|Title:||Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG||Authors:||Chang H.-C.
|Issue Date:||2013||Journal Volume:||9||Journal Issue:||8||Start page/Pages:||2010-2020||Source:||Molecular BioSystems||Abstract:||
Sodium-coupled glucose co-transporters SGLT1 and SGLT2 play important roles in intestinal absorption and renal reabsorption of glucose, respectively. Blocking SGLT2 is a novel mechanism for lowering the blood glucose level by inhibiting renal glucose reabsorption and selective SGLT2 inhibitors are under development for treatment of type 2 diabetes. Furthermore, it has been reported that perturbation of SGLT1 is associated with cardiomyopathy and cancer. Therefore, both SGLT1 and SGLT2 are potential therapeutic targets. Here we report the development of a non-radioactive cell-based method for the screening of SGLT inhibitors using COS-7 cells transiently expressing human SGLT1 (hSGLT1), CHO-K1 cells stably expressing human SGLT2 (hSGLT2), and a novel fluorescent d-glucose analogue 1-NBDG as a substrate. Our data indicate that 1-NBDG can be a good replacement for the currently used isotope-labeled SGLT substrate, 14C-AMG. The Michaelis constant of 1-NBDG transport (0.55 mM) is similar to that of d-glucose (0.51 mM) and AMG (0.40 mM) transport through hSGLT1. The IC50 values of a SGLT inhibitor phlorizin for hSGLT1 obtained using 1-NBDG and 14C-AMG were identical (0.11 μM) in our cell-based system. The IC50 values of dapagliflozin, a well-known selective SGLT2 inhibitor, for hSGLT2 and hSGLT1 determined using 1-NBDG were 1.86 nM and 880 nM, respectively, which are comparable to the published results obtained using 14C-AMG. Compared to 14C-AMG, the use of 1-NBDG is cost-effective, convenient and potentially more sensitive. Taken together, a non-radioactive system using 1-NBDG has been validated as a rapid and reliable method for the screening of SGLT1 and SGLT2 inhibitors. ? 2013 The Royal Society of Chemistry.
|ISSN:||1742206X||DOI:||10.1039/c3mb70060g||SDG/Keyword:||4 chloro 7 nitrobenzofurazan; 6 deoxy N (7 nitrobenz 2 oxa 1,3 diazol 4 yl)aminoglucose; 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose; drug derivative; fluorescent dye; glucosamine; glucose; phlorizin; SLC5A1 protein, human; SLC5A2 protein, human; sodium glucose cotransporter 1; sodium glucose cotransporter 2; 4 chloro 7 nitrobenzofurazan; fluorescent dye; glucosamine; glucose; molecular probe; sodium glucose cotransporter 1; sodium glucose cotransporter 2; animal; article; cell strain COS1; chemistry; Chlorocebus aethiops; CHO cell; Cricetulus; drug antagonism; gene expression; genetics; human; kinetics; metabolism; molecular probe; spectrofluorometry; synthesis; transport at the cellular level; analogs and derivatives; antagonists and inhibitors; CHO cell line; COS 1 cell line; molecular probe; 4-Chloro-7-nitrobenzofurazan; Animals; Biological Transport; Cercopithecus aethiops; CHO Cells; COS Cells; Cricetulus; Fluorescent Dyes; Gene Expression; Glucosamine; Glucose; Humans; Kinetics; Molecular Probes; Phlorhizin; Sodium-Glucose Transporter 1; Sodium-Glucose Transporter 2; Spectrometry, Fluorescence; 4-Chloro-7-nitrobenzofurazan; Animals; Biological Transport; Cercopithecus aethiops; CHO Cells; COS Cells; Cricetulus; Fluorescent Dyes; Gene Expression; Glucosamine; Glucose; Humans; Kinetics; Molecular Probes; Phlorhizin; Sodium-Glucose Transporter 1; Sodium-Glucose Transporter 2; Spectrometry, Fluorescence
|Appears in Collections:||藥學系|
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