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

Abstract

5-hydroxytryptamine (5-HT) is a key regulator of muscle contraction in parasitic flatworms. In Schistosoma mansoni, the myoexcitatory action of 5-HT is effected through activation of a serotonergic GPCR (Sm.5HTR_L), prioritizing pharmacological characterization of this target for anthelmintic drug discovery. Here, we have examined the effects of several aporphine alkaloids on the signaling activity of a heterologously expressed Sm.5HTR_L construct using a cAMP biosensor assay. Four structurally related natural products - nuciferine, D-glaucine, boldine and bulbocapnine - were demonstrated to block Sm.5HTR_L evoked cAMP generation with the potency of GPCR blockade correlating well with the ability of each drug to inhibit contractility of schistosomule larvae. Nuciferine was also effective at inhibiting both basal and 5-HT evoked motility of adult schistosomes. These data advance our understanding of structure-affinity relationships at Sm.5HTR_L, and demonstrate the effectiveness of Sm.5HTR_L antagonists as hypomotility-evoking drugs across different parasite life cycle stages.

Keywords: 5-HT; Methoxyisoquinoline; Natural products; Schistosomiasis.

Graphical abstract

Fig. 1

Aporphine alkaloid natural products are Sm.5HTR_Lantagonists. (A) Structures of apomorphine, a semi-synthetic aporphine, and four naturally occurring aporphines containing methoxyquinoline substructure (nuciferine, D-glaucine, boldine and bulbocapnine). (B) Effects of aporphines on Sm.5HTR_L dependent cAMP generation. HEK293 cells co-transfected with the 22-F cAMP biosensor and Sm.5HTR_L were first treated with the either DMSO vehicle control (open circles) or the indicated compound (solid circles, 5 μM added at solid triangle). After 30 min, 5-HT (0.8 μM, grey triangle) was added. Following stabilization of the 5-HT response, forskolin (20 μM, open triangle) was added to each well. (C) Dose-response curves show inhibition of 5-HT (0.8 μM) evoked cAMP generation in the presence of increasing concentration of individual aporphines.

Fig. 2

Aporphine alkaloids inhibit schistosomule contractility. (A) Schistosomule body length traces were recorded over 1 min for untreated schistosomules (left), 5-HT (10 μM) treated schistosomules (middle), and schistosomules exposed to both 5-HT and the Sm.5HTR_L antagonist methiothepin (10 μM, right). (B) Quantification of body contractions for basal conditions (white bars), addition of 5-HT (10 μM, black bars), and 5-HT plus increasing doses of indicated aporphine (grey bars).

Fig. 3

Correlation of inhibition of Sm.5HTR_Lwith inhibition of schistosomule mobility. Measured IC₅₀ for blockade of Sm.5HTR_L by different aporphine derivatives (ordinate) plotted against measurements of IC₅₀ for inhibition of schistosomule contractility (abscissa). Compounds: 1 = nuciferine, 2 = D-glaucine, 3 = boldine, 4 = apomorphine and 5 = bulbocapnine. Data are fitted to a linear equation (y = bx+a), slope = 1.06 ± 0.11 (standard error).

Fig. 4

Aporphine alkaloid inhibition of adult schistosome movement. Effects of prioritized compounds on male and female adult schistosome worms. Basal movement of (A) male and (B) female worms treated with either DMSO (‘ctrl’) or the indicated compound (10 μM, 2 h). *p < 0.05, **p < 0.01. Effect of aporphine antagonists on 5-HT evoked stimulation of (C) male and (D) female adult schistosome movement (expressed as fold change over basal movement for DMSO control cohort).