Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems.

Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.

Osmotic water transport with glucose in GLUT2 and SGLT.

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na(+)-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na(+)-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.