[en] Background
Succinic acid (SA), a metabolic component that takes part in the citric acid cycle, has been described as the cognate agonist for the orphan receptor SUCNR1 (GPR91). This receptor belongs to the G Protein-Coupled Receptor family (GPCR), that play an essential role in regulating many physiological functions and represent 30% of targets for currently marketed drugs. Several studies on KO models suggested different roles for SUCNR1 through its inactivation. Nevertheless, the characterization of the pharmacology and physiology of SUCNR1 is limited by the lack of small molecules used as pharmacological tools.
Methods
In order to identify ligands modulating SUCNR1 Gαi activity, we adapted a specific cAMP assay based on a modified luciferase fused with cAMP binding domain (Glosensor® promega corporation). Upon cAMP binding, conformational changes induce luminescent signal. This sensitive assay was carried out in a real time measurements fashion. It was compatible with the screening of chemical libraries. We utilized a SOSA library based on the principle that active compounds might have an activity on new targets at high concentration.
Results
We performed a primary agonist screening on 1280 compounds of a SOSA library at two different concentrations (100 and 10µM) with a cAMP assay (Z’=0,4-0,6). We selected 114 out of them that were characterized at least by an increase (or decrease) of 20% of the luminescent signal from HEK293.SUCNR1 cells. These compounds were subjected to a secondary screening performed in triplicates. Results analysis provided 16 putative modulators of SUCNR1 Gαi activity. We followed a similar strategy in another screening to find candidates with an antagonist profile.
In parallel, we docked a part of the ZINC database ‘‘lead-like’’ molecules against SUCNR1 protein built by homology modelling from the β2-adrenergic receptor. This virtual screening has sorted 30 compounds with substantial theoretical affinity. They are currently undergoing functional evaluation in the cAMP assay.
Conclusions
We set up a real-time luminometric cAMP assay for SUCNR1. We selected a dozen of putative modulators of SUCNR1 that were selected for thorough evaluation. Furthermore, we obtained 30 potentially active compounds from a virtual screening that are assayed for functional activity at the receptor. The pharmacology of hits is currently characterized with different models and assays.
