Ergot Alkaloids (Re)generate New Leads as Antiparasitics

Abstract

Praziquantel (PZQ) is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT) rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors.

Fig 1. 5-HT reverses PZQ action on regenerative polarity.

(A) Regenerative phenotypes (scored after 7 days) from excised trunk fragments (top, boxed region) exposed to either PZQ alone (75μM, 24hrs), or PZQ (75μM) and 5-HT (250μM, 24hrs). Original anterior of the worm oriented to the left, head structures are arrowed. Scalebar, 4mm. (B) Dose response relationship for the effect of PZQ on regenerative bipolarity. (C) Reversal of PZQ-evoked bipolarity (75μM, 24hrs) by simultaneous incubation with 5-HT but not other neurotransmitters (dopamine (DA), octopamine (OCT), epinephrine (EPI), norepinephrine (NE), at a final concentration of 250μM). The analogue O-methylserotonin (O-MT) was used at a final concentration of 100μM. **, p <0.01.

Fig 2. Analysis of serotonergic receptors in Dugesia japonica.

(A) Unrooted maximum likelihood tree (PhyML 500 bootstrap replicates) of predicted D. japonica serotonin GpCR protein sequences (Dataset A and B in S1 Text) shows the distribution of 17 planarian receptors in S1-like (red), S4-like (blue) and S7-like clades (green). C. elegans sequences used for comparison are CeSER1a (O17470, SER1), CeSER4 (G5EGH0, SER4) and CeSER7a (Q22895, SER7). Previously cloned planarian 5-HTR sequences (*) are renamed as follows: S7.1 = 5HTLpla4 [23], DtSER1 [25]; S7.2 = 5HTLpla1 [23]; S7.3 = 5HTLpla2 [23]; S7.4 = DjSer7 [22]; S7.5 = 5HTLpla3 [23]. (B) Pie chart showing relative abundance of 5-HT GpCR transcripts as reflected by FPKM values. S7.1 represented the most abundant 5-HT receptor (~44% of all transcripts) and S7 the most abundant clade (~70% of all transcripts). (C) Effect of RNAi against individual S7 receptors on drug-evoked bipolarity. Data are expressed as the proportion of two-headed worms evoked by submaximal PZQ in RNAi worms (75μM) relative to a control RNAi cohort (Smed-six-1 RNAi). Data are not presented for S7.8 (the least abundant S7 receptor) as attempts to amplify this sequence were unsuccessful. Data represent mean± standard error of 3 to 7 independent knock-down cycles (*, p<0.02). Inset, qPCR analysis of S7.1 expression levels following RNAi relative to control RNAi cohorts (**, p<0.002). (D) Effect of RNAi targeting individual S7 receptors on intact worm mobility. Minimal intensity projection image represents motion of 10 worms over a 2 minute period within an illuminated watchglass. Scalebar, 25mm.

Fig 3. Effect of various ergot alkaloids on planarian regeneration and schistosomule contractility.

(A) Effect of different ergot alkaloids on planarian regenerative polarity. Compounds caused either bipolar regeneration (‘two-head’, solid) or no-headed regenerants (open). Illustrative phenotypes are indicated (left) resulting from exposure to LY215840 (top) or ergotamine (bottom). Structures of individual compounds (‘a’ through ‘l’) are indicated. Indole ring modifications are highlighted in red, and shared structure with 5-HT shown in blue. Concentrations used for regenerative assay (24/48hrs) were: ‘a’ (75μM), ‘b’ (1.5μM), ‘c’ (25μM), ‘d’ (1μM), ‘e’(10μM), ‘f’ (2μM), ‘g’ (1μM), ‘h’ (1μM), ‘I’ (5μM), ‘j’ (10μM), ‘k’ (10μM), ‘l’ (10μM). (B) Effect of the same ergot alkaloids on contractile activity of schistosomules, with compounds grouped as in (A). Decreased (solid) and increased (open) mobility are expressed relative to controls (set as ‘1’). Body length versus time plots (left) were resolved for individual schistosomules treated with small molecules. Example traces are shown for LY215840 (top) and ergotamine (bottom), relative to control. Drug concentrations (30 minute exposures) were: ‘a’ (10μM), ‘b’ (10μM), ‘c’ (50μM), ‘d’ (50μM), ‘e’(25μM), ‘f’ (25μM), ‘g’ (ND = not done), ‘h’ (0.5μM), ‘I’ (10μM), ‘j’ (5μM), ‘k’ (1μM), ‘l’ (0.5μM). Data for PZQ and bromocriptine are from [20].

Fig 4. Bipolarizing compounds inhibit planarian mobility.

(A) Top, reversible effect of PZQ on worm morphology (right) compared with control (left). Bottom, effect of PZQ on mobility of intact worms (75μM, 10mins incubation). Images show minimal intensity projections for 10 worms over 2 minutes. Scalebar, 25mm. (B) Dose response relationship for effect of PZQ on planarian mobility. Inset, minimal intensity projections at indicated doses (μM). (C) Antagonism of acute PZQ-evoked mobility defects in intact worms (75μM) by co-incubation with O-MT but not other neurotransmitters (all at 100μM). *, p < 0.01 relative to PZQ (dashed line). PZQ-evoked mobility defects are reversible on solution exchange (‘wash’). (D) Top, effects of bipolarizing ergot compounds on planarian mobility after 10min exposure. Bottom, quantification of the mobility effects evoked by different ergot alkaloids. Doses: PZQ (75μM), bromocriptine (2μM), LY215840 (1μM), metergoline (2μM), nicergoline (10μM). *, p < 0.01 relative to control.

Fig 5. PZQ and 5-HT action against schistosomules.

(A) Mobility of schistosomules (contractions per minute) exposed to the serotonergic agonist o-methylserotonin (O-MT, 1μM), praziquantel (PZQ, 50nM), or co-treated with PZQ (50nM) plus O-MT (1μM). (B) Measurement of schistosomule morphology changes in response to PZQ, as measured by the ratio of the worm’s average body length (‘L’) to width (‘W’) as depicted. (C) Phenotypes of schistosomules from experiments shown in B. *, p < 0.01 PZQ relative to O-MT + PZQ. **, p < 0.001 PZQ relative to control.

Fig 6. Proposed model of PZQ action.

PZQ action in planarians has previously been shown to depend on Ca_v1A and tyrosine hydroxylase (TH) functionality (red, [18,20]). Activity of this pathway is opposed by Ca_v1B and tryptophan hydroxylase functionality (blue, [18,20]). We propose these pathways differentially regulate second messenger (cAMP) action, muscle function and—in planarians—‘head’ versus ‘tail’ specification. Here, we demonstrate the role of serotonergic antagonists as phenocopying PZQ action. This is most conservatively explained by functional antagonism of the opposing Cav1A/dopaminergic pathway, however the possibility that PZQ directly targets flatworm bioaminergic receptors as an ergomimetic cannot be excluded until heterologous expression of platyhelminth GpCRs is optimized.