Kinetic profiling an abundantly expressed planarian serotonergic GPCR identifies bromocriptine as a perdurant antagonist

Abstract

The diversity and uniqueness of flatworm G protein coupled receptors (GPCRs) provides impetus for identifying ligands useful as tools for studying flatworm biology, or as therapeutics for treating diseases caused by parasitic flatworm infections. To catalyse this discovery process, technologies optimized for mammalian GPCR high throughput screening need be transposed for screening flatworm GPCRs. Here, we demonstrate the utility of a genetically encoded cAMP biosensor for resolving the properties of an abundantly expressed planarian serotonergic GPCR (S7.1R). Application of this methodology resolved the real time kinetics of GPCR modulation by ligands and demonstrated a marked difference in the kinetic action of antagonists at S7.1R. Notably, bromocriptine caused a protracted inhibition of S7.1R activity in vitro and a protracted paralysis of planarian movement, replicating the effect of S7.1R in vivo RNAi. The lengthy inhibition of function caused by bromocriptine at this abundantly expressed GPCR provides a useful tool to ablate serotonergic signaling in vivo, and is a noteworthy feature for exploitation as an anthelmintic vulnerability.

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

“…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. Here, we apply the cAMP biosensor to expand upon this growing understanding of structure-activity relationships at Sm.5HTR L by examining the effects of several aporphine alkaloids containing methoxyisoquinoline moieties.…”

“…Knockdown of this receptor, expressed across the parasite life cycle (Protasio et al., 2012), caused hypoactive motor phenotypes in schistosomules and adult worms consistent with the myoexcitatory role of 5-HT (Patocka et al., 2014). Knockdown of the Sm.5HTR L homologue in free living planarians ( Dj- S7.1R, see companion paper, Chan et al., 2016a) also causes hypomobility (Chan et al., 2015). …”

Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca 2+ channel in neuromuscular function and regeneration

“…Animals were scored for their ability to escape into the shaded perimeter within five minutes. For generalized monitoring of worm mobility, planarians were tracked within an illuminated watchglass as previously described (Chan et al, 2016). Regenerative assays were performed using 5 day-starved worms (n>30) in pH-buffered Montjuïch salts (1.6mM NaCl, 1.0mM CaCl 2 , 1.0mM MgSO 4 , 0.1mM MgCl 2 , 0.1mM KCl,1.2mM NaHCO 3 , pH 7.4 buffered with 1.5mM HEPES) and regenerative polarity scored after 1 week.…”

Anthelmintics: From discovery to resistance II (San Diego, 2016)

“…They point out that the SNPs identified may have effects on gene expression, function and resistance selection. Two papers from the laboratory of Jonathan Marchant (Chan et al., 2016a & b) on serotonergic G protein coupled receptors (GPCRs) in flatworms comment on their diversity of flatworm GPCRs for identifying ligands to treat parasitic flatworm infections. They illustrate a novel approach using a genetically encoded cAMP biosensor to resolve the properties of an expressed serotonergic GPCR (S7.1R).…”