A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors

Abstract

Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets (‘target selection’) and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics.

Pharmacological profiling an abundantly expressed schistosome serotonergic GPCR identifies nuciferine as a potent antagonist

“…To profile the activity of Sm.5HTR L , a cAMP-dependent luciferase reporter (pGloSensor-22F) was co-expressed. Sm.5HTR L is G s coupled in this system, permitting a rapid and robust read out of 5-HT evoked cAMP generation in live cells (Chan et al., 2016b). …”

“…In this movement assay, exogenous 5-HT stimulates contractility, and knockdown of Sm.5HTR L (Patocka et al., 2014), or application of the known Sm.5HTR L antagonist methiothepin (Fig. 2A (Chan et al., 2016b)) reversed this effect.…”

“…Schistosomules were obtained by mechanical transformation from shed cercariae as described in (Chan et al., 2016b). Briefly, cercariae were shed by exposure to light (1.5 h), vortexed in 50 mL conical tubes (3 × 45secs, each separated by 3 min on ice), and tails were removed by gradient centrifugation (24 ml Percoll, 4 ml 10X EMEM, 1.5 ml penicillin-streptomycin, 1 ml of 1M HEPES in 0.85% NaCl, 9.5 ml distilled water) at 500 g/15 min at 4 °C.…”

“…number NR-34792) and sacrificed 6–8 weeks post-infection. Adult S. mansoni were recovered from the mesenteric vasculature by portal perfusion (Chan et al., 2016b). Mice were anesthetized in a CO 2 chamber, sacrificed by cervical dislocation, and perfused with sodium citrate (25 mM).…”

“…This approach revealed properties and a pharmacological divergence of Sm.5HTR L from the closest human GPCR homolog (Hs.5HTR 7 ) that further enhanced the appeal of Sm.5HTR L as a chemotherapeutically vulnerable node for anthelmintic development (Chan et al., 2016b). The chemical screening data demonstrated a potent, and long lasting inhibition evoked by the ergot alkaloid bromocriptine at Sm.5HTR L (evidenced also for the planarian S7.1R in the companion paper (Chan et al., 2016a)), and highlighted several Sm.5HTR L antagonists with dimethoxyisoquinoline substructures.…”