Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems

Sina Schmidl,Jun-Yong Choe,Mislav Oreb,Tudor I Oprea,Oleg Ursu,Cristina V Iancu

doi:10.1038/s41598-021-93063-5

Abstract

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.

Highlights

Human glucose transporters (GLUTs), proteins of the SLC2 gene family, facilitate the diffusion of hexoses into the cell and play a pivotal role in glucose homeostasis[1]
Depending on which side of the cell membrane the substrate cavity opens to, GLUTs have two major conformations captured by the crystal structures of some isoforms and GLUT bacterial homologs[5,12,13,14,15]
In silico ligand screening requires a structural model for the protein target

Summary

Introduction

Human glucose transporters (GLUTs), proteins of the SLC2 gene family, facilitate the diffusion of hexoses into the cell and play a pivotal role in glucose homeostasis[1]. The 14 GLUT isoforms present in humans show an amino acid identity of 19–65% (homology of 42–81%)[5] but differ in substrate specificity, affinity, and tissue distribution[6]. According to their sequence similarities, three classes of GLUTs have been d efined[7] with GLUT1-4 representing Class I, GLUTs 5, 7, 9, and 11 in Class II and GLUTs 6, 8, 10, 12, and 13 forming Class III6,7. Inactivating mutations in the GLUT2-encoding gene lead to the rare but severe Fanconi–Bickel s yndrome[26] Patients suffering from this autosomal, recessive disease show very diverse symptoms[26], and its treatment is challenging due to the lack of effective drugs[27]. Identifying GLUT2-specific ligands might provide new ways to explore the basis of substrate specificity among GLUT members[31]

Methods

Results

Conclusion