Identification of novel small molecules as potential SGLT2 inhibitors through combined virtual screening and experimental validation.

The sodium-glucose co-transporter 2 (SGLT2) plays an important role in mediating glucose reabsorption within the renal filtrate and regulating blood glucose levels, which makes it a well-known target for diabetes mellitus. A number of SGLT2 inhibitors (SGLT2i) have been established as important antidiabetic drugs, and research on new SGLT2i with high affinity is ongoing. Herein, 101 compounds were screened from a compound library (approximately 16,000 compounds) using a virtual screening workflow that integrated various docking programs, pharmacophore modeling, and druggability filter. To verify the results of virtual screening, we established a HK-2 cell model with d-glucose derivative 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) for measuring glucose uptake via SGLT2. 12 candidate compounds were selected and purchased for subsequent experimental validation. Among these, 3 non-glycoside compounds significantly inhibited the 2-NBDG uptake in a dose-dependent manner and their IC₅₀ values for SGLT2 were 71.43 μM, 72.66 μM, and 91.44 μM, respectively. Mechanism studies demonstrated that all 3 compounds significantly downregulated SGLT2 level and activated silent information regulator 1 (SIRT1) expression in high-glucose-induced cell injury models. These findings confirmed the ability of these compounds to bind to SGLT2 and also revealed their potential mechanisms in regulating oxidative stress and metabolism. Furthermore, molecular dynamics simulation indicated the high binding stability of SGLT2 and 3 compounds complexes during a 100-ns simulation period. In conclusion, our results identified 3 potential SGLT2i and preliminarily elucidated their mechanism of action, which lays the foundation for the development of novel and potent non-glycoside SGLT2i in future.