title: Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG.
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, (14)C-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 (14)C-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 (14)C-AMG. Compared to (14)C-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.
authors: School of Pharmacy, National Taiwan University, 12F, No. 1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan.
authors: Hung-Chi
authors: HC
authors: Chang
authors: Su-Fu
authors: SF
authors: Yang
authors: Ching-Chun
authors: CC
authors: Huang
authors: Tzung-Sheng
authors: TS
authors: Lin
authors: Pi-Hui
authors: PH
authors: Liang
authors: Chun-Jung
authors: CJ
authors: Lin
authors: Lih-Ching
authors: LC
authors: Hsu
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D015870:Gene Expression; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000378:metabolism; D010695:Phlorhizin / Q000737:chemistry / Q000494:pharmacology; D051269:Sodium-Glucose Transporter 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051297:Sodium-Glucose Transporter 2 / Q000235:genetics / Q000378:metabolism; D000077203:Sodium-Glucose Transporter 2 Inhibitors; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; C495674:SLC5A1 protein, human; C089180:SLC5A2 protein, human; D051269:Sodium-Glucose Transporter 1; D051297:Sodium-Glucose Transporter 2; D000077203:Sodium-Glucose Transporter 2 Inhibitors; C045400:6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose; D010695:Phlorhizin; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; D005944:Glucosamine