Chang Hung-Chi, Yang Su-Fu, Huang Ching-Chun, Lin Tzung-Sheng, Liang Pi-Hui, Lin Chun-Jung, Hsu Lih-Ching
School of Pharmacy, National Taiwan University, 12F, No. 1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan.
Mol Biosyst. 2013 Aug;9(8):2010-20. doi: 10.1039/c3mb70060g. Epub 2013 May 8.
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.
钠偶联葡萄糖共转运蛋白SGLT1和SGLT2分别在葡萄糖的肠道吸收和肾脏重吸收中发挥重要作用。阻断SGLT2是通过抑制肾脏葡萄糖重吸收来降低血糖水平的一种新机制，目前正在研发选择性SGLT2抑制剂用于治疗2型糖尿病。此外，有报道称SGLT1功能紊乱与心肌病和癌症有关。因此，SGLT1和SGLT2都是潜在的治疗靶点。在此，我们报告了一种基于细胞的非放射性方法的开发，该方法使用瞬时表达人SGLT1（hSGLT1）的COS-7细胞、稳定表达人SGLT2（hSGLT2）的CHO-K1细胞以及一种新型荧光d-葡萄糖类似物1-NBDG作为底物来筛选SGLT抑制剂。我们的数据表明，1-NBDG可以很好地替代目前使用的同位素标记的SGLT底物（14）C-AMG。1-NBDG转运的米氏常数（0.55 mM）与d-葡萄糖（0.51 mM）和AMG（0.40 mM）通过hSGLT1转运的米氏常数相似。在我们基于细胞的系统中，使用1-NBDG和（14）C-AMG获得的SGLT抑制剂根皮苷对hSGLT1的IC50值相同（0.11 μM）。使用1-NBDG测定的著名选择性SGLT2抑制剂达格列净对hSGLT2和hSGLT1的IC50值分别为1.86 nM和880 nM，这与使用（14）C-AMG获得的已发表结果相当。与（14）C-AMG相比，使用1-NBDG具有成本效益、方便且可能更灵敏。综上所述，使用1-NBDG的非放射性系统已被验证为一种快速可靠的筛选SGLT1和SGLT2抑制剂的方法。
