Development of Type II Glucose Transporter Inhibitors: Phloretin as a GLUT-2 Screening Template from In Silico Modeling to In Vitro Assessment

Background/Objectives: Hepatocellular carcinoma (HCC) exhibits enhanced glycolytic activity, primarily facilitated by Class I glucose transporters (GLUTs), particularly GLUT-2. Phloretin, a natural polyphenol, is known to modulate glucose transport; however, its isoform-specific interactions and functional impact on HCC metabolism remain unclear. This study compared phloretin’s inhibitory effects on glucose uptake in HCC cells versus normal liver cell models and assessed its binding affinity across Class I GLUTs using molecular docking. Methods: Cytotoxicity was evaluated in HepG2 (HCC) and THLE-2 (normal hepatocyte) cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays to determine biologically relevant concentrations. Glucose uptake at sub-cytotoxic levels was quantified using the fluorescent analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose. To elucidate the molecular mechanism, in silico docking simulations were performed to compare the binding affinities of phloretin, glucose, and reference inhibitors (glutor and cytochalasin B) with the outward-facing conformations of GLUT-1 through GLUT-4. Results: Phloretin induced dose- and time-dependent cytotoxicity, with HepG2 cells exhibiting significantly higher sensitivity than THLE-2 cells. Functionally, phloretin markedly reduced glucose uptake in HepG2 cells, whereas THLE-2 cells showed minimal inhibition. Molecular docking revealed that phloretin occupies the central substrate-binding cavity of Class I GLUTs, forming its most stable interaction network with GLUT-2. Conclusions: These results demonstrate that phloretin selectively inhibits glucose uptake in liver cancer cells, likely through its high-affinity interaction with GLUT-2. Collectively, these findings highlight phloretin’s potential as a metabolic therapeutic agent and support GLUT-2 as a viable target for HCC intervention.