A Preliminary Study On The Mechanisms Of Novel Small Molecule GLP-1R Agonist S6 Stimulating Insulin Secretion In Rats

Objective:To study and verify the effect of a novel small molecule GLP-1R agonist S6 on insulin secretion and investigate its underlying electrophysiological mechanism in primary cultured rat pancreaticβ-cells.Methods:1)Pharmacophore-based virtual screening combined with molecular docking to screen small molecular compounds.2)The changes in intracellular calcium concentration were detected by the FLIPR^?fluorescence imaging detection system to identify the activation effect of the test compound to GLP-1R.3)The effect of S6 on insulin secretion under different conditions was observed.4)Changes in intracellular Ca²⁺concentration after administration of S6 was detected by Calcium imaging.5)The effects of S6 on voltage-dependent calcium（VDCC）channels,voltage-dependent potassium（Kv）channels and action potential duration from isletβcells under different conditions were detected by Whole cell Patch Clamp techniques.6)The targeting effect of S6 on GLP-1R was observed by CETSA.Results:1)5809 compounds were picked out from the ZINC small molecule database through virtual screening combined with molecular docking.Followed by screening based on the physicochemical properties of oral compounds,the final 108 compounds meet the requirements.30 commercial compounds were finally purchased.2)16 out of30 teste compounds showed GLP-1R agonistic effects,of which S6 had the potent agonistic effect.3)Insulin secretion results showed that S6 augmented the insulin secretion under high glucose conditions in rats.4)Calcium imaging techniques showed a dramatic increase in intracellular Ca²⁺after treatment ofβcells with S6.5)The results of patch clamp experiments showed that S6 prolonged action potential duration,decreased the current density of Kv channels in pancreaticβ-cells.6)Patch clamp experiments showed that S6 had no direct effect on voltage-depended calcium channels.7)Insulin secretion and calcium imaging technology collectively indicted that S6 elevated the intracellular Ca²⁺via the releasing of calcium store.8)The expression of GLP-1R showed a downward trend with increasing temperature.CETSA results showed that the expression of GLP-1R in S6 intervention group was higher than control at the same temperature.9)S6-induced insulin secretion was blocked by the GLP-1R antagonist Exendin（9-39）.10)The results of patch clamp experiments showed that Exendin（9-39）reversed the inhibition of S6 on Kv channels.11)Calcium imaging showed that S6-induced[Ca²⁺]i elevations was eliminated by Exendin（9-39）.Conclusion:S6 stimulates insulin secretion only in the context of high glucose concentrations in a glucose-regulated manner,which suggests that while effective in reducing hyperglycemia,S6 maximum minimized the risk of hypoglycemia.The electrophysiological mechanism of S6-mediated insulin secretion is that S6 activates GLP-1R,inhibiting Kv channels and prolonging action potential duration,then prolonging the opening time of voltage-dependent calcium channel,leading to the increase of intracellular calcium concentration,and finally triggering insulin secretion.In addition,S6 also promotes the increase in[Ca²⁺]i through the release of intracellular calcium pool.In summary,the results show the insulin promoting effect of S6,as a small molecule GLP-1 receptor agonist（GLP-1RA）,and provide insight for further research in this GLP-1RA field.