title: Structural selectivity of human SGLT inhibitors.
abstract: 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-ethoxyphenyl)methyl]phenyl}-D-glucitol], two structural analogs, and the aglycones 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 ≈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 [(1S)-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)) ≈ 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 hSGLT1. The aglycones of phlorizin and dapagliflozin were poor inhibitors of both hSGLT2 and hSGLT1 with K(i) values > 100 μM. 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.
authors.affiliation: Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1751, USA.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Hummel
authors.forename: Chuan
authors.initials: C
authors.lastname: Lu
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Chiari
authors.initials: C
authors.lastname: Ghezzi
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Hirayama
authors.forename: Donald D F
authors.initials: DD
authors.lastname: Loo
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kepe
authors.forename: Jorge R
authors.initials: JR
authors.lastname: Barrio
authors.forename: Ernest M
authors.initials: EM
authors.lastname: Wright
mesh_terms: D001559:Benzhydryl Compounds; D001692:Biological Transport / Q000502:physiology; D003920:Diabetes Mellitus / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D015394:Molecular Structure; D018408:Patch-Clamp Techniques; D010695:Phlorhizin / Q000737:chemistry / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D051297:Sodium-Glucose Transporter 2 / Q000737:chemistry / Q000378:metabolism; D000077203:Sodium-Glucose Transporter 2 Inhibitors; D013379:Substrate Specificity
chemical_list: D001559:Benzhydryl Compounds; D005960:Glucosides; D051269:Sodium-Glucose Transporter 1; D051297:Sodium-Glucose Transporter 2; D000077203:Sodium-Glucose Transporter 2 Inhibitors; C529054:dapagliflozin; D010695:Phlorhizin; D005947:Glucose