A Fluorescence-Based Assay to Probe Inhibitory Effect of Fructose Mimics on GLUT5 Transport in Breast Cancer Cells

Rapid cell divisionand reprogramming of energy metabolism aretwo crucial hallmarks of cancer cells. In humans, hexose traffickinginto cancer cells is mainly mediated through a family of glucose transporters(GLUTs), which are facilitative transmembrane hexose transporter proteins.In several breast cancers, fructose can functionally substitute glucoseas an alternative energy supply supporting rapid proliferation. GLUT5,the principal fructose transporter, is overexpressed in human breastcancer cells, providing valuable targets for breast cancer detectionas well as selective targeting of anticancer drugs using structurallymodified fructose mimics. Herein, a novel fluorescence assay was designedaiming to screen a series of C-3 modified 2,5-anhydromannitol (2,5-AM)compounds as d-fructose analogues to explore GLUT5 bindingsite requirements. The synthesized probes were evaluated for theirability to inhibit the uptake of the fluorescently labeled d-fructose derivative 6-NBDF into EMT6 murine breast cancer cells.A few of the compounds screened demonstrated highly potent single-digitmicromolar inhibition of 6-NBDF cellular uptake, which was substantiallymore potent than the natural substrate d-fructose, at a levelof 100-fold or more. The results of this assay are consistent withthose obtained from a previous study conducted for some selected compoundsagainst F-18-labeled d-fructose-based probe 6-[F-18]-FDF, indicating the reproducibility of the current non-radiolabeledassay. These highly potent compounds assessed against 6-NBDF openavenues for the development of more potent probes targeting GLUT5-expressingcancerous cells.

Automated summary

Rapid cell division and reprogramming of energy metabolism are crucial characteristics of cancer cells. In breast cancer, fructose can substitute glucose as an alternative energy source. GLUT5, the fructose transporter, is overexpressed in breast cancer cells, making it a valuable target for cancer detection and selective targeting of drugs. A novel fluorescence assay was used to screen and identify highly potent C-3 modified compounds as d-fructose analogues, providing avenues for the development of more potent probes targeting GLUT5-expressing cancer cells.