Development and Validation of a Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Background

Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive ‘genome to drug’ lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies.

Methodology/Principal Findings

We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed), relevant to industrial (384-well microtiter plate compatibility) and academic (96-well microtiter plate compatibility) settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative.

Conclusions/Significance

The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species further offers an opportunity for great strides to be made against additional neglected tropical diseases of biomedical and veterinary importance.

Author Summary

With only one effective drug, praziquantel, currently used to treat most worldwide cases of schistosomiasis, there exists a pressing need to identify alternative anthelmintics before the development of praziquantel-resistant schistosomes removes our ability to combat this neglected tropical disease. At present, the most widely adopted methodology used to identify promising new anti-schistosome compounds relies on time consuming and subjective microscopic examination of parasite viability in response to in vitro schistosome/compound co-culturing. In our continued effort to identify novel drug and vaccine targets, we detail a dual-fluorescence bioassay that can objectively be used for assessing Schistosoma mansoni schistosomula viability in a medium or high- throughput manner to suit either academic or industrial settings. The described methodology replaces subjectivity with sensitivity and provides an enabling technology useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that results obtained from these quantifiable in vitro screens would prioritize the most effective anti-schistosomal compounds for follow-up in vivo experimentation. This highly-adaptable dual-fluorescence bioassay could be integrated with other methods for measuring schistosome phenotype and, together, be used to greatly accelerate our search for novel anthelmintics.

Related collections

Most cited references 18

Record: found
Abstract: found
Article: not found

The Schistosoma japonicum genome reveals features of host-parasite interplay.

(2009)

Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis, which is a significant cause of morbidity in China and the Philippines. Here we present a draft genomic sequence for the worm. The genome provides a global insight into the molecular architecture and host interaction of this complex metazoan pathogen, revealing that it can exploit host nutrients, neuroendocrine hormones and signalling pathways for growth, development and maturation. Having a complex nervous system and a well-developed sensory system, S. japonicum can accept stimulation of the corresponding ligands as a physiological response to different environments, such as fresh water or the tissues of its intermediate and mammalian hosts. Numerous proteases, including cercarial elastase, are implicated in mammalian skin penetration and haemoglobin degradation. The genomic information will serve as a valuable platform to facilitate development of new interventions for schistosomiasis control.

0 comments Cited 260 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Thioredoxin Glutathione Reductase from Schistosoma mansoni: An Essential Parasite Enzyme and a Key Drug Target

Angela Kuntz, Elisabeth Davioud-Charvet, Ahmed A Al-Sayed … (2007)

Introduction Schistosomiasis (also known as bilharzia)—infection with the helminth parasites in the genus Schistosoma—remains an important infection in many tropical areas, especially Africa. More than 200 million people have schistosomiasis, with 20 million exhibiting severe symptoms. Recent analyses suggest that the morbidity due to schistosomiasis is grossly underestimated [1], resulting in an estimated 280,000 deaths annually in sub-Saharan Africa alone [2]. Since the mid-1980s praziquantel (Figure S1) has been the drug of choice for schistosomiasis; effectively it is currently the only choice available. Artemether has shown promise as a new drug for schistosomiasis, targeting larval parasites more effectively than praziquantel, which is primarily effective against adult parasites [3]. However, the use of artemether for schistosomiasis should be restricted so that its use as an antimalarial compound is not put at risk from the development of possible drug resistance in the malaria parasite. With the exception of the artemisinin-based drugs, no new drugs have been introduced for schistosomiasis after praziquantel, and prior drugs have ceased to be produced or are ineffective [4]. Furthermore, resistance to oxamniquine, which is effective against S. mansoni, has been reported [5], thereby reducing its potential usefulness. Research for new antischistosome drugs is limited by the difficulty of working with the parasite and the low priority the pharmaceutical industry generally places on tropical diseases. Currently more than 100 million people are being treated for schistosomiasis with praziquantel [2]; they are rapidly reinfected and must be retreated on an annual or semiannual basis. If praziquantel-resistant parasites develop, treatment for schistosomiasis will be in a crisis state. Adult S. mansoni parasites reside in the mesenteric veins of their human hosts, where they can survive for up to 30 years [6]. Living in an aerobic environment, worms must have effective mechanisms to maintain cellular redox balance. Additionally, worms must be able to evade reactive oxygen species generated by the host's immune response. In most eukaryotes there are two major systems to detoxify reactive oxygen species, one based on the tripeptide glutathione (GSH) and the other based on the 12 kDa protein thioredoxin (Trx). In both systems reducing equivalents are provided by NADPH via dedicated oxidoreductase flavoenzymes. Glutathione reductase (GR) reduces glutathione disulfide (GSSG) and drives the GSH-dependent systems [7,8], whereas Trx reductases (TrxR) are pivotal in the Trx-dependent system (Figure 1) [9]. In addition to providing protection against oxidative damage, the Trx and GSH systems also play important roles in cell proliferation, redox regulation of gene expression, xenobiotic metabolism, and several other metabolic functions [8,9]. Because of the diverse functions of the TrxR- and GR-dependent pathways, the two oxidoreductases have been identified as promising targets for drug development for many diseases, including malaria, trypanosomiasis, and cancer [9,10]. Figure 1 Redox Pathways in Mammals and S. mansoni In mammals (upper pathway), electrons from NADPH are transferred to an oxidoreductase flavoenzyme, either thioredoxin reductase (TrxR) or glutathione reductase (GR). Electrons are then transferred from the oxidoreductase flavoenzyme to the appropriate electron carrier, either oxidized thioredoxin (Trx-S2) or glutathione disulfide (GSSG) converting them to reduced thioredoxin (Trx-[SH]2) or glutathione (GSH), respectively. Trx-(SH)2 and GSH then supply reducing equivalents for a number of different reactions, including those that are glutaredoxin (Grx)-dependent. In S. mansoni (lower pathway), TrxR and GR are replaced with a unique oxidoreductase flavoenzyme, TGR, which provides reducing equivalents for Trx-, GSH- and Grx-dependent reactions. It was recently discovered that in S. mansoni, specialized TrxR and GR enzymes are absent, and instead replaced by a unique multifunctional enzyme, thioredoxin glutathione reductase (TGR) (Figure 1) [11]. This reliance on a single enzyme for both GSSG and Trx reduction suggests that the parasite's redox systems are subject to a bottleneck dependence on TGR. The amino acid sequence and domain structure of schistosome TGR has similarities to mammalian forms of TrxR and GR, with an additional amino-terminal extension of a glutaredoxin (Grx) domain of ~110 amino acids with a typical CPYC active site [11]. Like all mammalian TrxR isoforms, S. mansoni TGR is a selenoprotein with a carboxyl-terminal GCUG active site motif, where “U” is selenocysteine (Sec). Sec is a highly reactive amino acid that gives unique properties to selenoproteins [12]. It is encoded by a dedicated UGA codon in the selenoprotein mRNA and is recoded from translational termination to Sec insertion by a translation machinery utilizing a specialized structural element in the 3′-untranslated region, the SECIS element, which is also found in the mRNA of S. mansoni TGR [11]. Given the importance of cellular redox systems and the biochemical differences between the redox metabolism of S. mansoni and its human host, we hypothesized that TGR could be an essential parasite protein and a potentially important drug target. To test this hypothesis, we used RNA interference (RNAi), characterized the recombinant selenoprotein, and screened inhibitory compounds, including two established antischistosomal drugs that are no longer commonly used, potassium antimonyl tartrate (PAT) and oltipraz (OPZ). Methods Parasite Preparation Percutaneous infection of outbred mice (NIH Swiss or Swiss-Webster) with S. mansoni cercariae (NMRI strain) obtained from infected Biomphalaria glabrata snails, perfusion of adult worms (6–7 wk) and juvenile worms (23 d) and preparation of schistosomula from cercariae were as described [13]. This study was approved by the Institutional Animal Care and Use Committee of Illinois State University (08–2002; Department of Health and Human Services animal welfare assurance number A3762–01). Recombinant Sec TGR Expression and Purification A bacterial-type SECIS element was fused to the TGR open reading frame [14] using PCR with the following oligonucleotides primers: forward 5′-catATGCCTCCAGCTGATGGAAC-3′ and reverse 5′-TCGCCAACGACTCCAATTATTAGCCAACGTCCAGACGTGGTTAGCAATTGGATACGCGGGcagctg-3′. The entire TGR ORF plus the SECIS element was subcloned into pET-24a using NdeI/SalI to release the insert. Recombinant TGR was subsequently expressed in the Escherichia coli strain BL21(DE3) in the presence of pSUABC [14] in LB medium supplemented with 20 μM flavin adenine dinucleotide, and otherwise conditions for optimal selenoprotein expression were followed as described [15]. Cultures were centrifuged, lysed by alternative freeze-thaw, resuspended in TE buffer and supplemented with 20 μM flavin adenine dinucleotide. The sample was sonicated and cellular debris pelleted at 25,000g at 4 °C for 25 min. The supernatant was collected and filtered through a 0.45 μm filter and brought to a final concentration of 200 mM NaCl. TGR was then purified on an adenosine 2′,5′-diphosphate agarose (Sigma, http://www.sigma-aldrich.com) column equilibrated with TE buffer. The column was washed with 50 ml of TE. TGR was eluted with 1 mM NADPH in TE essentially as described [14,15]. The TGR sample (1/50 volume cell culture) was applied to a 2.5 ml column, which was then washed with 50 ml of TE after which TGR was eluted in ten 1 ml fractions of 1 mM NADPH in TE. Protein purity was >95% as determined by SDS-PAGE, and TGR concentration was determined from the flavin adenine dinucleotide absorption (ɛ463 = 11.3 mM−1cm−1). The pure protein was dialyzed against PBS and stored at −80 °C. Overall yield was ~10 mg of purified protein per liter of culture. TGR Enzymatic Assays Assays were performed at 25 °C in 0.1 M potassium phosphate (pH 7.4), 10 mM EDTA using 100 μM NADPH unless otherwise stated. The insulin assay and the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) assays were used to determine the TrxR activity of TGR. The DTNB assay [16] contained 3 mM DTNB, and the initial increase in A412 during the first 3 min was recorded upon enzyme addition. One enzyme unit in the DTNB reduction assay was defined as the NADPH-dependent production of 2 μmol of 2-nitro-5-thiobenzoic acid per minute using ɛ412 nm = 13.6 mM−1 cm−1. The insulin assay [17] mixture contained 1 mg/ml insulin and 10 μM schistosome Trx1 [18], and enzyme activity was monitored by observing the decrease in A340 during the first 3 min due to consumption of NADPH (ɛ340 nm = 6.22 mM−1 cm−1). GR activity was determined with 100 μM GSSG [19]. Grx activity was determined by monitoring the consumption of NADPH at 340 nm during the GSH-dependent reduction of 8 mM β-hydroxyethyl disulfide (HED) using 1 mM GSH coupled with of 0.6 units of yeast GR (Sigma) [20]. The inhibitory action of both PAT and OPZ was addressed by varying substrate and inhibitor concentrations as follows: DTNB 50, 100, 400, 1000 μM; GSSG 10, 25, 50, 100 μM; PAT 10, 30, 75 nM; OPZ 25, 50, 100 μM. The inhibitory constants (K i) were determined using Equation 1 [21]: where v maxapp is the apparent v max at different inhibitor concentrations and corresponds to the value of the 1/v intercept on the reciprocal plot. Initial inhibitor screens were conducted in 200 μl volumes in 96 well plates with 50 μM inhibitor, 100 μM NADPH, and 5 nM TGR in 0.1 M potassium phosphate (pH 7.4), 10 mM EDTA with a preincubation step of 15 min. Aurothioglucose was obtained from Research Diagnostics (http://www.researchd.com); aurothiomalate, menadione, methylene blue, naphthazarin, plumbagin, praziquantel, PAT, and safranin were from Sigma; OPZ was a gift from Dr. D. Cioli; auranofin (AF) was a gift from Dr. C.F. Shaw; and all other compounds were prepared as described in the references in the appropriate Tables. Upon addition of another aliquot of NADPH (final NADPH concentration of 100–200 μM) and 3 mM DTNB the activity was measured during the first 3 min by monitoring the A412. Residual activity was compared to controls incubated with equal volumes of inhibitor solvent. Compounds exhibiting >85% inhibition were analyzed in detail to determine IC50 values using the assays described above and including a 15 min preincubation step of TGR plus inhibitor and 100 μM NADPH. All assays were done in triplicate. Kinetic Analysis TGR activity with the following substrates was determined: GSSG, GSH, HED, S. mansoni Trx1 [18], DTNB, H2O2, t-butyl hydroperoxide, L-cysteine, sodium selenite, lipoic acid, lipoamide, alloxan, dehydroascorbic acid, and ubiquinone by monitoring the oxidation of NADPH. In all assays the reactions were performed in 1 ml 0.1 M potassium phosphate (pH 7.4), 10 mM EDTA using 100 μM NADPH and 20 nM TGR. Kinetic parameters were determined using Lineweaver–Burk plots using KaleidaGraph 4 (Synergy Software, http://www.synergy.com) least squares best fit of data and varying concentrations of one substrate while maintaining the concentration of other substrates. In the TrxR assays, substrate concentrations ranged from 2.5 to 200 μM NADPH and from 0.25 to 15 μM S. mansoni Trx1 or from 100 to 4,000 μM DTNB. To determine kinetic parameters for GR activity of TGR, substrate concentrations varied from 7.5 to 100 μM GSSG and from 10 to 100 μM NADPH. Grx activity was determined by using 25 to 4,000 μM HED, 10 to 100 μM GSH, and 1 to 100 μM NADPH. All assays were done in triplicate. Inhibitor Studies on Cultured Worms AF was dissolved in DMSO and added to freshly perfused worms in DMEM to 10 μM. Control worms were treated with equal amounts of carrier. Worms were subsequently observed for unpairing, motility, and mortality, and were collected at the indicated time intervals for analysis. Worms were homogenized by sonication in PBS and homogenates were assayed for TrxR and GR activities as described above. Activities of the control enzymes GSH peroxidase (GPx) and lactate dehydrogenase (LDH) were determined using published methods [22,23] with 1 mM sodium azide, 3.0 units of yeast GR (Sigma), 1 mM GSH, 0.02 mM DTT, 0.0007% (w/v) H2O2, and 100 μM NADPH in the GPx assay and 10 μM sodium pyruvate and 100 μM NADPH in the LDH assay. A replicate experiment was performed in which the worms were homogenized in 1% picric acid and the ratio of GSH:GSSG was determined as described [24]. Each assay was done in triplicate and each experiment was done three times. Inhibitor Studies on Infected Mice Mice (14-wk-old females; C57BL/6 in experiment 1, NIH-Swiss in experiment 2) infected with 60 S. mansoni cercariae were injected intraperitoneally with 6 mg/kg of AF twice daily for 9 d, beginning 7 wk post infection. Infected control mice were injected with equal amounts of carrier. Mice were perfused 1 wk after the final dose of AF and worm burdens were determined. RNA Silencing A 500-nucleotide fragment (bp 1364–1866) of S. mansoni TGR was amplified by PCR (forward primer 5′-CTATTTGGCTAGACGTCTGT-3′ and reverse primer 5′-AATACAGTTTCCTTCCCGTT-3′) and cloned into PCRII-TOPO vector (Invitrogen, http://www.invitrogen.com). The resulting construct was linearized using XhoI (for SP6 RNA polymerase transcription) and SacI (for T7 RNA polymerase transcription). RNA transcription was conducted according to manufacturer's instructions (Ambion, http://www.ambion.com). A 280-bp, nonschistosome dsRNA used for irrelevant dsRNA negative controls was synthesized from the PCRII-TOPO vector using T7 or SP6 RNA polymerases as described above. Cercariae were mechanically transformed to schistosomula by vortexing, and bodies were separated from tails by Percoll gradient centrifugation as described [13]. Immediately after transformation, TGR dsRNA or irrelevant dsRNA (each at 54 μg/ml) was added to ~500 schistosomula in 50 μl water followed by incubation at 37 °C for 30 min. After addition of 250 μl of RPMI-1640 medium, 300 U/ml penicillin, and 175 μg/ml streptomycin, parasites were incubated at 37 °C in 5% CO2 atmosphere for 4 d. Reduced O2 tension was obtained by filtering media and purging with 95% N2, 5% CO2 for 20 min before use. Parasites were cultured during the course of the experiment in the appropriate gaseous mix at 37 °C in a sealed modular incubator chamber (Billups-Rothenberg, http://www.brincubator.com) flushed with the same gaseous mix. On day 2 of the experiment, 50 μl of fresh media was added. Schistosomula were assessed daily for viability by microscopy. For each time point at least 1,500 parasites were scored as alive or dead in each experiment (three replicates of 500 each), and each experiment was repeated three times. Total RNA was harvested from parasites after the course of an experiment by TRI reagent, following the manufacturer's instructions (Sigma). Complementary DNA was synthesized using 1 μg of RNA, 1 μl of oligo dT (500 μg/ml), and 1 μl of 10 mM dNTP mix in a 10 μl reaction. The mixture was heated to 65 °C for 5 min and quickly chilled on ice. Then 4 μl of first-strand buffer, 2 μl of 0.1 M dithiothreitol, and 1 μl of RNaseOut (Promega, http://www.promega.com) were added to the mixture. The mix was incubated for 2 min and then 1 μl of Thermoscript reverse transcriptase (200 U) (Invitrogen) was added and incubated for 50 min at 42 °C. The reaction was heated at 70 °C for 15 min to inactivate the enzyme. The resulting single-stranded cDNAs were used as templates in PCR reactions using gene-specific primers to amplify TGR (forward 5′-CTATTTCCGTAGACGTCTGT-3′ and reverse 5′-AATACAGTTTCCTTCCCGTT-3′) or GAPDH (forward 5′-GTTTTGGTCGTATCGGGAGA-3′ and reverse 5′-ATGCGTTAGAAACCACGGAC-3′). Cytotoxicity Assay Cytotoxicity assays were performed using sulforhodamine B to determine cellular protein content as described [25]. Briefly, myeloma cell line SP2/0 was cultured in 96-well microtiter plates containing 0.2 ml of RPMI-1640 per well at a cell density of 1,000 per well at 37 °C in 5% CO2. Cells were treated with drug concentrations (or drug carrier alone) and exposure times as indicated. After treatment, cells were fixed with 10% TCA at 4 °C for 1 h. Fixed cells were rinsed to remove fixative and then stained in 0.4% (w/v) sulforhodamine B (Sigma) in 1% acetic acid for 30 min. After washing with 1% acetic acid and dye extraction in 10 mM Tris (pH 10.5), plates were read at A564nm. The A564nm of drug-treated cells was compared to carrier-only-treated cells. The treatments were done in triplicate and the experiments repeated three times. Statistical Analysis The significance of the reduction in worm burdens after AF treatment was determined by two-tailed Student t-test. Results Biochemical and Kinetic Analysis of Recombinant S. mansoni TGR Because it is a selenoprotein, it is not possible to directly express recombinant schistosome TGR in Escherichia coli. However, after we added a bacterial-type selenocysteine insertion sequence element to the open reading frame we successfully expressed TGR, using the strategy previously employed for production of mammalian TrxR [14,15]. The purified recombinant TGR demonstrated substantial activity with a broad range of substrates, combining the characteristic activities of mammalian TrxR, GR, and Grx. The specific activities were 10.2 U/mg with DTNB, 2.2 U/mg with Trx (coupled to insulin reduction), 7.2 U/mg with GSSG, and 9.9 U/mg in a Grx assay coupled to HED. In addition, TGR could also reduce sodium selenite, H2O2, tert-butyl hydroperoxide, alloxan, lipoic acid, and lipoamide. It could not, however, reduce dehydroascorbic acid or ubiquinone. Table 1 summarizes the kinetic parameters of the enzyme with these different substrates. Table 1 Kinetics of Recombinant S. mansoni TGR with Different Substrates The enzymatic properties of recombinant parasite TGR differed from those of mammalian TGR [26]; schistosome TGR had greater GSSG reduction activity relative to Trx reduction (GSSG:Trx ratio of 4:1), whereas native mouse TGR had the reverse proportions (1:2), and S. mansoni TGR had 6-fold higher Grx activity than mouse TGR (Table 2). The activity and kinetic constants of recombinant S. mansoni TGR were similar to those reported for mammalian TrxR [26] and for human GR (Table 2) [27,28], although its activity was lower than that of human GR. Table 2 Comparison of the Kinetic Properties of Human TrxR1, TGR, and GR to Recombinant S. mansoni TGR Inhibition Studies on Recombinant S. mansoni TGR In order to assess the potential for inhibition of TGR we screened several structurally diverse series of compounds (Figure S1) known to act as inhibitors of disulfide reductases from various organisms. Screening was performed with a TGR assay using DTNB, including a 15 min preincubation of recombinant enzyme, inhibitor, and NADPH before addition of substrate (Table 3). Compounds exhibiting >85% inhibition in the initial screen were analyzed further to determine IC50 values for GR, TrxR, and Grx activities of TGR using the appropriate assays with a 15 min preincubation (Table 4). In order to determine if compounds showed selective inhibition of TGR compared to human TrxR and GR, the IC50 values for TGR were compared to those of human TrxR and GR either reported in literature or determined here (Table 4). Table 3 Primary Screen of NADPH-Dependent Oxidoreductase Flavoprotein Inhibitors and Antischistosome Drugs as Inhibitors of S. mansoni TGR Table 4 IC50 Values of TGR Inhibitors with 15-Minute Preincubation Assays The gold complexes AF, aurothioglucose, and aurothiomalate, which are efficient inhibitors (i.e., with low K i/IC50 values) of mammalian TrxR [29,30], were found also to be efficient TGR inhibitors. AF was the most potent inhibitor of TGR, with IC50 values in the low nanomolar range. Aurothioglucose had a less inhibitory effect on the reduction of GSSG and Grx activity (HED-coupled assay) than on the reduction of DTNB, whereas the other two gold compounds inhibited to a similar extent all three principal activities of TGR (Table 4). This indicates that the Grx activity, presumably catalyzed by the N-terminal Grx domain of the enzyme [11], could be dissociated from the other activities of the enzyme, which should be catalyzed by the C-terminal GCUG motif. This interpretation is corroborated by the fact that a C-terminally His-tagged form of the enzyme lacking the Sec residue was previously found to lack TrxR or GR activity, while still supporting Grx activity [8]. Platinum drugs (e.g., RMA19 and RMA35) are also irreversible inhibitors of mammalian TrxR [31,32]. RMA35, but not RMA19, was here a potent inhibitor of TGR; it had the opposite effect with human TrxR [31], indicating that specific structural features can be exploited for the design of selective TGR inhibitors. The most active Mannich base, inhibitors of plasmodial and kinetoplastid flavoenzymes [33,34], was 3-DAP, with low micromolar IC50 values against TGR. The naphthoquinones juglone [35] and naphthazarin [36] and several of their derivatives are subversive substrates of human TrxR. Among the naphthoquinones, LS826 [37] behaved as a selective TGR inhibitor, with an IC50 of 8 μM, but as a poor inhibitor of the human enzymes. While the GR inhibitor M5 [38,39] had no effect on TGR, naphthazarin and its derivatives JD155 and JD159 [36] inhibited TGR, but not selectively. Tricyclic aromatics, which are inhibitors of Plasmodium falciparum growth and human GR [40,41], were inactive as inhibitors of TGR. Similar to the interactions of mammalian TrxR with several quinones [35], inhibitory compounds may function as both substrates and inhibitors (i.e., subversive substrates) of TGR. The interaction of schistosome TGR with naphthazarin was found to exhibit this type of effect, with naphthazarin both acting as substrate (Figure 2A) and leading to time-dependent inhibition (Figure 2B and 2C). Figure 2 Naphthazarin Is a Substrate and Inhibitor of TGR (A) Activity of recombinant S. mansoni TGR with naphthazarin as a substrate was determined by NADPH consumption under steady-state conditions. (B) Time-dependent inhibition of TrxR activity of TGR by naphthazarin with DTNB as a substrate. Concentrations of naphthazarin are 0.5 μM (♦), 1 μM (█), 2.5 μM (▴), 5 μM (×), and 10 μM (+). (C) Time-dependent inhibition of TGR activity with GSSG. Concentrations of naphthazarin are 0.5 μM (♦), 1 μM (█), 2.5 μM (▴), 5 μM (×), and 10 μM (+). We examined three antischistosomal drugs [42,43], praziquantel, OPZ, and PAT. Both PAT and OPZ were here efficient, noncompetitive inhibitors of TGR (Figure 3); the K i values (± standard deviation) for PAT were 4.86 ± 0.46 nM (TrxR activity) or 7.22 ± 4.96 nM (GR activity), and for OPZ were 18.1 ± 1.1 μM (TrxR activity) or 33.8 ± 6.0 μM (GR activity). The widely used antischistosomal drug praziquantel displayed no inhibitory activity against TGR. Figure 3 Inhibition of S. mansoni TGR by PAT and OPZ Reciprocal plots showing inhibition of the TrxR and GR activities of TGR by PAT (A) and OPZ (B). Both are noncompetitive inhibitors, altering the Vmax while the K m for substrates remains constant. The apparent Vmax for various concentrations of inhibitors were used to calculate K i values. Concentrations of PAT are no drug (♦), 10 nM (), 30 nM (▴), and 75 nM (×); and of OPZ are no drug (♦), 50 μM (), 75 μM (▴), and 100 μM (×). Concentrations of DTNB ranged from 50 to 1,000 μM (TrxR activity) and of GSSG from 10 to 100 μM (GR activity). Inactivation of S. mansoni TGR by OPZ followed pseudo-first order reaction kinetics. A semi-logarithmic plot of the fraction of noninhibited enzyme activity ln(v i/v 0) versus incubation time yielded linear curves with increasing slopes, equivalent to the apparent rate constant of irreversible inhibition (k obs) in GR (Figure 4A) and in TrxR (Figure 4C) assays. The k obs values determined for S. mansoni TGR inactivation by OPZ ranged from 16.3 × 10−3 min−1 to 355.5 × 10−3 min−1 for GR activity and from 11.0 × 10−3 min−1 to 58.9 × 10−3 min−1 for TrxR activity, within the log-linear range of the inhibition curve. When the derivation described by Kitz and Wilson [44] for irreversible inactivation was applied to the experimental data, only at low inhibitor concentration, the secondary plot expressing k obs as a function of inhibitor concentration followed Equation 2: where K I represents the dissociation constant of the inhibitor, and k i is the first-order rate constant for irreversible inactivation. In both secondary plots (Figure 4B and 4D) showing inactivation of S. mansoni TGR at low OPZ concentrations, a rough estimation of k i, the resulting half-time value (t 1/2), and K I, were 0.04 min−1, 17.3 min, and 32.8 μM in the GR assay, and 0.06 min−1, 11.5 min, 140 μM in the TrxR assay, respectively. The resulting second-order rate constant values of ki /K I were 7.1 M−1 s−1 and 20.3 M−1 s−1 for inactivation of, respectively, GR and TrxR activities by OPZ. At higher inhibitor concentrations, the curves were not hyperbolic, but became polynomial, suggesting that more than one binding site of OPZ is involved in the presence of reacted and unreacted enzymes species in mixture. A double reciprocal replot of k obs versus [I] did not fit to a linear relationship (unpublished data). In close accord with observations made in recent studies on the inactivation of human glutathione reductase [45] or of human thioredoxin reductase [31], the inactivation of S. mansoni TGR by OPZ likely involves distinct enzyme populations, i.e., the enzyme species reduced at the flavin for inhibitor reduction, or the major two-electron reduced form with a dithiol in the active site(s) for alkylation. Although covalent inactivation of the enzyme was observed in both GR and TrxR assays, the kinetics of inactivation indicated that OPZ interacts in a complex way with the multifunctional enzyme S. mansoni TGR. Figure 4 Time-Dependent Inactivation of S. mansoni TGR by OPZ The time dependency of inactivation of S. mansoni TGR (15 pmol/μl) was revealed by determining the residual GR (A) and TrxR (C) activities of enzyme in the presence of 100 μM NADPH and inhibitor at 0, 2, 4, 10, and 30 min incubation periods. OPZ concentrations were 0 (•), 25 (□), 50 (♦), 75 (Δ), and 100 (▴) μM. All incubation mixtures included 2% final DMSO. The k obs data as a function of inhibitor concentration from the GR (B) or TrxR assay (D) versus OPZ concentrations followed a polynomial equation (not shown), suggesting that more than one binding site of OPZ is involved in the presence of different enzyme species of reacted and unreacted enzymes in mixture during the course of the inactivation process. The error bars in (B) and (D) represent ± the standard errors of the k obs values estimated separately at each inhibitor concentration, as obtained from nonlinear regression analysis. Inhibitor Studies on Cultured Worms Because of the potent effect of AF on recombinant S. mansoni TGR activity, we also analyzed its effect on worm pairing (female worms are present in copula in the male gynocophoral canal, and worms will remain paired for several days in optimal axenic culture) and worm viability (as evidenced by muscle contraction and parasite movement). Incubating worms with 10 μM AF resulted in unpairing of male and female worms after 1 h, 83% mortality after 6 h, and 100% mortality after 9 h of treatment; control worms remained paired and active throughout the treatment. When compared to control worms, TrxR and GR activities of TGR in worm homogenates were nearly 100% inhibited after 1 h of treatment with 10 μM AF (Figure 5A). In contrast, the activities of control enzymes, LDH and GPx, showed no significant deviation from controls (Figure 5A). The ratio of GSH:GSSG was also determined at each time point. The control worms displayed a relatively constant ratio of ~18:1, while in AF-treated worms a gradual oxidation of glutathione was detected as early as 1 h of AF treatment. After 6 h of treatment the GSH:GSSG ratio had decreased by 85% to 2.6:1 (Figure 5B). Figure 5 The Activity of Auranofin against Cultured Schistosoma mansoni Each point is the average of three independent experiments ± the standard deviation. (A) Specific enzyme activities of TrxR, GR, LDH, and GPx in worm homogenates from control worms (solid lines) and worms treated with 10 μM AF (dashed lines). (B) Ratio of GSH:GSSG in worm homogenate from control worms (solid line) and worms treated with 10 μM AF (dashed line). Each assay was done in triplicate and each experiment was done three times. The error bars show the standard deviation of the three replicate experiments. Likewise, larval, skin stage parasites, and juvenile liver stage parasites (unpublished data) are killed in less than 10 h by AF at concentrations as low as 5 μM (Figure 6A). For comparison, a mammalian cell line tolerated AF at concentration as high as 100 μM for 5 d, while 5 μM AF led to adult worm death in 24 h (Figure 6B). A similarly large differential in toxicity between adult worms and mammalian cells for PAT was also found (Figure 6C). Figure 6 Survival of Schistosomula, Adult Worms, and Myeloma Cells in the Presence of Auranofin or Potassium Antimonyl Tartrate (A) Cultured schistosomula treated with 0 μM AF (dashed line); AF 0.5 μM (♦); 1 μM (*); 2 μM (x); 5 μM (▴); 10 μM (). (B) Survival of adult Schistosoma mansoni worms at 24 h (dashed line), myeloma cells at 24 h (); and myeloma cells at 5 d (♦) at given concentrations of AF. (C) As in (B) in the presence of given concentrations of PAT. Each treatment was done in triplicate and each experiment was done three times. Error bars show the standard deviation of the three replicate experiments. In Vivo Inhibition Studies To further investigate the potential of TGR as a drug target, we analyzed the possible effects of AF treatment on the survival of worms in mammalian hosts. In preliminary studies, mice infected with S. mansoni were administered 6 mg/kg AF twice daily for 9 d, which is a safe dose for healthy mice [46], beginning 7 wk after infection (when adult worms are present in the mesenteric venules) followed by a 1 wk rest. Mice were then perfused and worms were collected and counted. In two independent experiments utilizing different mouse strains, the AF-treated mice had a 59% and 63% decreases in worm burden compared to control mice (Table 5). Table 5 Effect of AF Treatment in Mice Experimentally Infected with S. mansoni RNAi Silencing of TGR In order to further analyze the importance of TGR for worm survival, we incubated schistosomula (larval parasites) with double-stranded TGR RNA to silence TGR expression. After 2 d of TGR dsRNA treatment, TGR activity (using DTNB as substrate) was reduced by 35% in parasites in aerobic culture (20% O2) and after 3 d of TGR dsRNA treatment, TGR activity was reduced by 61% or 63.5% in parasites cultured anaerobically or aerobically, respectively. A marked decrease in TGR mRNA after 3 d of treatment was also seen (Figure 7A). The RNAi silencing of TGR led to substantial decreases in parasite survival; approximately 92% of dsRNA-treated parasites were dead after 4 d of treatment both in aerobic and anaerobic conditions (Figure 7B). Control parasite survival was over 95% in both aerobic and anaerobic growth conditions. After 3 d, TGR dsRNA-treated parasites had darker bodies with internal vacuoles and did not move, while all irrelevant dsRNA-treated parasites showed contractile movement and had clear bodies (Figures 8 and Figure S2). Treatment of cultured parasites with sublethal levels of AF and TGR dsRNA showed an additive effect significantly accelerating the killing of schistosomula compared to RNAi or AF treatments alone (Figure 7C). Schistosomula treated with both TGR dsRNA and 2 μM AF had 49.8% ± 2.8 % survival after 1 d compared to 91.3% ± 1.1% for RNAi alone and 93.7% ± 1.1% for 2 μM AF alone (p < 0.001), and after 2 d survival in the combination treatment was 8.9% ± 1.5% compared to 66.1% ± 1.4% for 2 μM AF alone and 76.1% ± 3.2% for RNAi alone (p < 0.001), with similarly significant results over the remainder of the 4 d time course. Praziquantel had no effect alone or in combination with RNAi. Figure 7 Survival of Schistosomula after RNAi Silencing of TGR Expression (A) Qualitative measure of TGR transcripts by reverse transcription PCR. The abundance of TGR mRNA was greatly reduced by dsRNA treatment while the control gene GAPDH was unaffected. (B) Parasites were cultured in the presence of double-stranded TGR RNA (dashed lines) or of double-stranded irrelevant RNA (solid lines) in 20% O2 (♦) or 0% O2 (). A 500-nucleotide fragment (bp 1364–1866) of S. mansoni TGR cloned into PCRII-TOPO vector was used to transcribe TGR dsRNA. Irrelevant, nonschistosome dsRNA used for negative controls was synthesized from the PCRII-TOPO vector using T7 and SP6 RNA polymerases. (C) Combination of RNAi and drug treatments. Solid lines are irrelevant dsRNA treatments and dashed lines are dsTGR treatments; no additions (♦), 2 μM praziquantel () and 2 μM AF (▴). At each time point, data comparing the combination treatment (2 μM AF + TGR dsRNA) to either 2 μM AF or TGR dsRNA alone were statistically significant (*p < 0.004). In each experiment for each time point at least 1,500 parasites were scored as alive or dead (three replicates of 500 parasites). The error bars represent the standard deviations of three independent experiments. Figure 8 Photomicrographs (100×) of Irrelevant dsRNA-Treated Schistosomula (left image) and TGR dsRNA-Treated Schistosomula (right image) after Three Days of Treatment All organisms in the left image are alive; parasites have different shapes, elongated, contracted, and curved during movement. In the right image, all of the parasites are dead and have roughly the same shape (no movement) and internal vacuoles (arrows). The bar represents 250 μm. Discussion In this study we have demonstrated that TGR is an essential protein for the survival of S. mansoni and that it meets all the major criteria of an important target for antischistosomal chemotherapy development. Silencing of TGR expression by RNAi lead to rapid parasite death, and auranofin, a specific chemical inhibitor of TGR, provides partial parasitological cures of infected mice. We have screened a number of TGR inhibitors and identified potential lead compounds for novel drug development. Furthermore, we demonstrated that TGR was likely a key target of some earlier therapies. It has been suggested that antioxidants play an important role in protecting adult S. mansoni worms from immune attack by the host [47]. Recent studies indicate that the enzymatic antioxidant pathway in S. mansoni is uniquely dependent on TGR [11]. In the parasite, TGR completely replaces more specialized TrxR and GR orthologs and, therefore, TGR functions in reducing both Trx and GSSG. This may lead to a bottleneck effect that should make TGR an attractive drug target. The results presented here strengthen this hypothesis. Recombinant S. mansoni TGR was here shown to possess substantial TrxR, GR, and Grx activity, with rates comparable to those reported for native TGR purified from mouse testis [26] and more than 40-fold more active than recombinant mouse TGR [48]. When S. mansoni TGR was first cloned, we proposed that the enzyme was a selenoprotein with a penultimate Sec residue, and that TGR should have enzymatic activity dependent on the C-terminal Sec-containing motif in analogy to mammalian TrxR and TGR isoforms [11]. This study, in which the enzyme was expressed as a selenoprotein, showed that hypothesis to be correct. It should be noted, however, that the Grx activity of the enzyme was sustained without the C-terminal motif, as shown here and previously [11]. Characterization of the parasite TGR revealed enzymatic properties that differed from those of mammalian TGR, TrxR, and GR. TGR was also identified as a multifunctional oxidoreductase with a remarkably wide substrate specificity, capable of directly reducing peroxides, selenium-containing compounds, and several important low-molecular-weight antioxidants, as well as Trx, DTNB, GSSG, and GSH-HED. However, TGR was unable to reduce dehydroascorbic acid or ubiquinone, compounds that are reduced by mammalian TrxR1 [49,50]. These findings show that TGR is likely to serve multiple functions in the parasite and is functionally distinct from the TrxR and GR orthologs of the human host. These substrate preferences might possibly be exploited for future TGR-directed anti-schistosome drug design. Several additional differences exist between schistosome TGR and mammalian TGR, TrxR1, TrxR2, or GR. In mammals, TGR is thought to serve a highly specialized function with expression restricted primarily to the testis [26,51]. Both TrxR1 and TrxR2 isoenzymes have a nearly ubiquitous distribution in mammalian tissues; TrxR1 is predominantly a cytoplasmic enzyme [52], while TrxR2 is mitochondrial [53]. GR is also widely distributed and is believed to be primarily cytoplasmic, although at least in yeast an alternatively translated isoform is targeted to the mitochondria [54]. For Schistosoma spp. it seems clear that TGR accounts for the complete combined Trx and GSSG reduction in the parasite. Moreover, schistosome TGR mRNA is alternatively spliced, producing both cytoplasmic and mitochondrial forms of the enzyme (LLC and DLW, unpublished data). Therefore, it appears that schistosome TGR fulfills all of the major functions of the mammalian TGR, TrxR1, TrxR2, and GR orthologs, emphasizing the importance of the parasite protein, and this alone should make it an attractive target for treatment of schistosomiasis. Because redox metabolism in other medically important parasitic platyhelminths is thought to be similar to S. mansoni [55], targeted inhibition of parasite TGR orthologs may also be a useful strategy to develop drugs against other parasitic infections. The previously used antischistosome drugs PAT and OPZ inhibited recombinant schistosome TGR, suggesting that the enzyme may already have served as a target protein for antischistosomal therapy. The dithiolethione compound OPZ reached Phase III clinical trials for schistosomiasis treatment [42] before it was withdrawn because of adverse side affects [56]. Although the mechanism of action of OPZ is not well defined, it has been reported to decrease GSH levels in adult S. mansoni worms [57]. Previous studies have shown that OPZ requires metabolite bioactivation both to display potent schistosomicidal activity and inhibition of the GSSG reduction activity [58]. The metabolism of OPZ in humans was investigated and the structures of active metabolites were identified (Figure 9). Metabolism of OPZ results in the production of a minor oxo analog, and the major dimethylated pyrrolopyrazine through the biological methylation of the intermediate pyrrolopyrazine thione. Two general hypotheses have been proposed to explain the mechanisms of OPZ activation [59]. The first suggests that OPZ acts through a bioactive metabolite following reaction with cellular thiols. The generation of the major methylated pyrrolopyrazine thione, an electrophilic intermediate species (structure 4, Figure 9), would lead to alkylation of protein thiols. While the disulfide bond of dithiolethiones is more difficult to reduce than a linear disulfide bond because of its aromatic character, the same pyrrolopyrazines can be obtained both by chemical (use of thiols like GSH) and electrochemical methods from OPZ. The greater reactivity of dithiols compared to monothiols with respect to reactions with dithiolethiones was determined to be due to an intrinsic enhanced reactivity of specific biological dithiol targets [60]. The second hypothesis also involves redox reactions from the major methylated pyrrolopyrazine thione (structure 4, Figure 9) in the presence of oxygen, contributing to a flux of reactive oxygen species [61,62]. Some disulfide intermediates (structure 5, Figure 9) displaying high schistosomicidal activity were shown to act as prodrugs of the pyrrolopyrazine thione (structure 4, Figure 9) [58]. Figure 9 Metabolism of Oltipraz Metabolism of OPZ results in the production of a minor oxo analog (2), and the major dimethylated pyrrolopyrazine (3) through the biological methylation of the intermediate pyrrolopyrazine thione (4). The action mechanism of OPZ against S. mansoni was also extensively investigated. The schistosomicidal activity of OPZ was correlated with glutathione depletion and a 2-fold lower GSSG reduction activity in S. mansoni, which was assigned to the action of a metabolite (having the general structure 5, Figure 9) rather than to OPZ itself [58]. Initially, GR was proposed to be responsible for GSSG reduction activity in S. mansoni and thus to be the target of OPZ. However, because there is no classical GR in this parasite, we checked the activity of OPZ as inhibitor of S. mansoni TGR and/or the prodrug of the species involved in decreased S. mansoni TGR activity in worms. Our results suggest that the antischistosomal effects of OPZ may have at least in part been through inhibition of schistosome TGR and that OPZ should be revisited to improve its toxicological profile. The first chemotherapy used for schistosomiasis was PAT, a trivalent antimonial that was administered to patients infected with S. haematobium beginning in 1917 [63]. PAT and other trivalent antimonials dominated chemotherapy for schistosomiasis until the introduction of praziquantel in the 1980s. Earlier studies suggested that the antischistosome action of trivalent antimonials is due to the selective inhibition of the parasite glycolytic enzyme phosphofructokinase (PFK) [64,65]. As found here, PAT is a noncompetitive TGR inhibitor with a K i value in the low nanomolar range, showing that PAT is by three orders of magnitude a better inhibitor of TGR than of PFK, which has an IC50 of 16 μM [65]. Combined with our other results, this suggests that the chemotherapeutic action of PAT is more likely to occur through inhibition of parasite TGR and than of PFK, or that inhibition of both enzymes provides a synergistic antiparasitic affect. We are currently carrying out investigations to test this hypothesis. Gold compounds may inhibit selenoproteins by targeting the Sec residue, and mammalian TrxR is a selenoprotein that is especially sensitive to such inhibition [29]. Auranofin, aurothiomalate, and aurothioglucose have all been used clinically for the treatment of rheumatoid arthritis [66]. Here we found that gold compounds are also potent inhibitors of schistosome TGR. It should also be noted that, although schistosomes express a GPx with a catalytically active Sec residue [67], GPx activity was not inhibited by AF (Figure 5). Furthermore, the inhibition of TGR by AF in cultured worms greatly reduced the GSH:GSSG ratio and resulted in rapid worm death. These results on cultured worms support the conclusion that TGR should be a prime target for antischistosomal drug therapy. The significant decrease in worm burden in infected mice treated with AF also supports this notion. The concentration of AF used to achieve this affect was well tolerated by the mice in this and previous studies [46], and it was reported previously that mammalian cells have over 95% viability when cultured in 10 μM AF [68]. The reasons for a generally low toxicity toward mammalian cells by AF are not known but could involve low uptake or efficient metabolic defense systems. While mammalian cells and the infected mouse hosts obviously tolerated the dosage of AF used in our studies, the results presented here show that larval, juvenile, and adult parasites are extremely sensitive to killing by AF. It should be noted that the toxic effects of AF occurred in the absence of exogenous oxidative stress (e.g., no added H2O2 or other oxidant) on the worms. Partial parasite clearance with AF is not an invalidation of S. mansoni TGR as an essential protein; factors such as the low in vivo bioavailability of AF might be the cause of the incomplete worm clearance. It is also important to emphasize that although the development of a schistosomiasis drug leading to partial cures is not a preferred goal, there are a number of factors associated with schistosome infections to consider. For example, unlike bacterial or protozoal infections, which require 100% cure rates or risk relapse, reduction of worm burdens by 50% would lead to significant decreases in pathology and morbidity associated with schistosomiasis, and no rebound in pathology would occur because adult worms do not multiply in their host. Furthermore, because praziquantel is highly effective and has low toxicity, it is essential to identify new drugs that can be used in combination with it to prevent parasites from developing resistance. Combination therapies may be useful to prevent praziquantel resistance even if only partial worm reductions were affected by the new drug alone. The effectiveness of artemisinin derivatives in combination therapy with praziquantel against schistosomiasis has been demonstrated [3], but concerns remain that the use of artemisinins for multidrug-resistant malaria treatment not be compromised. Our results indicate that praziquantel does not inhibit TGR; therefore, it is reasonable to assume that drugs targeting TGR will have a different mechanism of action than praziquantel. Unlike the more selective action of praziquantel against larval and adult parasites [69,70] and artemisinin against juvenile parasites [71], respectively, inhibition of TGR led to both larval and adult parasite death. Development of drugs targeting parasite TGR may thus provide the opportunity for prophylactic therapies. We propose that the screen for inhibitors of TGR as initiated here be continued and that it may lead to the development of novel drugs with potent antischistosomal effects. In this context it should be noted that the kinetics of inhibition of TGR may be rather complex, as discussed above for OPZ. Further studies are therefore needed with the different inhibitors already identified in the present study for a characterization of their interactions with TGR. With TGR being a probable important drug target for treatment of schistosomiasis, is it an essential protein for the parasite? The RNAi treatments silencing TGR expression that led to significant decreases in parasite survival indicate that it is. The TGR dsRNA treatment led to a significant decrease in both TGR mRNA and enzymatic activity, while treatment with irrelevant dsRNA had no effect. Genome sequence analysis, although not complete, represents ~95% coverage of the S. mansoni genome. Analysis of the genome sequence indicates that TGR is a single-copy gene (unpublished data) and no other activity is present in worms for Trx and GSH reduction [11]. Parasite death, which occurred in either aerobic or anaerobic conditions, was presumably due to pleiotropic effects of silencing TGR. Furthermore, combined treatment of schistosomula with TGR dsRNA and AF showed significant synergistic effects, suggesting that both act by decreasing TGR activity. Identification of the exact mechanism for the lethal effect of silencing the enzyme requires further experiments, as do the effects of inhibitors, which are not necessarily solely due to loss of enzyme activity, because the inhibited enzyme may also have a gain of function leading to cell death [72]. To conclude, in this report we have provided the first, to our knowledge, comprehensive biochemical analysis of a TGR selenoprotein from any organism, showing that it has remarkably wide substrate specificity and may be effectively inhibited by a number of low-molecular-weight compounds. Furthermore, using both inhibitors and RNA silencing we have shown that TGR is an essential schistosome protein, and we believe that our results have validated it as a key drug target for treatment of schistosomiasis. Supporting Information Alternative Language Abstract S1 Translation of the Abstract into Arabic by Ahmed A. Sayed (30 KB DOC) Click here for additional data file. Alternative Language Abstract S2 Translation of the Abstract into French by Jean Dessolin (29 KB DOC) Click here for additional data file. Figure S1 Chemical Structures of Compounds Used in This Study as Inhibitors of TGR of S. mansoni (107 KB PDF) Click here for additional data file. Figure S2 Photomicrographs (100×) of Irrelevant dsRNA-Treated Schistosomula and TGR dsRNA-Treated Schistosomula after Three Days of Treatment Schistosomula treated with irrelevant dsRNA are shown in the image on the left; those treated with TGR dsRNA are on the right. All organisms in the left image are alive; parasites have different shapes, elongated, contracted, and curved during movement. In the right image, all of the parasites are dead and have roughly the same shape (no movement) and internal vacuoles (arrows). The bar represents 330 μm. (7.5 MB EPS) Click here for additional data file.

0 comments Cited 96 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

Drug Discovery for Schistosomiasis: Hit and Lead Compounds Identified in a Library of Known Drugs by Medium-Throughput Phenotypic Screening

Maha-Hamadien Abdulla, Debbie S Ruelas, Brian Wolff … (2009)

Introduction Treatment and control of the flatworm disease, schistosomiasis, relies on a single drug, praziquantel (PZQ). Since the first clinical trials in the late 1970's [1], PZQ has proven safe and effective against all three major forms of the disease, and today, declining costs make the drug more affordable, currently at around 7–19 US cents per 600 mg tablet [2]. A single oral dose of 40–60 mg/kg is sufficient to achieve cure rates of 60–90% [3] while facilitating patient compliance, especially among children. Clinically relevant and widespread resistance, despite occasional and isolated incidences [4], has yet to occur. This fortuitous situation stands in contrast to the situation for some other ‘neglected tropical diseases’, (NTDs; [5]) for which antiquated and often toxic drugs must be parenterally administered over a number of days or weeks and which increasingly have problems associated with drug resistance [6]. Thankfully, concerted pharmaceutical discovery efforts via ‘public-private partnership’ (PPP) consortia [7],[8] are ongoing to address this desperate situation and robust ‘drug pipelines’ have been established which, hopefully, should yield new therapies over the next ten to 15 years. All of this recent activity has bypassed schistosomiasis, due in part to the tremendous success of PZQ. Yet, reliance on a single drug to treat a population of over 200 million people infected and over 700 million people at risk over three continents [9] seems particularly perilous when considering the threat of drug resistance. Also, PZQ is not without problems. Principal among these is its relative inactivity against migratory juvenile and sub-adult worms [10],[11] meaning that, for effective treatment and sustainable control, PZQ must be given on a regular basis. Thus, recent discussions, as part of treatment landscape for human helminthiases in general [12], have focused on reawakening the need to search for alternatives to PZQ, including the development of combinations of drugs incorporating PZQ [13],[14]. The latter option, if more difficult and costly to develop, has the longer term benefit of extending the availability of PZQ while hindering the onset of resistance to this most valuable of drugs. Only to a limited extent has the underlying rationale for inquiry of anti-schistosomal compounds (and anthelmintics in general) involved detailed knowledge of the molecular drug target or mechanism of action although there are some notable advances, e.g., inhibition of redox [15] and proteolytic enzymes [16], and heme aggregation [17]. The relative lack of validated molecular drug targets for this parasite is in stark contrast to those underpinning entire drug development portfolios of PPPs tackling other infectious diseases of global import such as malaria and the trypanosomiases [7],[8]. Hopefully, this paucity of targets can be better addressed with the recent availability of the draft genomes of both Schistosoma mansoni [18] and S. japonicum, and the first attempts to prioritize those targets ([19]; TDR Drug Targets Prioritization Database [20]). More common in schistosome drug discovery has been the complementary approach of phenotypic (whole organism) screening in vitro (usually with adult worms) and/or animal models of disease to measure compound efficacy [21]. These strategies are usually without specific knowledge of the target and/or mechanism of action (e.g., [22],[23]), or for which bioactivity has been characterized in other parasitological or biomedical settings [24],[25],[26]. They are of proven value. For example, PZQ was first developed as a veterinary cestocide before being tested in an animal model of schistosomiasis [27] and long before data regarding its mechanism of action was gathered. However, the pace of discovery with these techniques is somewhat slow, relying on a small number of research groups expert in handling the complex schistosome life cycle and working with both finite yields of parasite (adult parasites must be harvested from mammalian hosts) and long screen timelines (it takes approximately 30 days for S. mansoni infections to become patent in the mouse model [28]). Here, we have taken an alternative approach to phenotypic screening by designing a three-component screen workflow built upon juvenile parasites (schistosomula) that are easily obtainable from the vector snails and in far greater numbers than adult parasites. The screen is formatted to 96-well microtiter plates thus providing increased throughput and improved interfacing with similarly formatted small molecule libraries maintained in-house at the UCSF Small Molecule Discovery Center (SMDC; http://smdc.ucsf.edu/). Adult parasite screens in vitro form the second component of the workflow that is completed with compound efficacy tests in a murine model of patent schistosomiasis. Two GO/NO GO filter points are strategically placed in the screen workflow to prioritize compounds more likely to meet the target product profile (TPP) for treatment of schistosomiasis and its demand for short course oral chemotherapy [29]. As constructed, the entire process is intended to streamline and accelerate the identification of hit compounds and chemistries in vitro, and leads in vivo. The screen workflow was inaugurated using a library of commercially available and chemically diverse compounds. Approximately 41% of the library comprises drugs already approved for human use thereby opening the possibility for repositioning (re-profiling or re-purposing) [30] chemical entities as novel anti-schistosomals. The same collections have already provided a number of leads against other parasites [31],[32],[33]. Drug repositioning offers shortened development timelines and decreased risk with compounds having already passed regulatory clinical trials with full toxicological and pharmacokinetic profiles [7],[30]. All of this adds to up to significant potential cost savings –important in the context of diseases afflicting the poor for which investment returns will be marginal. The results accrued from the inaugural screen are promising in that a number of potent anti-schistosomal single compounds and chemical classes have been identified in vitro, some of which elicit demonstrable anti-schistosomal effects in the murine model of disease. Importantly, these and future data arising from the screen workflow are made public via various online portals to allow those interested examine and mine the outputs and, hopefully, identify their own opportunities for NTD drug development. Methods Maintenance of the S. mansoni life cycle A Puerto Rican isolate is maintained in the laboratory using the intermediate snail host, Biomphalaria glabrata and the Golden Syrian hamster Mesocricetus auratus (5–6 weeks old; Simonsen labs) as the definitive host. Animals were maintained and experiments carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at UCSF. Infections with S. mansoni are initiated by subcutaneous injections of 800–1000 cercariae. At 6–7 weeks post-infection (p.i.), hamsters are euthanized with peritoneal (i.p.) injections of 50 mg/kg sodium pentobarbital and adult worms harvested by reverse perfusion of the hepatic portal system [34] in RPMI 1640 medium (Invitrogen, Carlsbad, CA). Preparation of schistosomula and adult parasites for the screen workflow Upon exposure to light, 50–100 snails that are patent with S. mansoni infection, are induced to shed cercariae into the surrounding water. Cercariae are cleaned and concentrated over a series of sieves using distilled water and allowed to stand on ice in a 50 mL polystyrene tube for 1 h. During this time, cercariae clump, settle to the bottom and stick to the inside surface of the tube. The water is poured off and replaced with 9 mL ice-cold ‘Incomplete’ Medium 169 ([35]; custom made at the UCSF Cell Culture Facility) that contains 1× penicillin-streptomycin solution. Cercariae are mechanically transformed into schistosomula by passing back and forth between two 10 mL syringes attached via a 22-gauge double-headed needle (adapted from [36]). After deposition into a 9 cm diameter Petri dish, cercarial heads are separated from tails by swirling in Incomplete Medium 169 and the lighter tails aspirated leaving the heads (schistosomula) settled in the center of the dish. Under sterile conditions, schistosomula are washed 3 times in Incomplete Medium 169 and allowed to settle over ice in a 1.5 mL microfuge tube. Parasites are kept on ice for up to 2 h prior to screening with compounds. As a note, Medium 169 is preferred over RPMI as a culture medium for schistosomula – worms survive with <10% mortality for up to 4 weeks whereas in RPMI, approximately 40–60% of the parasites die within 3 days with continued mortality out to two weeks (Ruelas and Caffrey, unpublished). Adult worms, perfused from hamsters, are washed 5 times in RPMI 1640 containing 1× penicillin-streptomycin solution and 10 µg/mL amphotericin B (both supplied by the UCSF cell culture facility). After 3 further washes in Incomplete Medium 169, parasites are maintained in ‘Complete’ Medium 169 (with the addition of 10% fetal bovine serum (FBS; HyClone, Logan, Utah) at 37°C and 5% CO2 for up to 24 h prior to screening with compounds. Compound storage and handling for the schistosomula component of the screen workflow The ‘Spectrum’ and ‘Killer’ compound collections, together comprising 2,160 (1,992 unique) compounds were purchased from Microsource Discovery Systems, Inc. (Gaylordsville, CT, USA; http://www.msdiscovery.com/). Information on both is available for download as .xls files from http://www.msdiscovery.com/spectrum.html and http://www.msdiscovery.com/killer.html, respectively. Together the library contains synthetic compounds, natural products and drugs of which 821 are FDA-approved [31]. The library is maintained as 1 and 5 mM stocks in 384-well plates and −80 C at the UCSF Small Molecule Discovery Center that is juxtaposed to the UCSF Sandler Center. For the first component of the screen workflow (see Figure 1 for schematic) involving primary screens of schistosomula, 96-well polystyrene dilution plates (Corning, MA) are prepared using a Matrix WellMate bulk dispenser and a Biomek FXp liquid handling system. To these plates, the FXp transfers 4 µL of 1 mM compound in neat DMSO from quadrants of the 384 well stock plates. Eighty compounds from each quadrant are transferred to each dilution plate leaving the outer two columns empty. The WellMate then dispenses 16 µL DMSO and 180 µL Incomplete Medium 169 to the dilution plates to yield 20 µM compound in 200 µL 10% DMSO. Finally, the FXp transfers 10 µL of diluted compounds to the 96-well screen plates followed by 180 µL of Complete Medium 169. Under sterile conditions, 10 µL of schistosomula (200–300 worms) maintained on ice are added manually so that the final concentrations of test compound and DMSO per well are 1 µM and 0.5%, respectively. The outer two columns (1 and 12) of each screen plate are kept empty for eventual manual addition of the anti-schistosomal compounds, PZQ and the cysteine protease inhibitor, K777 [16], each at 1 and 5 µM. Plates are maintained at 37°C in a 5% CO2 atmosphere. 10.1371/journal.pntd.0000478.g001 Figure 1 Workflow for phenotypic screening of S. mansoni. The workflow was prosecuted with the Microsource Discovery Inc.'s “Spectrum” and “Killer” collections that together comprise 2,160 (1,992 unique) compounds, including 821 drugs approved for use with humans (http://www.msdiscovery.com/). The goal was to interface this parasite with the 96-well plate-formatted small molecule libraries available at the UCSF Small Molecule Discovery Center (SMDC; http://smdc.ucsf.edu/) and common elsewhere, thereby accelerating throughput and facilitating screen automation. Schistosomula are placed at the apex of a three-component workflow that subsequently incorporates screens against adult parasites in vitro and finally an animal model of infection to measure in vivo efficacy. Times at which phenotypes were recorded in vitro are indicated in hours and days Two GO/NO GO workflow filters allow for prioritization of the ‘hit’ compounds in vitro. The numbers of ‘hits’ generated at various points in the workflow are indicated in bold typeface. Data arising from each of the three screening components are posted online as a flat file at The Sandler Center's ‘Low Hanging Fruit” website (http://pathology.ucsf.edu/mckerrow//fruit.html), and in a cross-searchable format, at the database maintained by Collaborative Drug Discovery (CDD Inc.; (http://www.collaborativedrug.com/). For smaller numbers of compounds, the workflow need not be hierarchically prosecuted, rather every compound can be screened against both schistosomula and adults. For confirmatory screens of schistosomula (Figure 1), consensus hits (details below) from the primary screen are ‘cherry picked’ using the Matrix WellMate and Biomek FXp. A custom protocol in Pipeline Pilot software (Accelrys, CA) generates an Excel file that is read by the FXp. This file is designed to randomly distribute hit compounds among wells containing only DMSO (‘dummy wells’) in the dilution plate. To start the liquid-handling procedure, the WellMate transfers 48 µL of neat DMSO into the inner 80 wells of 96-well polystyrene dilution plates. From 5 mM stocks in neat DMSO, the Span 8 arm on the FXp then transfers 2 µL of consensus hit compounds (or DMSO from a plate containing 100% DMSO) into the dilution plates. To complete the dilution plates, 150 µL of Incomplete Medium 169 are added to each well. From this dilution plate, 4 µL are transferred into the 96-well screen plates followed by 186 µL of Complete Medium 169. Under sterile conditions, 10 µL schistosomula (200–300) are then added manually to yield final concentrations of 1 µM and 0.5% for test compound and DMSO, respectively. Compound handling for the adult component of the screen workflow For the second component of the screen workflow involving screening of adult S. mansoni (Figure 1), the FXp Span 8 transfers 4 µL 5 mM hit compounds in neat DMSO into 96-well polystyrene dilution plates. The hits are distributed randomly in the first few rows of these plates, but with fewer dummy DMSO wells to accommodate the smaller 24-well screen plates. Then, 96 µL of neat DMSO are added to this plate using the WellMate bulk dispenser. From this, 10 µL of the diluted compounds are added manually to the 24-well screen plates and immediately mixed with 0.99 mL of Complete Medium 169 to prevent evaporation of DMSO. Under sterile conditions, adult worms (4–8 pairs) are manually added in 1 mL Complete Medium 169. Final concentrations of test compound and DMSO are 1 µM and 0.5%, respectively. Phenotype scoring and compound concentration Two screen analysts spent approximately four weeks testing the Microsource ‘Killer Collection’ with both schistosomula and adult parasites in order to familiarize themselves with the types of phenotypes arising and their changes as a function of time. Phenotypes were scored using a Zeiss Axiovert 40 C inverted microscope and ×10 and ×2.5 objective lens for schistosomula and adults, respectively. Screening analysts are blind to the compound identities which are not disclosed until the conclusion of each of the schistosomular and adult components of the workflow. As a further precaution against subjective bias, each screen analyst visually scores and characterizes phenotype ‘hits’ in isolation. Both analysts then compile ‘consensus hits’. In those cases for which a consensus cannot be reached the compounds in question are scheduled for re-screening. With repetition, the failure rate to identify consensus hits was decreased to less than 5% per plate for both the schistosomula and adult components of the workflow. It was also during the four week training period that a decision was reached on the compound concentration at which the Microsource collections would be screened. Initial testing of the Killer collection at 10 and 5 µM with schistosomula yielded too many hits (average of 25 and 15% of the 80 compounds per plate, respectively) to subsequently perform, in a reasonable time-frame, in vitro screening with the more limiting adult parasites. At 1 µM, however, an average 10% hit rate was achieved. For primary screens of schistosomula, phenotypes were monitored after 7 d a time frame considered long enough to record the development of any potentially relevant phenotype (Figure 1). For confirmatory screens with schistosomules, phenotypes were scored after 24 h and 7 days in order to identity fast-acting compounds and re-confirm the data from the primary screen, respectively. For adult screens, phenotypes were monitored after 7 and 24 h, and, thereafter, daily up to 4 days (Figure 1). GO/NO GO filters in the screen workflow Two GO/NO GO filters are positioned in the screen workflow in order to prioritize which compounds go forward (Figure 1) based upon the TPP for schistosomiasis treatment and its demand for short course oral therapy [29]. The first filter, placed between the schistosomular and adult components of the workflow, prioritizes compounds yielding phenotypes by 24 h and removes compounds (where data are available) that are clearly toxic and/or unsuitable for oral administration. The second GO/NO-GO filter, upon completion of the adult screen component, prioritizes those hit compounds for tests in the murine model of schistosomiasis mansoni. This prioritization is more complex than the first filter as a number of parameters must be simultaneously considered. Primary emphasis is placed on the time to appearance of the phenotype plus the severity of that phenotype, e.g., fast-acting ‘death’ phenotypes (<24 h) are most preferred. Other factors influencing the decision include clinical indication (if known) for the compound including undesirable side-effects (e.g., hormones disallowed; psychoactives less preferred); oral bioavailability (preferred over other routes of administration) and data on acute toxicity (e.g., LD50 oral (p.o.), i.p., and/or intra-venous (i.v.)). Finally, where compounds with similar chemistries are represented more than once, a single example is initially considered for tests in the mouse model. Murine model of schistosomiasis mansoni The third and final component of the S. mansoni screen workflow (Figure 1) entails infections of 4–6 week-old Swiss Webster mice (Simonsen Laboratories) with S. mansoni that are initiated by subcutaneous injection of 140 cercariae. Experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at UCSF. Groups of 4 or 5 mice are used per treatment. Commencing on day 42 p.i. when S. mansoni infections are patent (i.e., when parasite eggs are present in feces) compound is administered once daily (QD) and/or twice daily (BID) for 4 days. This time period is considered sufficient to record any compound efficacy given that the desired TPP for any new-anti-schistosomal calls for short course therapy [15],[29]. Compound is administered p.o. (vehicle is 2.5% Cremophor EL unless otherwise stated) or, i.p., when data on oral bioavailability are not to hand. The amount of compound to administer is guided by available LD50 values for acute toxicity, in which case compound is given close to that value in order to determine whether a therapeutic window exists. Overt toxicity of compounds (e.g., death and behavioral changes) is assessed daily during and after treatment until the time of euthanasia. At 55 days p.i., mice are euthanized with an i.p. injection of 0.05 mg/g sodium pentobarbital, and adult worms perfused as described above for hamsters. Compound efficacy in vivo is measured as described [16] using a number of criteria and is compared to that of the anti-schistosomal drug, PZQ, as a ‘gold standard.’ The criteria include the parasitological parameters of numbers of male and female worms recovered by perfusion, and hepatic egg burdens. Also, the amelioration of pathology as evidenced by decreased liver and spleen weights is recorded. Attention is also paid to worm size upon recovery. To recover eggs trapped in liver, whole livers (or the caudate liver lobe, see below) from individual mice are excised, weighed and digested in 0.7% porcine trypsin in PBS for 1 h at 37°C on an orbital shaker. Eggs are sedimented at 4°C and counted under a dissecting microscope as described previously [37]. Statistics Data for worm and hepatic egg counts, and organ weights, were compiled on a per mouse basis and median values calculated per treatment group. All data were subjected to the Mann-Whitney nonparametric test to determine any statistical differences in egg and worm burdens, and organ pathologies between treated and untreated control mice. As an expedient alternative in some experiments, particularly in those cases where worm burdens were not dramatically decreased, hepatic egg counts were calculated as an average per treatment group rather than per mouse. To do this, the caudate liver lobe from individual mice was excised, pooled per treatment group and weighed prior to trypsin digestion and counting of eggs. The single value arising was then calculated with respect to the total liver weight in the group and then divided by the number of mice in the group. Results Phenotype classification for schistosomula During initial testing of the Microsource ‘Killer Collection’, it became clear that schistosomula display different and often multiple phenotypes that change over time. Eventually, we could consistently ascribe six phenotypes to worms under chemical insult relative to control worms exposed to 0.5% DMSO (Figure 2; Table S1). The phenotype terms we employed range from the obvious (‘dead’, ‘overactive’, and ‘rounded’) to the more sublime (‘dark’, ‘slow’), yet, nonetheless, clearly distinguishable from DMSO controls. An example of the overactive phenotype is shown in Video S1 which should be compared with normal worm movement displayed in Video S2. The last phenotype, ‘degenerate but mobile’, describes those cases in which the worms are clearly motile yet severely disrupted in morphology. An example of this phenotype is produced by PZQ, used as a ‘gold standard’ schistosomicide throughout the screen workflow. PZQ initially elicited an overactive phenotype (observed within 10 mins) that progressed to a combination of ‘overactive/degenerate but mobile’ by 7 days (Figure 2C). In contrast, the cysteine protease inhibitor, K777 [16], also used as a standard compound, had a more progressive effect; “slow/dark” by 3 days leading to ‘dark/slow/dead’ by 7 days (Figure 2B). 10.1371/journal.pntd.0000478.g002 Figure 2 Examples of the different and multiple phenotypes manifest by schistosomula exposed to chemical insult. Worms were exposed for 7 days to 1 µM of either the schistosomicidal cysteine protease inhibitor, K777 (B), or the current chemotherapy, PZQ (C), and compared to controls with DMSO alone (A). Phenotypes ascribed are ‘dark/dead’ for K777 and ‘overactive/degenerated but mobile’ for PZQ. Images were captured using a Zeiss Axiovert 40 C inverted microscope (10× objective) and a Zeiss AxioCam MRc digital camera controlled by AxioVision 40 version 4.5.0.0 software. Scale bar = 0.2 mm. Phenotype classification for adult worms Similar to schistosomula, adult worms could manifest multiple and changing phenotypes in response to chemical insult (Figure 3; Table S2). There was some overlap in the phenotypes classified compared to schistosomula: ‘dead’, ‘dark’, ‘slow’ and ‘overactive’ remained relevant whereas ‘rounded’ and ‘degenerate but mobile’ did not. Additional, adult-specific phenotypes were: ‘tegumental blebbing’ (teg. bleb.) to document damage to the surface (tegument) of the adult; ‘sexes separated’ (sex sep.), whereby the male and female worms become unpaired, and the self-evident ‘shrunken.’ For example, PZQ, elicited ‘shrunken/dark/slow’ phenotypes (observed within 2 min of addition of 1 µM PZQ) that progressed to ‘shrunken/dark/sexes sep/teg. bleb/dead’, by 4 days (Figure 3C). In contrast, K777 had a more progressive effect; ‘slow/dark’ by 2 days leading to ‘dark/slow/sexes sep/on sides/dead’ by 4 days (Figure 3B). 10.1371/journal.pntd.0000478.g003 Figure 3 Examples of the different and multiple phenotypes manifest by adult S. mansoni exposed to chemical insult. Worms were exposed for 2 days to 1 µM of either K777 (B) or PZQ (C) and compared to controls with DMSO alone (A). Phenotypes ascribed are ‘slow/sex separated/on sides’ for K777 and ‘slow/dark/shrunken/on sides/sex separated/tegumental blebbing’ for PZQ. Arrows point to female worms. Images captured using a Zeiss 2000-C Stemi inverted microscope mounted over a Diagnostic Instruments Transmitted Light Base and a Zeiss AxioCam MRc digital camera controlled by AxioVision 40 version 4.5.0.0 software. Scale bar = 0.7 mm. Schistosomula primary and confirmatory screens Of the 1,992 unique compounds comprising the Microsource Spectrum and Killer collections, 118 yielded phenotypes (termed ‘hits’ representing 5.9% of the total) in the schistosomula primary screen component of the workflow after 7 days at a concentration of 1 µM. The compound names, structures, therapeutic uses and phenotypes identified are listed in Table S1. The majority of these (105) were returned as hits in the confirmatory schistosomula screen component after 7 days and, of these, 61 (3.1% of all the compounds screened), were fast-acting, i.e., phenotypes were recorded at the 24 h time-point (Table S1). When the Microsource Spectrum and Killer collections are broken down into their component drug classes (Table 1, Table S3), and using the 7 d confirmatory screen data, the greatest percentage of hits per class, as might be expected, was for the anthelmintics (29%). Within this group the known anti-schistosomals PZQ and hycanthone were identified, as were other anthelmintics such as niclosamide, bithionol and pyrvinium pamoate (Table S3). Examples of other drug classes returning percentage hits greater than 10% are the antibiotics, fungicides, antineoplastics, dopaminergics and seratonergics. 10.1371/journal.pntd.0000478.t001 Table 1 Phenotypic hits for S. mansoni schistosomula as a function of compound class. Compound Class (Number of compounds per class) Primary hits (after 7 days) Confirmatory hits (after 7 days) Confirmatory hits (after 24 hours) Adrenergic (40) 2 2 1 Analgesic (35) 0 0 0 Anesthetic (17) 1 0 1 Anthelmintic (24) 7 7 5 Antibiotic (178) 20 18 11 Antihistamine (14) 0 0 0 Antihyperlipidemic (16) 2 2 0 Antihypertensive (25) 1 0 1 Antiinflammatory (48) 1 1 1 Antineoplastic (77) 10 10 4 Antioxidant (12) 0 0 0 Antiprotozoal (31) 4 4 2 Antiviral (11) 1 1 0 Cholinergic (64) 4 3 3 Diuretic (18) 0 0 0 Dopaminergic (38) 7 6 5 Estrogen/progesterone (33) 2 1 1 Fungicide (29) 6 5 3 GABAergic (13) 0 0 0 Glucocorticoid (27) 0 0 0 Herbicide (31) 1 1 0 Insecticide (25) 3 2 1 Muscle relaxant (13) 1 1 1 Seratonergic (38) 7 6 6 Other (480) 11 10 4 Unknown (655) 27 25 11 TOTAL (1,992) 118 105 61 See Table S3 for details of the compounds assigned to each drug class. Upon completion of the first (schistosomular) component of the screen workflow and bearing in mind the target drug profile for schistosomiasis [29], the first of two GO/NO-GO filters was enacted (Figure 1; Table S1). Fast-acting compounds (phenotypes by 24 h) were prioritized and those clearly toxic or otherwise inappropriate for oral use were removed. Accordingly, all but four (phenylmercuric acetate, thimerosal, benzalkonium chloride and homidium bromide) of the 61 fast-acting compounds were prioritized for in vitro tests against adult parasites Apparent structure activity relationship (SAR) for tricyclic psychoactive drugs inducing an ‘overactive’ phenotype in schistosomula During the first component of the screen workflow it became clear that certain tricyclic psychoactive compounds within the Microsource collections, notably the phenothiazines and dibenzazepines, elicited a striking “overactive” phenotype that lasted for the 7 day duration of the experiment. To verify the result, a mini-screen incorporating the tricyclic psychoactives and structurally related compounds was set up over three logs of concentration; 1.0, 0.1 and 0.01 µM (Table S4). The overactive phenotype was confirmed at 1.0 µM for the same compounds and extended to 0.1 µM, but not 0.01 µM. Examination of structural-activity relationships (SAR) in the phenothiazine and dibenzazepine classes indicated the importance of an unsubstituted propyl side chain possessing a terminal dimethylamine function (Figure 4; Table S4). Even subtle alteration of this pharmacophore, such as the introduction of a branching methyl substituent, led to abrogation of the phenotype (compare promazine and trimeprazine in Table S4). More dramatic modification of the side chain (e.g., shortening, introduction of terminal piperazine or piperidine moieties) similarly abrogated the overactive phenotype. The trend held whether carbon was substituted for nitrogen at position 10 of the phenothiazine (or at position 11 of the dibenzazepine) or whether the tricyclic core was altered internally or substituted. 10.1371/journal.pntd.0000478.g004 Figure 4 SAR for the phenothiazine and dibenzazepine classes of psychoactive drugs eliciting the ‘overactive’ phenotype in schistosomula. See Table S4 for individual compounds and Supplementary Videos 1 and 2 to compare phenotypes between ‘overactive’ and control worms. Adult screen Of the 57 compounds passing the first GO/NO GO filter and pursued in the second (adult) component of the screen workflow, 30 were hits (1.5% of the 1,992 compound total) after the maximal screen time of 4 days at 1 µM (Table S2). Seventeen and 10 compounds generated phenotypes by the 7 h and 24 h time points, respectively. Included in this set was the antibiotic, anisomycin, and the anthelmintics PZQ (worms visibly contracted and shrank within seconds of adding 1 µM), niclosamide, pyrvinium pamoate and bithionol. Three compounds, including a former chemotherapy of schistosomiasis, hycanthone, generated phenotypes after 2 days. To prioritize candidates for tests in the murine model of schistosomiasis, a second GO/NO GO filter was implemented utilizing a greater number of considerations than for the first filter (Figure 1; Table S2). As for the first filter, due provision was made for the TPP for schistosomiasis therapy: fast-acting compounds (phenotypes within 7 and 24 h) were prioritized but with a preference now for those that generated the most severe phenotypes, e.g., “teg. bleb” and “dead”. Prioritizations were counter-balanced by available knowledge of compound efficacy, toxicity, and side-effects. Thus, bithionol was deprioritized due to its lack of efficacy in schistosomiasis patients [38] and celastrol due to its toxicity in mice (death within one day of a single 10 mg/kg i.p. dose (R. Swenerton, unpublished data). Likewise, rhodomyrtoxin B displays low LC (lethal concentration)50 values of between 2 and 20 µM against Hep-G2 (human hepatocellular carcinoma) and MDA-MB-231 (human mammary adenocarcinoma) cell lines [39] and was, therefore, not considered further. The tricyclic psychoactive compounds (e.g., chlorpromazine, imipramine) were also deprioritized at this stage given that the less severe ‘overactive’ phenotype that appeared by 2 h was either transient (lasting only until the 24 h time point) or without progression to a more severe phenotype(s). Also, their psychoactivity and associated side effects, e.g., sedation, detracted from their immediate consideration. In all, therefore, five compounds were prioritized for efficacy tests in the model of murine schistosomiasis: two antibiotics, anisomycin and lasalocid sodium; two natural products, diffractaic acid and gambogic acid; and the helminthicide/molluscicide, niclosamide (Figure 5A, Table S2). 10.1371/journal.pntd.0000478.g005 Figure 5 Structures of compounds prioritized for efficacy tests in the murine model of schistosomiasis mansoni. (A) Hit compounds arising from the in vitro components of the screen workflow and (B) analogs of niclosamide currently marketed as veterinary anthelmintics or as an intestinal anti-protozoal in humans (nitazoxanide) and tested in the murine model of schistosomiasis. Murine model For the third and final component of the screen workflow, all compounds were administered orally in 2.5% Cremophor EL (unless otherwise stated) either once (QD) and/or twice daily (BID) for 4 days to mice with patent S. mansoni infections (42 days p.i.). The antibiotic, anisomycin, at 100 mg/kg p.o. QD, had no significant effect on male or female worm burdens (Figure 6A), yet decreased hepatic egg burdens by 36% (calculated as an averaged single value for the treatment group). Neither liver nor spleen weights were significantly different from those of the untreated control group (Figure 6B and C). Increasing to a BID administration resulted in toxicity; all mice died between 3 and 10 days after the commencement of treatment. The ionophoric antibiotic, lasalocid sodium, was better tolerated by mice. Significant decreases in male (44%) and female (41%) worm counts were measured at 100 mg/kg QD and BID, respectively (Figure 6A). For egg burdens, reductions of 39 and 55% (calculated as averaged single values per treatment group) were measured QD and BID, respectively. Lasalocid sodium also significantly improved organ pathology compared to controls (Figure 6B and C). 10.1371/journal.pntd.0000478.g006 Figure 6 Effect of lasalocid sodium and anisomycin on male and female worm burdens, and organ pathology (as measured by weight) in mice infected with S. mansoni. Compounds were administered orally QD or BID at the doses indicated for 4 days 42 days after infection with 140 S. mansoni cercariae. Points represent data from individual treated or untreated (control) infected mice. The horizontal bars represent median values. Significance (p) values are indicated where p≤0.05. Egg burdens were calculated as single values per treatment group (see text for details). For the Usnea lichen metabolite, diffractaic acid poor solubility in aqueous media or vehicle prevented the ability to accurately gavage mice. Therefore, i.p. administration in 50 µL 100% DMSO over a range of doses (10, 40 and 100 mg/kg) was performed to determine compound efficacy while also observing for overt toxicity. No decrease in worm or egg burdens was measured at the lower doses, whereas at 100 mg/kg, all mice died within 7 days of the cessation of treatment (data not shown). Gambogic acid, a xanthone isolated from various species of the Garcinia tree, was recently shown to be non-toxic in rats after oral administration every other day for 13 weeks at 30 and 60 mg/kg in 2% carboxymethylcellulose-sodium [40]. Using the same vehicle at 100 mg/kg QD, no effects on worm or egg burdens were recorded (data not shown). The final compound, niclosamide (2′5-dichloro-4′-nitrosalicylanilide), a molluscicide and intestinal helminthicide, is poorly absorbed across the intestinal wall. Therefore, we obtained from Bayer a wettable powder formulation of the compound (marketed as Bayluscide WP 70) that is better absorbed (in rats about a third of an oral dose [41]). However, in tests with both niclosamide formulations at 100 mg/kg BID, no effects on worm or egg burdens were noted (data not shown). Further, niclosamide was ineffective at 100 mg/kg BID in two additional vehicles (2% Tween80/7% ethanol and 6% PEG 4000/2%Tween 80/7% ethanol). In a final attempt to demonstrate efficacy, i.p. administration of niclosamide at 100 mg/kg BID in 100 µL 25% DMSO was without effect – upon dissection of mice the compound was noted to adhere as a solid mass at the injection site on the inner side of peritoneal membrane, indicating that much of the compound had not been absorbed. Screening niclosamide analogs in the murine model Based on the strong in vitro efficacy measured for niclosamide against both schistosomula (Table S1) and adults (Table S2), and notwithstanding its lack of in vivo efficacy, we searched for structurally related compounds that are commercially available and have demonstrated oral efficacy against helminths or protozoa. Three salicylanilides, closantel, oxyclozanide, and rafoxanide, and the nitrothiazolyl-salicylamide, nitazoxanide (Figure 5B) were purchased. The salicylanilides are well-established drugs used in the agribusiness sector as helminthicides, including against liver fluke disease caused by Fasciola hepatica [42]. They have also displayed variable efficacies in experiments with farm animals harboring agriculturally important Indian schistosome species such as Schistosoma incognitum and Schistosoma nasale ([43],[44]). Nitazoxanide (marketed as Alinia) is approved for the treatment of diarrhea caused by Cryptosporidium parvum and Giardia lamblia and has shown efficacy against human fascioliasis hepatica [45]. Accordingly, we judged there to be sufficient precedent and data available to move these compounds straight into our mouse disease model. The oral efficacy of these drugs were compared to the ‘gold standard’ drug, PZQ. When administered at 100 mg/kg p.o. QD for 4 days commencing at 42 days p.i., PZQ significantly decreased male (91%) and female (87%) worm burdens (Figure 7A) and these were associated with a decreased hepatic egg load (60%; Figure 7B) and improved organ pathology (Figure 7C and D). The decrease in egg burden was not considered significant, however, due to the low load recorded for one of the control mice. The few worms surviving treatment and recovered by perfusion were the smallest seen in all of the in vivo experiments and some were physically damaged (not shown). By comparison, BID administration of the salicylanilides, closantel and oxyclozanide, at 100 mg/kg yielded less pronounced effects on worm burdens (only oxyclozanide significantly decreased female loads by 53%; Figure 7A) and egg burdens were not affected (Figure 7B). However, worms recovered after oxyclozanide treatment were smaller than controls (not shown) and organ pathology (significantly so for the spleen) was also improved (Figure 7C and D). The third salicylanilide tested, rafoxanide, at either 100 mg/kg QD or BID, caused mouse mortality within 5 days of the cessation of treatment, however, this seemed not to be due to systemic toxicity per se but rather an accretion of drug in the stomach that caused gastric blockage. At 50 mg/kg QD (i.e., half the dose of PZQ) all mice survived. The drug was the most effective of the niclosamide analogs tested significantly decreasing male (56%) and female (50%) worm loads (Figure 7A). Also, worms recovered were smaller than controls (not shown). Egg counts were decreased by 49%, but as noted for PZQ above, the value was not significant due to an outlier control mouse with a particularly low hepatic egg count. Rafoxanide was as effective as PZQ in improving organ pathology (Figure 7C and D). The final niclosamide analog tested, nitazoxanide, was without effect on worm burdens at 100 mg/kg QD and BID (Figure 8A) but significantly improved organ pathology BID (Figure 8B and C). Nitazoxanide also decreased egg outputs by 34% (calculated as a single averaged value per treatment group). 10.1371/journal.pntd.0000478.g007 Figure 7 Effect of closantel, oxyclozanide, rafoxanide and PZQ on male and female worm burdens, egg burdens and organ pathology in mice infected with S. mansoni. Compounds were administered orally for 4 days 42 days after infection with 140 S. mansoni cercariae. Doses administered were: 100 mg/kg BID for closantel and oxyclozanide, 50 mg/kg QD for rafoxanide and 100 mg/kg QD for PZQ. Points represent data from individual treated or untreated (control) infected mice. The horizontal bars represent median values. Significance (p) values are indicated where p≤0.05. 10.1371/journal.pntd.0000478.g008 Figure 8 Effect of nitazoxanide on male and female worm burdens, and organ pathology in mice infected with S. mansoni. Compound was administered orally at the doses indicated for 4 days 42 days after infection with 140 S. mansoni cercariae. Points represent data from individual treated or untreated (control) infected mice. The horizontal bars represent median values. Significance (p) values are indicated where p≤0.05. Egg burdens were calculated as single values per treatment group (see text for details). Discussion Compared to the high profile activity supporting the development of novel anti-protozoal and anti-infective therapies [7],[8] the pace of drug discovery for anti-schistosomals (and anthelmintics in general) is slow. For schistosomiasis, a number of mutually suppressive factors are responsible. Perhaps foremost is the success and clinical reliability of PZQ that have dampened investment in a dedicated drug development pipeline, a situation in stark contrast to malaria and protozoal NTDs for which drug toxicity and/or increasing drug resistance fuel a number of multinational PPP programs to identify new therapeutics [7]. Other contributing factors are; the relatively small number of groups involved in anti-schistosomal discovery, the need to maintain a complex life-cycle that generates finite parasite yields and the long identification and development time lines associated with phenotypic (whole organism) screens as traditionally prosecuted in animal models and/or in vitro with adult worms. Despite these drawbacks, phenotypic screening has successfully identified PZQ and other vital anthelmintics (e.g., albendazole and ivermectin) that are in medical use today. Most often, the compounds originated in the animal health sector as part of its discovery programs to identify veterinary anti-parasitics [46],[47]. For this report, we have designed a phenotypic screen process by introducing a three-component workflow (Figure 1) that places S. mansoni schistosomula at its apex. The intent is to streamline and accelerate the identification of anti-schistosomal compounds by interfacing the helminth with the microtiter plate (96- and/or 384-well) formatted compound libraries and associated robotic liquid handling systems now standard in industry and many academic institutions, and routinely employed to screen the more tractable protozoan parasites [7]. Given their small size (∼200×60 µm) schistosomula are readily adaptable to the 96-well plate format and survive for 7 days with less than 10% mortality under the conditions described. Also, they are quickly and easily transformed from the invasive cercariae that are harvestable in their tens of thousands on at least a weekly basis from vector snails. Both points are immediately attractive and conducive to designing a higher throughput screen workflow. The alternative adult parasite is too large for 96-well plate formatting and can only be harvested from vertebrate hosts (e.g., mice and hamsters) in more limiting numbers, and entails considerable expenditure associated with animal procurement and maintenance. That stated, adults are not omitted entirely from the screening process but are placed downstream of schistosomula when the number of compounds to be tested is more manageable – a consideration also of importance for the final component of the workflow involving the animal model of schistosomiasis. The Microsource collections of 2,160 compounds were prosecuted at a throughput of 640 compounds/month for the primary schistosomular screen component. With one full-time technician and an associate analyst it took 20 weeks to complete the in vitro screening of the collections against schistosomula and adults. Efforts to at least double the screening capacity of the first component of the screen workflow are being studied, for example, through the employment of additional staff and expansion of our in-house S. mansoni life-cycle. Also, screen formatting to 384-well plates is being considered together with the complete automation of both the liquid handling of the parasite and phenotype identification and categorization. Data accrued from each component of the screen workflow is available as a flat file online at the UCSF Sandler Center's ‘Low Hanging Fruit’ website http://www.sandler.ucsf.edu/fruit.html and at http://www.collaborativedrug.com/, a database that can be mined across compounds and parasites to identify molecules and chemistries of interest. Both sites are continually updated as screening campaigns are concluded and it is hoped that the data will contribute to drug discovery efforts for schistosomiasis and other NTDs. The descriptive approach employed here to annotating the dynamic responses of this metazoan parasite to chemical stimuli differs necessarily from the single end-point fluorometric or colorimetric assays, now routine for high-throughput assays of single-celled organisms and with which a rigorous quantification of a live versus death ratio is relatively facile [7]. Given the traditionally slower compound throughput for schistosome screening, the demand for marker dyes or reagent-based kits has simply not been present with visual-based scoring systems being the norm [21],[22],[23]. Our attempts to incorporate nuclear dyes (e.g., propidium iodide and DAPI) as a quantitative marker of cell death in schistosomula did not correlate with the clear deleterious action of some compounds observed under bright field microscopy (Caffrey, unpublished data). Often dyes were simply excluded from crossing the schistosome tegument regardless of worm condition. Thus, our decision to visually classify phenotypes, though potentially prone to subjectivity, turned out to be a consistent semi-quantitative approach as employed, i.e., using blind consensus determination of bioactivity by trained analysts familiar with the parasite's phenotypic manifestations (see discussion below). Further, it might be argued that the workflow, because of its simplicity, and without the need for expensive kits or reagents, is more adaptable to a greater variety of discovery settings. Nevertheless, we are aware that any attempt to improve the quantitative rigor of hit identification and classification should be a primary goal. Accordingly, we are examining a number of automated time-lapse image capture platforms to improve efficiency and accuracy, including the ability to record phenotypes too subtle to be observed with the human eye. In addition to increased throughput and improved automation, the logistical decision to commence the screen workflow with the schistosomulum stage has both potentially advantageous and disadvantageous consequences. Of advantage is that the workflow may identify compounds that are active against both immature and adult stages of parasite, or, at least, against immature parasites. This is important in the context that the current chemotherapy, PZQ, is markedly less effective against the immature (migratory and sub-adult) parasite compared to mature egg-laying adults [10],[11],[48]. Thus, the identification and development of a small molecule prophylaxis for individuals harboring immature parasites, such as in areas of higher transmission, would be of considerable value. By extension, the opportunity to develop a combination (possibly synergistic) therapy with PZQ to decrease the threat of resistance to the latter may also be facilitated by the present screen workflow that commences with the schistosomular stage rather than adults. The concept of a PZQ-based combination therapy based on reciprocal drug efficacy against immature and mature parasites has already shown value with the artemisinin class of compounds ([14] and references therein). A possible disadvantage of the current screening approach is the potential for missing compounds that are inactive against schistosomula, yet, nevertheless, might have yielded interesting phenotypes against adults worms. We accept this possibility as part of the overall goal to streamline and accelerate the identification of anti-schistosomal compounds. We would emphasize that, where smaller compound collections are concerned, the screen workflow can be conducted in a non-hierarchical manner whereby every compound is tested against both schistosomula and adults. Whether a compound is a hit or not or whether it passes or fails the GO/NO GO criteria as implemented here, all screen data are made publicly available for (re)interpretation. As to the choice of compound collections maintained at the UCSF SMDC (http://smdc.ucsf.edu/) to initiate the screen workflow, the Microsource Spectrum and Killer collections seemed appropriate for a number of reasons. First, the collections comprise a tractable set of 1,992 unique compounds, so that with a modest throughput the first and second components of the workflow were complete within 20 weeks. Secondly, the collections have a track record of yielding novel leads against other parasites including Plasmodium falciparum [31],[33] and Trypanosoma brucei [32]. Finally, the collections contain a chemically diverse set of natural and synthetic small molecules, 41% (821 compounds) of which are drugs already FDA-approved. From a drug-repositioning standpoint, this is particularly attractive because of the existence of clinical data (e.g., adsorption, distribution, metabolism, excretion and toxicity (ADMET)) that could contribute to fast-tracking these compounds as anti-schistosomals, especially as the compounds are off-patent and without intellectual property concerns. Of the 118 compounds identified as hits and phenotypically classified after 7 days of incubation in the primary schistosomular screen component, 105 were confirmed. Likewise, for the adult component of the workflow, repeated tests with compounds resulted, in most cases, in the same phenotypes. Thus, our blinded consensus approach to visually recording bioactivity provided reasonable reproducibility. In further support of the strategy, known schistosomicides, including PZQ and hycanthone, were, without fail, identified and consistently characterized, as were other anthelmintics, such as bithionol and niclosamide. Importantly, direct visual observation allowed us to identify and record the multiple and changing phenotypes that are possible with schistosomes and, not least, discover an apparent SAR for tricyclic psychoactive compounds primarily focused on the structure of the side chain. As yet the molecular target(s) of the dibenzazepine and phenothiazine drugs in question is unknown. It is possible that the ‘overactive’ phenotype is not neuroreceptor-mediated but perhaps a result of membrane interference (depolarization?). Nonetheless, it is interesting that, over a two log-fold concentration, compounds designed to interact with different ligand-gated receptors in humans nevertheless yield the same phenotype in the parasite, suggesting that a single parasite receptor or a discrete subset of receptors may be the target. By mining the available genome sequence information for S. mansoni [18],[19], one might envisage RNA interference of candidate cholinergic, dopaminergic or seratonergic receptors in an effort to modulate the overactive phenotype. This would prove the hypothesis that these compounds share a receptor and aid the development of an SAR-based drug discovery program. Both GO/NO GO filters in the workflow were designed in consideration of the TPP demanded for new anti-schistosomal drugs that employs the current therapeutic, PZQ, as a gold-standard. The bar is high - PZQ decreases worm burdens by between 60 and 90% in a single oral dose [3],[48]. Criteria of speed of appearance, phenotype severity (death preferred) and oral suitability were balanced with clinical data on dosage and safety. As interpreted for this report, the second GO/NO GO filter removed a number of compounds and compound classes that elicited striking phenotypic effects. Among these were the tricyclic psychoactive compounds. The ‘overactivity’ they elicited may yet prove therapeutically significant, perhaps by disrupting the parasite's migratory program or its ability to remain in position within the host. Targeting neurotransmission (if that indeed is the mechanism in schistosomes) is a successful chemotherapeutic strategy for other helminths ([49]). We will examine the efficacy of these compounds in the murine disease model at low doses either alone or in combination with PZQ. As implemented, the second GO/NO GO filter prioritized five (niclosamide, anisomycin and lasalocid sodium, diffractaic acid and gambogic acid) of the 30 hit compounds identified in the second (adult) component of the screen workflow for tests in the animal model of schistosomiasis. All five quickly kill (within hours) both schistosomula and adults at 1 µM in culture. Also, LD50 toxicity data are available against which a dosing regimen can be prepared and four of the five top hits (excepting diffractaic acid) can be administered orally. In the murine model of disease, anisomycin and lasalocid sodium demonstrated varying parasitological efficacies and amelioration of hepatic and splenic pathology. Though not as effective as PZQ, we consider the identification of these novel in vivo anti-schistosomal activities as proof that the screen as conceptualized (w.r.t. drug-repositioning) and implemented can identify potentially interesting and chemically diverse compounds. Such compounds might be employed for therapy as is or as leads for further derivatization (e.g., anisomycin is chemically relatively simple) in order to improve bioactivity while reducing toxicity. The point is further underscored with the niclosamide analogs. Though niclosamide and its wettable powder formulation were ineffective in the mouse model of disease, niclosamide's rapid and severe in vitro bioactivity encouraged us to search for other salicylanilide analogs. We identified a number that are well-established in the veterinary sector and possess better oral bioavailability with systemic anthelmintic activity, including against related trematode parasites [42]. The often significant in vivo efficacy of these drugs in both the parasitological and pathological parameters measured, particularly with rafoxanide, encourage further study of the salicylanilides as a source of anti-schistosomal leads. These investigations are underway. In conclusion, as a central component in a pre-clinical drug discovery pipeline for schistosomiasis, we have developed a partially automated phenotypic screen workflow of increased throughput. All data arising are posted and updated online and work continues to improve automation, rigor and throughput. As currently performed, the workflow has already identified a diversity of hit compounds and chemistries in vitro, as well as lead compounds orally bioactive in a short time frame commensurate with the TPP for chemotherapy of schistosomiasis [29]. Critically, the drug-repositioning dimension with the availability of clinical data for many of these hits and leads can be leveraged to optimize further compound development. Accordingly, screens of other libraries containing known drugs are ongoing. Given the possibility of the emergence of resistance to the current PZQ monotherapy, and because any strategic planning for therapy of infectious diseases should incorporate provisions for drug combinations, our future studies will focus on the in vivo performance of the present and future lead compounds, either alone or in combination, including with PZQ. Supporting Information Table S1 Phenotypes recorded upon primary and confirmatory screening of the Micosource ‘Spectrum’ and ‘Killer’ Collections at 1 µM in vitro against S. mansoni schistosomula. (3.31 MB XLS) Click here for additional data file. Table S2 Phenotypes recorded against S. mansoni adults at 1 µM in vitro with those hit compounds that arose in screens against schistosomula. (0.05 MB XLS) Click here for additional data file. Table S3 Compounds yielding phenotypes in S. mansoni schistosomula as a function of drug class. (0.40 MB XLS) Click here for additional data file. Table S4 Apparent structure activity relationship for tricyclic pyschoactive compounds (e.g., dibenzazapines and phenothiazines) within the Microsource collections that generate the ‘overactive’ phenotype in S. mansoni schistosomula at 1.0 and 0.1 µM. (0.11 MB XLS) Click here for additional data file. Video S1 S. mansoni schistosomula demonstrating the ‘overactive’ phenotype after 7 d in vitro in the presence of 1 µM imipramine. (6.53 MB MOV) Click here for additional data file. Video S2 Control S. mansoni schistosomula incubated for 7 d in vitro. (7.04 MB MOV) Click here for additional data file.

0 comments Cited 90 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Negl Trop Dis

Journal ID (publisher-id): plos

Journal ID (pmc): plosntds

Title: PLoS Neglected Tropical Diseases

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Print): 1935-2727

ISSN (Electronic): 1935-2735

Publication date Collection: July 2010

Publication date (Electronic): 27 July 2010

Volume: 4

Issue: 7

Electronic Location Identifier: e759

Affiliations

[1]Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom

Swiss Tropical Institute, Switzerland

Author notes

* E-mail: krh@ 123456aber.ac.uk

Conceived and designed the experiments: EP IWC KFH. Performed the experiments: EP. Analyzed the data: EP. Wrote the paper: EP IWC KFH.

Article

Publisher ID: 10-PNTD-RA-0935R3

DOI: 10.1371/journal.pntd.0000759

PMC ID: 2910722

PubMed ID: 20668553

SO-VID: d29fc118-04a3-4ae8-9e12-55fe5a9b92da

Copyright © Peak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 26 February 2010

Date accepted : 8 June 2010

Page count

Pages: 12

Comments

Comment on this article

scite_

Cited by 50

See all cited by

Most referenced authors 345

See all reference authors

Development and Validation of a Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability

Read this article at

Abstract

Background

Methodology/Principal Findings

Conclusions/Significance

Author Summary

Related collections

SDG 3: Good Health and Well-Being

Most cited references 18

The Schistosoma japonicum genome reveals features of host-parasite interplay.

Thioredoxin Glutathione Reductase from Schistosoma mansoni: An Essential Parasite Enzyme and a Key Drug Target

Drug Discovery for Schistosomiasis: Hit and Lead Compounds Identified in a Library of Known Drugs by Medium-Throughput Phenotypic Screening

Author and article information

Contributors

Journal

Affiliations

Author notes

Article

History

Page count

Categories

Comments

Comment on this article

Similar content 8

Cited by 50

Most referenced authors 345