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.

Imaging by atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells.

The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. This cochlear amplification is thought to be generated by the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. Although a fructose transporter, GLUT-5, was initially proposed to be this protein, a later study identified the gene of the motor protein distributed throughout the OHC plasma membrane. This protein has been named "prestin." However, although previous morphological studies by electron microscopy and atomic force microscopy (AFM) found the lateral wall of OHCs to be covered with 10-nm particles, believed to be motor proteins, it is unknown whether such particles consist only of prestin or are a complex of GLUT-5 and prestin molecules. To determine if the 10-nm particles are indeed constituted only of prestin, plasma membranes of prestin-transfected and untransfected Chinese hamster ovary (CHO) cells, which do not express GLUT-5, were observed by AFM. First, the cells attached to a substrate were sonicated so that only the plasma membrane remained on the substrate. The cytoplasmic face of the cell was observed by the tapping mode of the AFM in liquid. As a result, particle-like structures were recognized on the plasma membranes of both the prestin-transfected and untransfected CHO cells. Comparison of the difference in the frequency distribution of these structures between those two cells showed approximately 75% of the particle-like structures with a diameter of 8-12 nm in the prestin-transfected CHO cells to be possibly constituted only by prestin molecules. Our data suggest that the densely packed 10-nm particles observed on the OHC lateral wall are likely to be constituted only of prestin molecules.