance. Based on that premise, Araújo et al.  treated infected mice with a single dose of clonazepam associated with praziquantel or oxamniquine. The results showed alterations in the oogram and a higher number of dead worms recovered from liver of mice treated with both clonazepam and praziquantel, in comparison with mice treated only with praziquantel. In another study, the association of praziquantel and oxamniquine with lovastatin was investigated
Thus, it is highly relevant to follow systematic procedures during drug screening. Here, we present a schematic workflow of the above‐mentioned methods and different life stages that could be included in drug screening experiments (Figure 1).
4. Establishment of an anti‐
Schistosoma mansonidrug screening platform
The absence of efficient alternative for schistosomiasis treatment demonstrates the need for new research involving the development of new schistosomicidal compounds. Accordingly, development of a
For drug screening in schistosomula, the methods of choice were: resazurin fluorescence assay, lactate quantitative analysis, and visual assay using propidium iodide staining (PI). In order to perform drug screening in adult worms, the movement analysis software WormAssay was employed.
The standardization for schistosomula drug screening was performed using 96‐well plate format with 100, 200, 400, 600, and 800 parasites per well. Schistosomula were submitted to three different treatments: parasites exposed to 0.1% dimethyl sulfoxide (DMSO; vehicle control); heat‐killed parasites (negative control); and parasites exposed to 20 μM of the sirtuin inhibitor Salermide (half maximal inhibitory concentration—IC50). Lancelot et al.  demonstrated that Salermide induces death and apoptosis of schistosomula, separation of adult worm pairs, as well as a reduction in egg laying.
The PI staining procedure was established by the use of 5 μg/mL of fluorophore into 96‐well culture plates containing 100 parasites and visualized in inverted fluorescence microscope. Our results have shown that mortality evaluation by phenotype observation in bright‐field light microscope was overestimated when compared to PI staining results. This result reinforces the subjectivity problem of bright‐field visual analysis since parasites that presented a dead phenotype (intracellular granularity and absence of movement) not always stain with PI, indicating their viability.
In viability assays using resazurin, we observed large variability between technical and biological replicates and a low value of relative fluorescence units (RFU). Mansour and Bickle  described RFU values >1000 in wells containing 100 schistosomula, and such high RFU values were possible only with 500 parasites per well in our assays conditions. Overall, using this fluorescence assay, we were unable to discriminate schistosomula killed by Salermide treatment from the vehicle control (0.1% DMSO). Issues in assay sensitivity measured by RFU values could be due to differences in the fluorescence reader platform, indicating that the assay can present a reproducibility issue. Limitations in this methodology such as low sensitivity and reliability, when compared to visual analysis, were described by the authors who proposed the application of resazurin as
Standardization of lactate quantitative analysis demonstrated low variability and significant differences between RFU values of schistosomula exposed to 20 μM Salermide from 0.1% DMSO. RFU values were similar to those described in the work of Howe and collaborators  confirming the method's reproducibility. Mortality of the parasites detected in the lactate quantitative analysis was confirmed by PI staining and observation under the microscope, in contrast to our previous results with resazurin assay. Considering the fluorescence‐based viability assays, lactate quantitative analysis has shown to be more reliable in our hands. However, the test may also be subject to interference, since some compounds crystallize in contact with culture medium and can emit fluorescence at the wavelength utilized (530 nm excitation/590 nm emission).
The use of viability assays that target schistosome metabolism is important for drug screening, since some drugs may reduce parasite viability but do not cause parasite death, and hence these parasites may not stain by PI. Therefore, we believe that the combination of these two approaches to evaluate parasite viability/mortality is a good strategy for drug screening as they are complementary methodologies.
Regarding adult worm drug screening, Howe and collaborators  proposed the use of one male adult worm per well exposed for 72‐h treatment with praziquantel and mefloquine, and detected a reduction in medium lactate. The use of only males and one parasite per well does not validate this methodology for drug testing in adult worms, as males and females may react differently to treatments and each individual has distinct susceptibility/metabolism. Therefore, the use of this method would require larger number of worms including both sexes.
The method of trematode movement analysis using impedance was described by Smout and collaborators  and was recently validated in
In our platform, eight adult worms were established as the minimum number of specimens to be analyzed. Most importantly, paired females and males should not be analyzed because a drug could be active in only sex, disturbing the movement analysis due to female presence in male gynaecophoric channel. The plates containing the worms are filmed for 1 min and 30 s every day for 10 days. The software is freely available for download, recognizes the wells, and provides the total of movement units for each. Moreover, handling is simple and our tests confirm its sensitivity and accuracy.
For validation of our anti‐
These data confirm the need to perform an assay aimed at identifying compounds capable of altering the parasite metabolism and, consequently, their viability, since the compound cannot cause parasite death, hindering the observation of drug effect in a visual assay. Clustering methodologies, life stages, and parasite strains could change the outcomes of large screening studies such as those using a single method and stage; for example, Li et al.  performed a screening of 59,360 thioredoxin glutathione reductase (TGR) inhibitors against
Our work demonstrates the need to use parallel and complementary methods, since only using PI, for example, a lower number of compounds would be selected as active and consequently, potential compounds would be excluded. In view of the foregoing methods, the contributions from studies aimed at identifying new therapeutic compounds against schistosomiasis could, perhaps, be most effective if they employ more than a single method for screening drugs and different parasite stages. The association of fluorescence with PI staining enables the selection of compounds capable of altering parasite viability and/or inducing mortality. Moreover, male and female adult worms develop and have different metabolisms, and thus, their susceptibility to a specific drug also differs. One clear example is the studies demonstrating that female adult worms are more sensitive to praziquantel than males [4, 42] and approximately 1341 genes are up‐regulated in female adult worms when compared to male . Regarding the different life stages, results demonstrate that some drugs, including praziquantel, are active solely in the mature stage of
These results indicate the need to employ different methods for drug screening as one can find a larger number of hits than when the tests are performed using only one method or in only one stage of the parasite. In addition, it substantiates the importance of using the selective approach to find active compounds, thus using a rational approach targeting previous validated targets, allowing direct design of specific compounds.
5. Validation of
S. mansonidrug targets
The most common strategies used in drug discovery are the empirical and the rational approaches. The first is based on testing various compounds randomly looking for biological activities. The second is also called the selective approach as it proposes identifying a biological target and then designing or looking for a specific inhibitor for that target. In this sense, there are many strategies to assess gene function in parasites in order to elucidate their role in development, mechanisms of drug resistance, and speculate its use as a parasite control method. Among them, we can mention comparative “omics,” RNA interference, heterologous complementation using model organisms (i.e.,
5.1. Histone‐modifying enzymes
Among the most studied targets are epigenetic modulators, and among them are regulators of chromatin epigenetic modifications, named histone‐modifying enzymes (HMEs), which act on the epigenome resulting in a change in the gene transcription profile. HMEs are involved in a wide range of reactions including methylation, demethylation, acetylation, deacetylation, phosphorylation, ADP‐ribosylation, deimination, sumoylation, ubiquitination, etc. Yet, recent findings have described a myriad of lysine modifications, among others: formylation, succinylation, crotonylation, and malonylation .
The enzymes involved in the insertion and removal of methyl groups are called histone methyltransferase (HMT) and histone demethylases (HDM) . The histone‐modifying enzymes involved in the insertion and removal of acetyl groups are called histone acetyltransferases (HAT) and histone deacetylases (HDAC and sirtuins). Acetylation of lysine residues and methylation of lysine and/or arginine residues in histones H3 and H4 tails are two changes of particular importance [47, 48]. The insertion of acetyl and methyl groups neutralizes the positive charge of histones, destabilizing the structure of nucleosome and allowing the DNA to separate from histones. This results in the facilitation of the access of transcription factors and RNA polymerase to the DNA stimulating gene expression. The removal of these groups has the opposite effect, increasing the positive charge, condensing chromatin, and thereby, repressing transcription . The heterochromatin is transcriptionally inactive when highly methylated at lysine 9 of histone H3 and not methylated at lysine 4, and hypoacetylated in histones H3 and H4 . It is important to highlight that HDACs and sirtuins also deacetylate other nonhistone substrates such as chaperones, peroxiredoxins, transcription factors, signaling mediators, and structural proteins (e.g., [51–55]).
These modifications allow specific combinations that affect the overall structure of chromatin and the transcription of genes, the so‐called histone code, which is, in many cases, conserved among organisms . Aberrant epigenetic states are often associated with human diseases such as cancer, inflammation, metabolic, and neuropsychiatric disorders, and thus HMEs are implicated and intensively studied as therapeutic targets in various diseases [56–59].
One of the most promising approaches for drug discovery among HMEs is the development of HDAC inhibitors, which targets the highly studied lysine deacetylases. These targets are also key for parasites, including schistosomes, which present, similarly to tumors, dependence on lactate fermentation as energy source, host independent growth, high metabolic activity, and host immune evasion through mimetism of molecules . In addition to schistosomes, HMEs have been highly explored as drug targets for parasitic diseases such as
Many different types of HDAC inhibitors (HDACi) are under development. The inhibitors targeting class I and II HDACs are classified into four families according to their structure: inhibitors containing short‐chain fatty acids (butyrate and the valproic acid—VPA), compounds derived from hydroxamic acid (the Trichostatin A—TSA and the acid‐suberoylanilide hydroxamic—SAHA or vorinostat), and the group of cyclic tetrapeptides and benzamides. Among these inhibitors, SAHA was approved by Food and Drug Administration (FDA) for use in adult individuals with cutaneous T‐cell lymphoma .
Some studies have shown that HDACi, such as TSA, triggers histone H4 hyperacetylation in
In addition to pan HDAC inhibitors, one international consortium has been focusing in strategic epigenetic druggable targets for diverse parasites . Recently, SmHDAC8 has risen as a promising target to treat schistosomiasis. First, SmHDAC8 was validated and proved to be essential for parasite infectivity, since parasites knocked down for SmHDAC8 were unable to normally develop in the mammalian host and showed, approximately, 50% reduction in oviposition . Structural analysis has also shown that HDAC8 of
To date, a way to expand the repertoire of specific Schistosoma, HDACi is utilizing a “piggyback” strategy, which accelerates the exploration for novel antischistosomal compounds. These strategies are founded in the principle of using structure‐based inhibitors, previously validated for other illnesses or other targets, to add a variety of chemical scaffolds and backbones, facilitating the development of selective inhibitors specifically aiming the schistosoma HMEs .
Besides deacetylases, HAT inhibitors and some derivatives of medicinal herbs, such as curcumin, also demonstrated their potential as inhibitors, since they induce hypoacetylation and lead tumor cells into apoptosis [70, 71]. Magalhães and collaborators  demonstrated the efficacy of curcumin in
Recent studies in
Studies using HMT and HDM inhibitors are less common, but some results have shown that the chloroacetyl derivative, allantodapsone, a PRMT1 (arginine methyltransferase) inhibitor, showed selective inhibition affecting the growth of tumor cells . Inhibitors of KDM1 (LSD1), a histone demethylase, are considered promising compounds for cancer therapy . Studies performed by our group, knocked down for PRMT3 and KDMs in schistosomula by RNA interference, show that these enzymes are important for
Due to the wide range of HME functions as “erasers” and “writers” of the epigenome, boosted by the use of histone and many nonhistone protein substrates, and taking into account the cellular localizations of these enzymes, it has been demonstrated, as expected, that they are essential and attractive targets for development of therapy for a number of infectious diseases, including schistosomiasis.
5.2. Protein kinases
Eukaryotic protein kinases (ePKs) participate in phosphorylation cascades that regulate diverse cellular processes. PKs are among the largest gene families in eukaryotes and have been extensively studied and considered potential targets for drug development. The development of PK inhibitors has culminated in the approval of some drugs for the treatment of various human diseases such as cancer and diabetes. Furthermore, PKs have gained interest as potential drug targets against many parasites, including
Four mitogen‐activated protein kinases (MAPKs) were studied by our group using double‐stranded RNA‐mediated interference to elucidate their functional roles. Mice were infected with schistosomula after gene knockdown, and the development of adult worms was observed. Andrade et al.  showed that c-Jun N-terminal kinase SmJNK participates in the maturation and survival of the parasite, associated with the presence of undifferentiated oocytes and damage in the adult male tegument. SmERK‐1 and SmERK‐2 are involved in egg production, since females were recovered with undeveloped ovaries and immature oocytes, and the infected mice harbored significantly fewer eggs. Furthermore, the Smp38 kinase seems to have an important role in the development and survival of parasites and in their protection against reactive oxygen species (unpublished data). Thus, we demonstrated that MAPK proteins are important for parasite survival
Guidi et al.  used RNA interference to investigate the function of 24 proteins in adult worms and schistosomula, and among those, kinases were included. For atypical protein kinase C (SmaPKC), knockdown resulted in decreased viability in both stages. Knockdown of polo‐like kinase 1 (SmPLK1) and p38 MAPK (Smp38) increased mortality only in larvae. The SmPLK1 inhibition with BI2536, a specific inhibitor, also increased mortality and interfered with egg production. Knockdown of SmPLK1 and SmaPKC also resulted in lower worm recoveries
Ressurreição et al.  reported that phosphorylation of PKC, ERK, and p38 MAPK kinases is modulated by light and temperature. Furthermore, in response to linoleic acid, these kinases appear to coordinate the release of components of the cercarial acetabular gland, and PKC and ERK, when activated, are located in putative sensory receptors in the tail, thus demonstrating the importance of PKC, ERK, and p38 MAPK signaling pathways in the mechanisms for host penetration.
As mentioned, PKs are conserved and widely studied in many organisms; therefore, a range of PK inhibitors is already available, which are valuable tools. The function of some kinases was studied by parasite exposure to these inhibitors to explore PK functions in
Knobloch et al.  used the inhibitor Herbimycin A to demonstrate that protein tyrosine kinases (PTKs) regulates gonad development and egg production through changes in gene expression of eggshell proteins, and suggested PTKs as novel anti‐
Imatinib, an Abl‐kinase‐specific inhibitor used in human cancer therapy, was tested against adult worms and caused effects on morphology and physiology of
Syk kinase (SmTK4) function was studied in adult worms using RNA interference and the specific inhibitor piceatannol. Prominent morphological changes in testes and ovaries were observed, demonstrating the role of SmTK4 in gametogenesis. In addition, the authors used yeast two‐/three‐hybrid library screenings and identified a Src kinase (SmTK6) acting upstream and a MAPK‐activating protein and a mapmodulin acting downstream of SmTK4 .
A set of commercial kinase inhibitors was tested by Morel et al.  in schistosomula and adult worms, conforming deleterious effects on parasite physiology, as well as the importance of kinases in parasite biology and reproduction. In that study, five protein kinase B (SmPKB or SmAkt) inhibitors were tested and three affected pairing and oviposition of adult worms, in addition cause mortality in larvae. These data, along with other studies , suggest that SmAkt is a key regulator of schistosoma reproduction processes .
The roles of protein kinases C (PKCs) and extracellular signal‐regulated kinases (ERKs) were studied through modulation of PKC and ERK activity by kinase activators and inhibitors in adult worms. Results have shown that this modulation induced worm uncoupling, suppressed egg output, male worm detachment, worm paralysis, and provoked sustained coiling. The authors also reported that praziquantel, the drug of choice for schistosomiasis treatment, induced activation of
5.3. Other targets
In addition to epigenetic modification, effectors and protein kinases, numerous proteins related to proteolytic, xenobiotic metabolism, redox processes, nucleotide biosynthesis and proteins involved in the nervous system of
Peptidases are enzymes that perform proteolytic reactions and peptide bond hydrolysis.
Prolyl oligopeptidases of the S9 family of serine peptidases have been investigated in
5.3.2. Xenobiotic metabolism
The biotransformation of xenobiotics involves pathways that can be divided into three phases: (I) oxidation, reduction, or hydrolysis of xenobiotics; (II) conjugation of metabolites with endogenous compounds; and (III) excretion of modified molecules through membrane‐bound transport proteins . The xenobiotic metabolism can be a promising area for drug development since it implicates mechanisms that the parasite uses to eliminate drugs or toxic compounds; additionally, it plays vital roles in providing essential molecules for parasite survival. Among the
The glutathione S‐transferase (GST) family, from biotransformation pathway phase II, has been extensively studied in schistosomes. The knockdown of SmGST26 and SmGST28 in sporocysts by dsRNA exposure increased their susceptibility to exogenous oxidative stress and to
The phase III membrane‐bound transport proteins are currently under study, and the ABC transporters are the most studied among them . The involvement of these ABC transporters such as P‐glycoprotein and multidrug resistance‐associated protein (SmDR1, SmDR2, SmMRP1, ABCA4, ABCB6, and ABCC10/MRP7) in drug susceptibility and development of drug resistance in schistosomes is clear, and this makes them excellent candidate targets for inhibitors that could potentiate the effect of existing drugs against schistosomes  or as new therapeutic targets themselves .
5.3.3. Redox mechanisms
Redox balance mechanisms are essential for schistosome worm survival, and differences between schistosome and human host redox networks were shown in previous studies (reviewed in Ref. ). The
5.3.4. Purine biosynthesis
The purine nucleotide
5.3.5. Neurotransmitter transporters
The inhibitory neurotransmitters, norepinephrine (NE) and dopamine (DA), are also present in
5.3.6. Neurotransmitter receptors
6. Future directions and new approaches
While schistosomiasis still has a high socioeconomic impact, with the total number of disability‐adjusted life years (DALY) lost to schistosomiasis estimated at 4.5 million per year, and treatment relies only on praziquantel since the early 1980s, drug discovery is still of great relevance. Our results with a
It is noteworthy that after identifying potential anti‐
This work has been supported by funding from the European Commission's Seventh Framework Programme for research, under Grant agreement no. 602080 (A‐ParaDDisE), CAPES Programa PCDD-Programa CAPES/Nottingham University (3661/2014), FAPEMIG (CBB-APQ‐00520‐13). GO received funds from CNPq (470673/2014‐1 and 309312/2012‐4) and is funded by CAPES (003/2014, REDE 21/2015), CNPq (304138/2014‐2), and FAPEMIG (PPM‐00189‐13).