Structural selectivity of human SGLT inhibitors.

摘要

Human Na~+-D-glucose cotransporter (hSGLT) inhibitors constitute the newest class of diabetes drugs, blocking up to 50% of renal glucose reabsorption in vivo. These drugs have potential for widespread use in the diabetes epidemic, but how they work at a molecular level is poorly understood. Here, we use electrophysiological methods to assess how they block Na~+-D-glucose cotransporter SGLT1 and SGLT2 expressed in human embryonic kidney 293T (HEK-293T) cells and compared them to the classic SGLT inhibitor phlorizin. Dapagliflozin [(1S)-1,5,5-anhydro-1 -C- {4-chloro-3- [(4-ethoxyphe-nyl)methyl]phenyl}-D-glucitol], two structural analogs, and the agly-cones of phlorizin and dapagliflozin were investigated in detail. Dapagliflozin and fluoro-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl} -4-F-4-deoxy-D-glucitol] blocked glucose transport and glucose-coupled currents with approx=100-fold specificity for hSGLT2 (K_i = 6 nM) over hSGLT1 (K_i = 400 nM). As galactose is a poor substrate for SGLT2, it was surprising that galacto-dapagliflozin [(15)-1,5-anhydro-1 -C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-galactitol] was a selective inhibitor of hSGLT2, but was less potent than dapagliflozin for both transporters (hSGLT2 K_i = 25 nM, hSGLT1 K_i = 25,000 nM). Phlorizin and galacto-dapagliflozin rapidly dissociated from SGLT2 [half-time off rate (t_(1/2, Off)) approx= 20-30 s], while dapagliflozin and fluoro-dapagliflozin dissociated from hSGLT2 at a rate 10-fold slower (t_(1/2, Off) >=180 s). Phlorizin was unable to exchange with dapagliflozin bound to hSGLT2. In contrast, dapagliflozin, fluoro-dapagliflozin, and galacto-dapagliflozin dissociated quickly from hSGLT1 (t_(1/2,Off) = 1-2 s), and phlorizin readily exchanged with dapagliflozin bound to hSGLTl. The aglycones of phlorizin and dapagliflozin were poor inhibitors of both hSGLT2 and hSGLT1 with K_i values > 100 muM. These results show that inhibitor binding to SGLTs is composed of two synergistic forces: sugar binding to the glucose site, which is not rigid, and so different sugars will change the orientation of the aglycone in the access vestibule; and the binding of the aglycone affects the binding affinity of the entire inhibitor. Therefore, the pharmacophore must include variations in both the structure of the sugar and the aglycone.