Non-competitive Blocking of Human Glut1 Hexose

28 29 The GLUT1 transporter has become an attractive target to block glucose uptake in malignant cells 30 since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines 31 are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains 32 unknown. Here, we use a combination of binding and glucose transport kinetic assays to analyze in 33 detail the effect of caffeine, pentoxifylline and theophylline on hexose transport in human erythrocytes. 34 The displacement of previously bound cytochalasin B revealed a direct interaction between the 35 methylxanthines and GLUT1. The methylxanthines behave as non-competitive blockers (KI values of 236 3 mM) in exchange and zero-trans efflux assays, whereas in mixed inhibition with a notable un37 competitive component is observed in zero-trans influx assays (KI values among 5-12 mM). These 38 results indicate that the methylxanthines do not bind to either the exofacial or endofacial D-glucose 39 binding sites, but instead, they interact at a different site accessible by the external face of the 40 transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline 41 disturbed D-glucose for binding to the exofacial substrate site. Interestingly, co-inhibition assays 42 showed that the methylxanthines bind to a common site on the transporter. We concluded that there is 43 a methylxanthine regulatory site on the external surface of the transporter, which is close but 44 distinguishable from the D-glucose external site. Therefore, the methylxanthine moiety may become an 45 attractive framework for the design of novel specific non-competitive facilitative glucose transporter 46 inhibitors. 47 48 49