Date Published: May 17, 2016
Publisher: Public Library of Science
Author(s): John D. Chan, John D. McCorvy, Sreemoyee Acharya, Malcolm E. Johns, Timothy A. Day, Bryan L. Roth, Jonathan S. Marchant, Timothy G. Geary.
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.
The neglected tropical disease Schistosomiasis is the most socioeconomically devastating helminth infection, and the second most burdensome parasitic infection behind malaria, infecting over 200 million people worldwide . Infected individuals are treated by the drug praziquantel (PZQ), the mainstay therapeutic for disease control.
In this study we executed a proof of principle pilot screen to evidence the feasibility of pharmacological profiling a flatworm GPCR in HTS format. The assay system employed relied on an genetically encoded luminescent biosensor . This is an appealing approach as this strategy is non-destructive and affords the ability to continually monitor the kinetics of cAMP generation from a single sample. Further by directly reporting cAMP levels, rather than transcriptional outcomes (e.g. cAMP reporter genes), this approach also reveals proximal receptor activity in real time to help discern how specific compounds are modulating GPCR activity. Acceptable Z’ scores were reliably obtained in 384 well format (Fig A in S1 Text), and we note the sensitivity of this approach has permitted responses from endogenous GPCRs to be resolved even in ultra-high throughput screening formats (3456-well plates). Obviously, this particular biosensor is suited only for Gs and Gi-coupled GPCRs, but the optimization of other biosensors—for example, genetically-encoded Ca2+ indicators , or reporters that are agonist independent —should aid HTS profiling of other flatworm GPCRs coupling to different second messenger cascades.