One technology that fulfills these requirements employs a bioluminescent cAMP reporter to monitor the activity of Gs and Gi-coupled GPCRs, marketed as GloSensor. lack of responsiveness to serotonergic ligands but robust cAMP generation in response to catecholamines acting on endogenous HEK293 cell GPCRs. (B) Response of HEK293 cells co-expressing Sm.5HTR and the F22 biosensor to various serotonergic and monoaminergic ligands previously identified to lack activity on endogenous Gs coupled GPCRs. Physique C. Comparison of ligand class specificities against Sm.5HTR and Hs.5HT7R. Categorized ligand specificities of individual compounds that block Sm.5HTR and Hs.5HTR7 from classification index of the screened library. While Sm.5HTR and Hs.5HTR7 show distinct selectivity profiles to the 23 and 31 ligands identified as hits, the broader classification of these ligands is similar. Physique D. Effect of Sm.5HTR antagonists Gadd45a on Sm.5HTRL. Luminescence response from Sm.5HTRL expressing HEK293 cells to 5-HT (EC80 dose = 0.8M) in the presence of indicated antagonists (10M). Data are shown relative to control samples unexposed to antagonist (black). Antagonist compounds screen encompass compounds from the GPCR library screen (grey), methoxy-isoquinolines (open), and ergot alkaloids (striped).Physique E. Toxicity test for screened compounds. (A) HEK293 cells transiently transfected with the F22 cAMP biosensor were incubated with test compounds (10M, 30 mins) and then assayed for forskolin (20M, 30 mins) evoked cAMP generation. Tested ligands showed no effects on luminescence signal values. The mitochondrial complex I inhibitor rotenone served as a positive control. (B) Resazurin reduction assay for cell viability of HEK293 cells exposed to test compounds (10M) and resazurin (10M) for 3 hours at 37C. Fluorescence was measured using a 560nm excitation/590nm emission filter set. Sodium azide was used as a positive control. Physique F. Effects of selected compounds on Sm.5HTR and Hs.5HT7R evoked cAMP Flubendazole (Flutelmium) generation. (A) Inhibition curves shown for the following methoxy-isoquinoline related compounds against Sm.5HTR (blue) and Hs.5HT7R (green): (i) rotundine (data reproduced from Fig 4A), (ii) palmatine, (iii) berberine and (iv) tetrebenazine. (B) Comparison of 5HTR selectivity between (i) the tetrandrine and (ii) berbamine, a structurally related compound.(DOCX) ppat.1005651.s001.docx (2.4M) GUID:?CCDE3DD8-CAEA-49A4-845C-F3019ADC6143 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Schistosomiasis is usually 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 usually challenging. Remarkably, even though over a quarter of Flubendazole (Flutelmium) 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 and validated for 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. Author Summary Parasitic flatworms express a diverse array of G protein coupled receptors, but our knowledge of their pharmacological profile is limited. No high throughput screen of a flatworm GPCR has been reported, even though these targets Flubendazole (Flutelmium) have precedent for high druggability and functionality in the chemotherapeutically vulnerable excitable cell niche. The goal of this study was to establish a method for profiling flatworm G protein coupled receptors that can be scaled to high content screening. Using a cAMP biosensor, we have performed a proof Flubendazole (Flutelmium) of principle miniaturized screen on a schistosome serotonergic GPCR that resolves new ligands that potently and selectivity block 5-HT receptor activity RNAi, allowed us to progressively define pathways engaged by PZQ [11C14]. These studies culminated in a model where PZQ acts as an ergomimetic [13] with PZQ efficacy regulated by the opposing functionality of dopaminergic Flubendazole (Flutelmium) and serotonergic neurons [11C14], known regulators of muscular activity, the tissue where planarian polarity determinants reside [15]. The serotonergic and dopaminergic G protein coupled receptors (GPCRs) engaged by activity of these bioaminergic neurons therefore represent potential downstream PZQ effectors. Their.