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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Inhibitor Binding in the Human Renal Low- and High-Affinity Na⁺/Glucose Cotransporters

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas (A.M.P., K.M.R.); and GlaxoSmithKline, Biochemical and Analytical Pharmacology, Research Triangle Park, North Carolina (S.A.K., C.D.S.)

The kidney contains two Na⁺/glucose cotransporters, called SGLT2 and SGLT1, arranged in series along the length of the proximal tubule. The low-affinity transporter, SGLT2, is responsible for the reabsorption of most of the glucose in the kidney. There is recent interest in SGLT2 as a target for the treatment of type II diabetes using selective inhibitors based on the structure of the phenylglucoside, phlorizin (phloretin-2'-β-glucoside). In this study, we examined the inhibition of -methyl-D-glucopyranose transport by phlorizin and a new candidate drug, sergliflozin-A [(2-[4-methoxyphenyl]methyl)phenyl β-D-glucopyranoside], in COS-7 cells expressing hSGLT1 and hSGLT2. Inhibition by phlorizin was competitive, with K_i values of 0.3 µM in hSGLT1 and 39 nM in hSGLT2. Inhibition by sergliflozin-A was also competitive, with K_i values of 1 µM in hSGLT1 and 20 nM in hSGLT2. Phloretin [3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; the aglucone of phlorizin] was a less potent inhibitor, with IC₅₀ values of 142 µM in hSGLT1 and 25 µM in hSGLT2. Site-directed mutagenesis of residues believed to be in the phlorizin binding site showed that only Cys610 is involved in inhibitor binding in the human transporters. Mutation of Cys610 in hSGLT1 to lysine resulted in an increased IC₅₀ for all inhibitors. In contrast, mutagenesis of the analogous Cys615 in hSGLT2 produced the opposite effect, a decrease in IC₅₀ for phlorizin and sergliflozin-A. The differences in the effects of the mutations between hSGLT1 and hSGLT2 suggest that this cysteine holds key residues in place rather than participating directly in inhibitor binding.

Address correspondence to: Dr. Ana M. Pajor, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555. E-mail: ampajor{at}utmb.edu