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, 10 (2), e1003942

'Death and Axes': Unexpected Ca²⁺ Entry Phenologs Predict New Anti-Schistosomal Agents


'Death and Axes': Unexpected Ca²⁺ Entry Phenologs Predict New Anti-Schistosomal Agents

John D Chan et al. PLoS Pathog.


Schistosomiasis is a parasitic flatworm disease that infects 200 million people worldwide. The drug praziquantel (PZQ) is the mainstay therapy but the target of this drug remains ambiguous. While PZQ paralyses and kills parasitic schistosomes, in free-living planarians PZQ caused an unusual axis duplication during regeneration to yield two-headed animals. Here, we show that PZQ activation of a neuronal Ca²⁺ channel modulates opposing dopaminergic and serotonergic pathways to regulate 'head' structure formation. Surprisingly, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. These divergent outcomes (death versus axis duplication) result from the same Ca²⁺ entry mechanism, and comprise unexpected Ca²⁺ phenologs with meaningful predictive value. Surprisingly, basic research into axis patterning mechanisms provides an unexpected route for discovering novel antischistosomal agents.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Biogenic amines differentially modulate PZQ evoked bipolarity.
(A) Anterior posterior (AP) polarity in normal (left, control) and PZQ-treated (75 µM, 48 hrs) D. japonica (right) after 7 days of regeneration. This result derived from incubation of trunk fragments (white box, amputation of head and tail structures) in drug-containing solution during early regeneration . (B) Diversity of flatworm neurotransmitters. Shading identifies different flatworm neurotransmitter families with branching reflecting molecular diversity. Key synthetic enzymes targeted by RNAi (white circles) were: CAT, choline acetyl transferase; PC2, prohormone convertase 2; GDC, glutamate decarboxylase; TH, tyrosine hydroxylase; HDC, histidine decarboxylase; TDC, tyrosine decarboxylase; TBH, tyramine-β-hydroxylase; TPH, tryptophan hydroxylase. (C) Effect of RNAi targeting neurotransmitter synthetic pathways on PZQ-evoked bipolarity, n≥3 independent trials. Abbreviations are as described in ‘B’. (D) Pharmacological screening of monoaminergic drugs revealed compounds that promote and inhibit head regeneration. Representative images of regenerative phenotypes observed using (i) PZQ (75 µM), (ii) bromocriptine (1 µM), (iii) dopamine (500 µM), (iv) apomorphine (750 nM), (v) mianserin (10 µM), (vi) reserpine (10 µM), (vii) fluoxetine (2 µM), (viii) 5-HT (1 mM), (ix) 8-OH DPAT (10 µM). In all cases, trunk fragments were treated for 48 hrs. (E) Penetrance of monoaminergics at evoking two-headed (black) or no-headed worms (open). (F) Bipolarity evoked by PZQ (solid) and bromocriptine (open) was antagonized by haloperidol (inset, co-incubation with 1.5 µM for 24 hrs). (G) Inhibition of PZQ-evoked bipolarity (90 µM, 24 hrs) by various concentrations of serotonergic ligands.
Figure 2
Figure 2. Analysis of drug action and selectivity in the planarian system.
(A) Similar kinetics of bromocriptine and PZQ-evoked bipolarity. Number of bipolar regenerants after exposure of trunk fragments to PZQ (75 µM, solid) or bromocriptine (1.5 µM, open) for the indicated durations. Fragments were amputated at t = 0. (B) Bromocriptine acts downstream of PZQ-evoked Ca2+ entry. Cav1A RNAi inhibits PZQ but not bromocriptine-evoked bipolarity. (C) Displacement of 3H-DA from planarian membrane fractions by various ligands, including dopamine (Ctrl), bromocriptine (10 µM), haloperidol (100 µM), and apomorphine (10 µM). Inset, 3H-DA displacement assay at various concentrations of bromocriptine, expressed as a fraction of total specific 3H-DA binding. Data represent average of at least three independent replicates. (D) Effect of reserpine and fluoxetine on 5-HT and dopamine levels in regenerating trunk fragments. Regenerating trunk fragments were exposed to of either reserpine (10 µM) or fluoxetine (10 µM) for 24 hrs prior to electrochemical HPLC analysis of 5-HT (open) and dopamine (closed) levels. Data represent analyses from multiple samples from at least two independent biological replicates, mean ± standard deviation.
Figure 3
Figure 3. Small molecules efficacious as antischistosomals miscue planarian AP polarity.
(A) Left, Schematic representation of ‘hits’ versus drug representation in Schistosoma mansoni drug screens. The number of drug hits in different functional classes (pie chart categories reflect LOPAC1280 pharmacological action ontology) were represented as appropriately scaled circles, allowing simple visual inspection of drug categories over/under represented as phenotypic outcomes. Blue circles highlight the top four drug categories (dopaminergics, serotonergics, Ca2+ signaling, phosphorylation) and red circles the proportional representation of other classes. For simplicity, drugs with ‘unknown’ mechanism of action classifications, and generalized anti-infective agents were not represented. Right, translation of hits (PKC, GSK3) in the phosphorylation category (*) from the schistosome screening literature to the planarian regeneration assay. (B) Images of regenerating worms after treatment with PKC modulators: (i) PMA (15 nM), (ii) 4-α PMA (inactive analog, 15 nM), (iii) OAG (100 µM), (iv) PDB (25 µM). (C) Involvement of PKC in PZQ-evoked bipolarity. Left, PKC inhibition using calphostin C (10 nM) attenuated PZQ (90 µM) evoked bipolarity. Middle, RNAi of DAGK potentiated low dose PZQ-evoked bipolarity (50 µM). Right, knockdown of a cPKC isoform attenuated PZQ-evoked bipolarity (90 µM). (D) Effect of Ca2+ on PMA-evoked bipolarity. Effects of indicated Ca2+ concentration on the bipolarizing ability of the PKC agonist PMA (solid squares, 15 nM) and the inactive analog 4-α-PMA (open squares, 15 nM). (E) The GSK-3 inhibitor ALP (5 µM) potentiated PZQ-evoked bipolarity (25 µM), while the GSK3 activator DIF-3 (1.75 µM) blocked PZQ action (50 µM).
Figure 4
Figure 4. Compounds that miscue planarian polarity regulate schistosomule contractility.
(A) Compounds transferred from planarian regenerative assay to schistosomule screen. (B) Top, image sequence showing periodic basal contractile activity of a schistosomule. Bottom, body length versus time plots for individual schistosomules treated with small molecules (low dose, grey; high dose, black). Drug concentrations (30 minute exposures) were: bromocriptine (BRM, 1 µM; 10 µM), praziquantel (PZQ, 1 µM; 10 µM), apomorphine (APM, 1 µM; 10 µM), mianserin (MSN, 5 µM; 10 µM), reserpine (RES, 10 µM; 50 µM), 5-HT (10 µM; 100 µM), fluoxetine (FLX, 1 µM; 10 µM), 8-OH DPAT (1 µM; 10 µM). (C) Dose-response relationship showing the effects of increasing concentrations of bromocriptine on schistosomule contractility. (D&E) Cumulative dataset from experiments such as those shown in ‘B’ for compounds active in the planarian regenerative bioassay parsed into compounds that (D) inhibit and (E) stimulate schistosomule contractility. Drug concentrations were the higher dose of values reported in (B). Dashed line, basal level of contractility.
Figure 5
Figure 5. Death and axes: phenologous responses evoked by PZQ in different organisms.
(A) Proposed model depicting phenology between PZQ-evoked outcomes in planarians (brown) and schistosomes (grey, adult worm depicted). In both organisms, we suggest PZQ evoked Ca2+ entry (blue) couples to dopaminergic signals that promote outcomes (green, head regeneration/paralysis) that are antagonized by serotonergic signals coupling to opposing phenotypes (red, tail regeneration/hyperactivity). (B) In planarians (bottom), a broad array of voltage-gated entry channels permits subfunctionalization of PZQ-evoked Cav1A activity (blue) to yield a physiological exploitable Ca2+ influx. In contrast, schistosomes (top) are more vulnerable to PZQ-evoked Cav1A activity, as these parasites possess a more limited repertoire of voltage-sensitive influx channels, lacking Nav and LVA Cav channels. Sequence identifiers - Dugesia japonica: Cav 1A (AEJ87267), Cav 1A (AEJ87268), Cav 2A (AEJ87269), Cav 2B (AEJ87270), Cav3 (AEJ87271), Nav1 (FY933419), Nav2 (FY957659). Schistosoma mansoni: Cav 1A (Smp_020270), Cav 1B (Smp_159990), Cav 2A (Smp_020170) & Cav2B (Smp_004730).

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