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.

Keywords: 5-HT; Ergot alkaloid; Flatworm; Praziquantel; cAMP.

Graphical abstract

Fig. 1

Heterologous expression of a planarian 5-HT receptor. (A) Comparison of mobility of cohorts of D. japonica flatworms with either a control gene or S7.1R targeted by in vivo RNAi. Left – minimal intensity overlay of images from a 2 min recording of respective RNAi cohorts (10 worms) within a watchglass. Right - Quantification of movement for 3 cohorts of 10 worms for each RNAi condition. Data analysed from (Chan et al., 2015). P value, ** = p < 0.01. (B) Kinetics of 5-HT (1 μM) evoked cAMP generation in HEK293 cells transfected with S7.1R and a cAMP dependent luciferase (black circles) or a cAMP-dependent luciferase alone (white circles). (C) Dose response relationship of effects of 5-HT concentration on peak luminescence amplitude in cells transfected with S7.1R and a cAMP dependent luciferase (black circles) or a cAMP-dependent luciferase alone (white circles). (D) 5-HT responsiveness (1 μM) of 7.1R expressing cells co-treated with bromocriptine (BRM, white circles) or cyproheptadine (CYPH, grey circles). Data are representative of n ≥ 3 assays, and represent mean ± s.d (B,C,D),or mean ± s.e.m. (A).

Fig. 2

Bromocriptine and cyproheptadine impair D. japonica movement. (A) Dose dependent inhibition of planarian movement following exposure (20min) to the indicated doses of bromocriptine (BRM, top), or cyproheptadine (CYPH, bottom). (B&C) Quantification of worm mobility in (B) bromocriptine or (C) cyproheptadine treated samples relative to controls. Data are representative of n ≥ 3 assays, and represent mean ± s.e.m.

Fig. 3

Bromocriptine effects a long lasting impairment of planarian movement. (A) D. japonica mobility before (pre) or at various time periods after a 20 min exposure (post drug, spanning time = ‘0’ to 1 week) to the S7.1R antagonists bromocriptine (BRM, 5 μM) and cyproheptadine (CYPH, 50 μM). (B&C) Quantification of images shown in (A) before and after drug washout. Each datapoint represents the mobility of a single worm (3 petri dishes, 10 worms in each dish). Plotted line connects the population mean for each condition. (D) Minimum intensity projections show movement profiles for worms treated with no drug (top), mianserin (middle; 10 μM, 20 min) and PZQ (bottom; 75 μM, 15 min), before drug exposure (left), during drug exposure (middle) and 2hrs after drug removal (right). (E) Effects of mianserin (10 μM) and praziquantel (100 μM) on 5-HT (1 μM) evoked cAMP signaling through the S7.1R. PZQ, unlike mianserin, did not block 5-HT action at the planarian S7.1R. Data are representative of n ≥ 3 assays, and represent mean ± s.e.m. P value, * = p < 0.05.

Fig. 4

Kinetics of movement inhibition evoked by S7.1R blockers. (A) Minimum intensity projections showing worm tracks prior to bromocriptine (5 μM, top) or cyproheptadine (50 μM, bottom) exposure (‘pre’), immediately after drug addition (‘0’) and at 5 min intervals thereafter. (B) Mean worm motility (3 petri dishes, 10 worms in each dish) from experiments as shown in (A) prior to, and after exposure to indicated drugs. Data are representative of n ≥ 3 assays, and represent mean ± s.e.m.

Fig. 5

Bromocriptine results in a long lasting impairment of S7.1R activity. (A) Outline of experimental workflow. HEK293 cells were co-transfected with S7.1R and a cAMP reporter and pre-incubated (60 min) with vehicle (DMSO), or S7.1R antagonist (bromocriptine, cyproheptadine; 10 μM), which was washed out prior to assaying for 5-HT responsiveness (90 min later). (B) Kinetics of 5-HT (1 μM) evoked cAMP generation in cells pre-treated with either vehicle control (DMSO, black circles), bromocriptine (10 μM, white circles) or cyproheptadine (10 μM, grey circles). (C) Quantification of 5-HT (1 μM) response (left) and forskolin (20 μM) response (right) for individual drug treatments. Data are representative of n ≥ 3 assays, and represent mean ± s.d (B) or mean ± s.e.m (C). P value, ** = p < 0.01.