Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides

Abstract

The specificity characteristics of transporters can be exploited for the development of novel diagnostic therapeutic probes. The facilitated hexose transporter family (GLUTs) has a distinct set of preferences for monosaccharide substrates, and while some are expressed ubiquitously (e.g., GLUT1), others are quite tissue specific (e.g., GLUT5, which is overexpressed in some breast cancer tissues). While these differences have enabled the development of new molecular probes based upon hexose- and tissue-selective uptake, substrate design for compounds targeting these GLUT transporters has been encumbered by a limited understanding of the molecular interactions at play in hexose binding and transport. Four new fluorescently labeled hexose derivatives have been prepared, and their transport characteristics were examined in two breast cancer cell lines expressing mainly GLUTs 1, 2, and 5. Our results demonstrate, for the first time, a stringent stereochemical requirement for recognition and transport by GLUT5. 6-NBDF, in which all substituents are in the d-fructose configuration, is taken up rapidly into both cell lines via GLUT5. On the other hand, inversion of a single stereocenter at C-3 (6-NBDP), C-4 (6-NBDT), or C-5 (6-NDBS) results in selective transport via GLUT1. An in silico docking study employing the recently published GLUT5 crystal structure confirms this stereochemical dependence. This work provides insight into hexose-GLUT interactions at the molecular level and will facilitate structure-based design of novel substrates targeting individual members of the GLUT family and forms the basis of new cancer imaging or therapeutic agents.

Tunable GLUT–Hexose Binding and Transport via Modulation of Hexose C‐3 Hydrogen‐Bonding Capabilities

“…Although the experiments above confirmed GLUT5 recognition of 2,5‐AM derivatives that were functionalized with a hydrogen bond donor at C‐3, they did not provide any evidence for their subsequent GLUT5‐mediated transport. Taking inspiration from readily quantified fluorescence experiments of hexose probes modified at C‐6 with the NBD fluorophore, we prepared the NB D ‐labeled probe 8 to assess uptake directly. A higher level of inhibition of [ 14 C]‐ d ‐fructose transport into EMT‐6 cells by 8 as compared with inhibition of [ 14 C]‐ d ‐glucose (Figures and ) suggests that this compound has a higher affinity for d ‐fructose transporters.…”

A comprehensive overview of substrate specificity of glycoside hydrolases and transporters in the small intestine

“…NBDM transport was not inhibited by d-glucose and d-glucosamine, pointing towards GLUT5 mediated transport. An interesting SAR was performed by Soueidan et al based on the synthesized C-6 NBD conjugates of both d-fructose and the C-3 (6-NBDP), C-4 (NBDT) and the C-5 (6-NBDS) epimers [127]. 6-NBDF showed rapid take up into MCF-7 and EMT-6 cell lines.…”

“…13 Radiolabelled (a) and fluorescent probes (b) used in GLUT5 mediated uptake studies. References used: ref 1 [120], ref 2 [121], ref 3 [122], ref 4 [123], ref 5 [124], ref 6 [125], ref 7 [126], ref 8 [127], ref 9 [128] for rapid detection in breast cancer models [128]. A significant difference was found in the cancerous cells MCF-7 and MCF10AneoT compared to normal breast cells MCF10A underlining their potential for rapid on-site high-throughput diagnostics.…”

Molecular Tools for Facilitative Carbohydrate Transporters (Gluts)

“…The availability of NBD as a fluorophore that does not impede Glut‐mediated uptake contributed to a direct evaluation of the impact that stereochemical factors have on fructose uptake. The C6‐NBD conjugates of fructose epimers (Scheme ) synthesized and evaluated by the West and Cheeseman team showed alteration in the mechanism of the uptake. Thus, in contrast to the C1‐analogue, 6‐NBDF (Scheme ) was found to be transported specifically through Glut 5.…”