The anthelmintic praziquantel is a human serotoninergic G-protein-coupled receptor ligand

Abstract

Schistosomiasis is a debilitating tropical disease caused by infection with parasitic blood flukes. Approximately 260 million people are infected worldwide, underscoring the clinical and socioeconomic impact of this chronic infection. Schistosomiasis is treated with the drug praziquantel (PZQ), which has proved the therapeutic mainstay for over three decades of clinical use. However, the molecular target(s) of PZQ remain undefined. Here we identify a molecular target for the antischistosomal eutomer - (R)-PZQ - which functions as a partial agonist of the human serotoninergic 5HT_2B receptor. (R)-PZQ modulation of serotoninergic signaling occurs over a concentration range sufficient to regulate vascular tone of the mesenteric blood vessels where the adult parasites reside within their host. These data establish (R)-PZQ as a G-protein-coupled receptor ligand and suggest that the efficacy of this clinically important anthelmintic is supported by a broad, cross species polypharmacology with PZQ modulating signaling events in both host and parasite.

Fig. 1

Resolution of stereoselective (R)-PZQ interaction with human 5-HT_2BR. Structures of the PZQ enantiomers: active, a antischistocidal (R)-PZQ (top, red) and b (S)-PZQ (bottom, blue). c Docking poses of R-PZQ (light red) and S-PZQ (light blue) in the human 5HT_2BR. Both isomers of PZQ dock favorably within the orthosteric pocket of the 5HT_2BR crystal structure (Protein Databank Code: 5TVN). The R-isomer shows a better predicted binding score by 0.6 kcal/mole partially due to a more favorable hydrogen bond with the backbone NH group of L209. d Displacement of ³H-LSD binding at human 5-HT_2BR by racemic PZQ ((±)-PZQ 10 µM, purple), (R)-PZQ (5 µM, red) and (S)-PZQ (5 µM, blue) in the PDSP primary screen (n = 5 for all measurements). e Complete binding displacement curves for ³H-LSD displacement at human 5-HT_2BR for SB206553 (black) and (±)-PZQ (purple, n = 5 for both ligands). f Functional Assays. Ca²⁺ flux assays showing responses from fluo-4 loaded Flp-In T-REx cells stably expressing 5-HT_2AR, 5-HT_2BR, or 5-HT_2CR at indicated concentrations of (±)-PZQ (purple, top), (R)-PZQ (red, middle) and (S)-PZQ (blue, bottom; n = 3 for each concentration) in cells expressing 5-HT_2AR, 5-HT_2BR, or 5-HT_2CR

Fig. 2

(R)-PZQ triggers Ca²⁺ release via 5-HT_2BR. a Representative fluorescence traces from a single experiment where single cells loaded with fluo-4-AM in a HEK293 cell line prior to (top), or after (bottom) 5-HT_2BR induction. Traces show fluorescence ratio following addition of indicated ligands (final concentrations (±)-PZQ (75 µM), 5-HT (100 nM) or ACh (100 µM)), or vehicle (DMSO, 0.05%). b Pseudocolored confocal images are displayed from the indicated times (‘1’–‘4’) on associated fluorescence traces as introduced in Fig. 1a. Scalebar, 80 µm. c Peak fluorescence ratio (F/F₀, where ‘F’ represents fluorescence at peak and ‘F₀‘ represents fluorescence at time = 0) from cumulative Ca²⁺ imaging after addition of vehicle, (±)-PZQ, 5-HT or ACh in uninduced (open bars) or induced cell lines (solid, hatched bars; n ≥ 3 independent inductions). Serotonergic antagonists (ritanserin (100 nM, final concentration), SB204741 (100 nM), or mesulergine (200 nM) were preincubated with induced cells prior to addition of (±)-PZQ (75 µM), 5-HT (10 nM), or ACh (100 µM). Data represent means ± s.e.m. of peak responses averaged from multiple cells (>20) from n ≥ 3 independent inductions. d Dose-response relationship for (±)-PZQ efficacy in cells induced for 5-HT_2BR expression (n = 6 for each concentration). e Confocal Ca²⁺ imaging assays with (±)-PZQ and resolved enantiomers in uninduced (top row) and 5-HT_2BR induced cell lines (bottom three rows). Experiments were performed with racemic PZQ ((±)-PZQ), and individual (S)-PZQ and (R)-PZQ stereoisomers (5 µM, final concentration). Scalebar, 80 µm. f Processed data set from experiments such as shown in e representing population mean ± s.e.m. (>20 cells) from n ≥ 3 independent inductions

Fig. 3

(R)-PZQ constricts mouse mesenteric vasculature. a Image of mesentery surrounding mouse intestine, showing blood vessel containing a schistosome worm-pair (inset). b Representative image of a surgically isolated piece of mouse mesenteric artery held under tension between two clamped wires. Scalebar, 1 mm. c Changes in tension triggered by varied manipulations. Left, sustained contraction of vessels in KPSS and 5-HT (10 µM). Right, tension in individual arteries incubated in the presence of vehicle (DMSO, open circle) or SB204741 (10 µM, closed circle), and challenged with (R)-PZQ (50 µM) or (S)-PZQ (50 µM). W, wash; complete media exchange. Preincubation of strips with SB204741 (10 µM) attenuated (R)-PZQ evoked vasoconstriction. d Cumulative data set from experiments reporting effects of ligands relative to KPS(S)-evoked contractile tension, concentrations of ligands: 5-HT (1 µM), (R)-PZQ (50 µM), (S)-PZQ (50 µM), SB204741 (10 µM). Replicate number for each measurement as indicated, where individual measurements reflect the response from a naïve artery isolated from different mice. Data are presented as mean ± s.d. Probability, p < 0.05 (*), p < 0.01 (**). e Schematic model (not to scale) depicting (R)-PZQ action on both parasite (contraction) and host vasculature (contraction, increased perfusion pressure) facilitating hepatic shift of worms