Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist S6 Stimulates Insulin Secretion From Rat Islets.

Glucagon-like peptide-1 receptor (GLP-1R) agonist-based therapeutics for type 2 diabetes mellitus have attracted worldwide attention. However, there are challenges in the development of small molecule GLP-1R agonists owing to the complexity of ligand recognition and signal induction mechanisms. Here, we attained S6 using virtual screening and fluorescent imaging plate reader (FLIPR)-based calcium assays. The purpose of this study was to identify and characterize S6, a novel small molecule GLP-1R agonist. Data from cellular thermal shift assay (CETSA) and Bio-Layer Interferometry (BLI) indicated that S6 could bind potently with GLP-1R. Radioimmunoassay data showed that S6 potentiated insulin secretion in a glucose-dependent manner and the insulinotropic effect was mediated by GLP-1R. Calcium imaging techniques suggested that S6 elevated the intracellular calcium concentration [(Ca2+)i] by activating GLP-1R. In patch-clamp experiments, we demonstrated that S6 inhibited voltage-dependent K+ (Kv) channels in a GLP-1R-dependent fashion. Besides, S6 significantly prolonged action potential duration but had no effect on voltage-dependent Ca2+ channels. In summary, these findings indicate that S6 stimulates glucose-dependent insulin secretion mainly by acting on GLP-1R, inhibiting Kv channels, increasing (Ca2+)i. This study will provide direction for the screening and development of novel small-molecule agents targeting GLP-1R in the future.

Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity.

The intracellular calcium concentration ([Ca(2+)]i) is an important readout for in vitro neurotoxicity since calcium is critically involved in many essential neurobiological processes, including neurotransmission, neurodegeneration and neurodevelopment. [Ca(2+)]i is often measured with considerable throughput at the level of cell populations with plate reader-based assays or with lower throughput at the level of individual cells with fluorescence microscopy. However, these methodologies yield different quantitative and qualitative results. In recent years, we demonstrated that the resolution and sensitivity of fluorescence microscopy is superior compared to plate reader-based assays. However, it is currently unclear if the use of plate reader-based assays results in more 'false negatives' or 'false positives' in neurotoxicity screening studies. In the present study, we therefore compared a plate reader-based assay with fluorescence microscopy using a small test set of environmental pollutants consisting of dieldrin, lindane, polychlorinated biphenyl 53 (PCB53) and tetrabromobisphenol-A (TBBPA). Using single-cell fluorescence microscopy, we demonstrate that all test chemicals reduce the depolarization-evoked increase in [Ca(2+)]i, whereas lindane, PCB53 and TBBPA also increase basal [Ca(2+)]i, though via different mechanisms. Importantly, none of these effects were confirmed with the plate reader-based assay. We therefore conclude that standard plate reader-based methods are not sufficiently sensitive and reliable to measure the highly dynamic and transient changes in [Ca(2+)]i that occur during chemical exposure.