GLUT2 shares characteristic motifs and high similarity with the other Class I representatives GLUTs 1, 3, and 47. In this group, it is the only transporter that also accepts fructose as a substrate6. This prompted us to test the identified GLUT2-inhibiting compounds against all Class I GLUTs and the Class II member GLUT5, a fructose-only transporter, to elucidate their specificity. Except for G2iF, which also inhibits other Class I GLUTs (although to a lesser extent: IC50 in GLUT2 = 7µM, IC50 in other Class I GLUTs ≥ 14 µM), and G2iI which inhibits GLUT5 (IC50 = 23 µM) less potently than GLUT2 (IC50 = 13 µM), the other nine GLUT2 ligands did not show a significant effect on the tested transporters (Fig. 3). Although so far unknown effects on other human transporters cannot be ruled out completely, these data indicate that, for the first time, very potent and specific GLUT2 inhibitors were identified.
A possible explanation for the high prevalence of GLUT2-selective inhibitors among the identified GLUT2 inhibitors may be that the inhibitors target the upper portion of the substrate cavity. All 11 inhibitors dock at the substrate cavity entrance, separately from the glucose binding site (Fig. 1B, Fig. 4). The substrate cavity base containing the glucose binding site is made up of residues mostly conserved in Class I GLUTs (Supplementary Fig. S1A). The cytosolic entrance of the substrate cavity surrounded by soluble loops, especially the large loop connecting the N- and C-domains of the transporter (GLUT2 amino acid residues 240–298), has much more variability in the protein sequence (Supplementary Fig. S1). Thus, the binding site location of the GLUT2 inhibitors is consistent with the GLUT2 selectivity exhibited by most of these ligands and suggests that targeting the entrance of the substrate cavity, whether in the inward- or outward-facing conformations, for ligand screening may increase the chances of producing GLUT-specific ligands. Also, given the separation between glucose and such inhibitor sites, bridging the two sites by attaching a glucosyl group to these types of inhibitors may substantially improve inhibitor potency while maintaining selectivity. Additionally, such compounds could help to crystallize GLUTs whose structures are yet unknown, including GLUT2, as the combination of substrate and inhibitor would greatly stabilize the transporter conformation.
A significant difference in the substrate sites between Class I GLUTs and GLUT5 is W420GLUT2 (W388GLUT1), conserved in Class I GLUTs but replaced by a smaller residue in Class II GLUTs (e.g., A396 in GLUT5). This substitution creates more space in the substrate cavity, changing the binding mode of ligands and substrate specificity31. For instance, GLUT5A396W mutant became a transporter of both glucose and fructose, while the wild-type can only transport fructose. Therefore, it is likely that GLUT2 inhibitors adopt different binding modes in GLUT5 than those described for GLUT2.
Analysis of the docked GLUT2 inhibitors from the virtual ligand screening (Fig. 4) suggests that L436, R280, A283 - residues not conserved in other Class I GLUTs or GLUT5 - may play a role in the selectivity of GLUT2 inhibitors. The equivalent substitutions of R280 in other GLUTs (S248GLUT1, S246GLUT3, K264GLUT4, D254GLUT5) would decrease or abolish this residue’s interactions with G2iA, G2iB, G2iC, G2iE, and G2iG (Fig. 4). A bulkier side chain in the position of A283 (M251GLUT1, M249GLUT3, L267GLUT4, E257GLUT5) could sterically interfere with the binding of G2iD, G2iG, G2iI, G2iJ, and G2iK. L436 (I404GLUT1, M402GLUT3, M420GLUT4, F412GLUT5) may be important in shaping the hydrophobic interactions in the binding pockets for G2iA, G2iB, G2iE, G2iH, and G2iI. The recognition of G2iF, the pan-Class I GLUT inhibitor, relies mostly on R244, a conserved residue in GLUT1-5. This sidechain has hydrogen bond and cation-p interactions with G2iF, suggesting that the guanidinium group’s position is critical. The quinoline nitrogen of G2iF makes a hydrogen bond with K249 (N217GLUT1, N215GLUT3, I233GLUT4, Q223GLUT5) and a weak interaction with D120 (N88GLUT1, N86GLUT3, Q104GLUT4, N94GLUT5). The equivalent substitutions in these positions for GLUT1-4 are still able to maintain interactions with G2iF. In GLUT4, the substitution of D120 with a glutamine residue would result in better interaction with G2iF, consistent with the lower IC50 of this inhibitor for GLUT4, relative to the other Class I GLUTs (Fig. 3).
With this study, we present a range of molecules that will serve as valuable tools to investigate the physiological role of GLUT2 in health and disease and may evolve to therapeutic drugs in GLUT2-related diseases. GLUT2 involvement in many important diseases has been described26,30,41,42. It is upregulated in several cancer types like pancreatic, hepatic, micropapillary, or colon cancer43. Inhibition of GLUT2 via the non-specific inhibitor phloretin has been shown to diminish tumor growth in colon cancer44 and hepatocellular carcinoma45. Although the significance of GLUT2 inhibition in these tissues has been demonstrated by siRNA experiments44,45 or comparison of cell lines with varying GLUT2 protein expression profiles44, involvement of other GLUTs, besides GLUT2, in these tumor tissues is probable and off-target effects by applying phloretin cannot be ruled out. The Class I GLUTs 1 and 3 are also overexpressed in many cancer types and related to elevated tumor growth and poor survival46. For cancer treatment, the non-specific inhibitor G2iF that inhibits Class I GLUTs but not GLUT5 might join phloretin as a putative drug45. Furthermore, substantial overexpression of the fructose transporters GLUT2 and GLUT5 lead to the hypothesis that certain cancer cells use fructose as a preferential carbon source43. In these cases, the here presented GLUT2/GLUT5 inhibiting compound G2iI might be a promising candidate in the combat against cancer and other high-fructose diet-related diseases47. Importantly, a potent and GLUT2-specific effector (e.g., G2iA) might further elucidate the particular role of GLUT2 in tumor pathogenesis and facilitate studies targeting GLUT2, thereby contributing to unravel complex cancer behavior further.
In healthy individuals, GLUT2 traffics to the apical side of the brush border membrane only after a meal, when glucose concentrations in the lumen are high, to support SGLT1 and accelerate glucose uptake26. In morbidly obese humans, a consistent location of GLUT2 at the apical membrane, even in fasting states, was observed and related to insulin resistance48. Because GLUT2 is a facilitator that transports glucose along a concentration gradient, the transporter’s continuous presence at the apical membrane enables transport from the blood through the basolateral membrane to the intestine. Higher blood glucose levels due to insulin resistance might result in higher glucose levels in the lumen in fasting states. The abnormal sugar supply supports bacterial growth and might interfere with a healthy gut microbiome48. Specific inhibition of GLUT2 could mitigate such pathologies. However, inhibition after a meal may have the opposite effect, with glucose malabsorption leading to higher glucose concentrations in the intestine, fueling bacterial growth49. An altered microbiome composition in mice with intestinal-specific GLUT2 deletion has been detected in previous studies49, supporting the gut microbiome as a possible field of application for GLUT2 inhibitors. Also, Schmitt et al. showed that GLUT2 deletion in the murine intestine causes favorable effects like improved glucose tolerance and diminished body weight gain49. This suggests that GLUT2 tailored inhibitors could lead to similar results and might be applied in morbidly obese patients or type 2 diabetic persons with beneficial health effects.
Interestingly, viral infections affect the expression of GLUT2. While the hepatitis C virus downregulates GLUT2 expression50, the transmissible gastroenteritis virus upregulates the transporter’s expression, enhancing intestinal glucose absorption, which promotes viral replication51. Hence, GLUT2 inhibition could assist in the containment of certain viruses. Clearly, the role of GLUT2 in the metabolic processes is highly complex and not fully understood. Therefore, the application of GLUT2-specific inhibitors also bears high risks as it might have not only beneficial but also adverse effects, and more studies are necessary to increase our level of knowledge. However, accessibility of specific GLUT2 inhibitors represents a tremendous advantage over less-specific GLUT inhibitors in developing drugs with a defined effective spectrum and lower side effects.
These compounds are valuable tools in the efforts of answering many open questions concerning GLUT2. For instance, it is still unclear how GLUT2 is mobilized in response to glucose in various cell types and different pathologies52. Possible players include the type of membrane lipids53, protein partners52,54, or glycosylation3. Distinct from other GLUTs, the extraordinary low affinity for glucose and fructose probably assigns special functions of glucose sensing23 and signaling18 to GLUT2, but the detailed molecular functions remain to be elucidated. Guillemain et al. showed that the intracellular loop between transmembrane domains 6 and 7 of GLUT2 is responsible for the glucose signal-transduction from the cell membrane to the nucleus via a complex mechanism involving further proteins18. However, the question remains if this signal cascade is triggered by GLUT2 mediated glucose transport or if other transporters are involved in sensing the sugar. With GLUT2-specific inhibition, glucose transport via GLUT2 and its function as signal transductor could be disentangled, helping to address this question. Also, GLUT2 plays a pivotal role in glucose sensing in brain astrocytes54, but the detailed mechanisms are unknown. Furthermore, the Fanconi-Bickel syndrome due to GLUT2 malfunction 30 has various symptoms that indicate yet undiscovered physiological roles for GLUT2, and the transporter’s role in certain cancer types remains unclear43. Future studies will benefit from the existence of a range of easily accessible GLUT2-specific inhibitors with varying affinities.