Differential Interactions of Nateglinide and Repaglinide on the Human -Cell Sulphonylurea Receptor 1

Differential Interactions of Nateglinide and Repaglinide on the Human ␤-Cell Sulphonylurea Receptor 1 Ann Maria K. Hansen,1 Inge T. Christensen,1 John Bondo Hansen,1 Richard D. Carr,1 Frances M. Ashcroft,2 and Philip Wahl1 Repaglinide and nateglinide represent a new class of plasma glucose levels potentiates GLUT2-mediated glu- insulin secretagogues, structurally unrelated to sulpho- cose uptake into the ␤-cell, and subsequent metabolism of nylureas, that were developed for the treatment of type glucose increases the ATP/ADP ratio, leading to closure of 2 diabetes. The inhibitory effect of these drugs was KATP channels, membrane depolarization, and opening of investigated on recombinant wild-type and mutant voltage-sensitive Ca2ϩ channels. The resulting increase in Kir6.2/SUR1 channels expressed in HEK293 cells. intracellular Ca2ϩ stimulates insulin secretion. Sulphonyl- Nateglinide and repaglinide dose-dependently inhibited ureas trigger the same series of events by blocking K whole-cell Kir6.2/SUR1 currents with half-maximal in- ATP channels directly. KATP channels are also found in a variety hibitory concentration (IC50) values of 800 and 21 nmol/l, respectively. Mutation of serine 1237 in SUR1 to of other tissues, including neurons, heart, and skeletal and tyrosine (S1237Y) abolished tolbutamide and nateglin- smooth muscle cells, where they play important physio- ide block, suggesting that these drugs share a common logical and pathophysiological roles (4). point of interaction on the SUR1 subunit of the ATP- The ␤-cell K channel is a hetero-octameric complex ؉ ATP sensitive K channel. In contrast, repaglinide inhibition of four inwardly rectifying potassium channel (Kir6.2) ؍ was unaffected by the S1237Y mutation (IC50 23 subunits, which form a tetrameric pore, and four regula- nmol/l). Radioligand binding studies revealed a single tory sulphonylurea receptor (SUR1) subunits (5,6). Sul- high-affinity binding site for [3H]repaglinide on mem- branes prepared from HEK293 cells expressing wild- phonylureas bind with high affinity to SUR1 to mediate closure of the Kir6.2 pore (7). Different SUR variants ؍ type (equilibrium dissociation constant [KD] 0.40 ؍ nmol/l) or mutant (KD 0.31 nmol/l) Kir6.2/SUR1 chan- confer different sensitivities to sulphonylureas and KATP nels. Nateglinide and tolbutamide displaced [3H]repa- channel openers on the Kir6.2 subunit; for example, tolbu- glinide binding to wild-type channels with IC50 values of tamide inhibits SUR1- but not SUR2-containing KATP chan- 0.7 and 26 ␮mol/l, respectively, but produced <10% nels (8,9). This accounts for the much lower potency of 3 displacement of [ H]repaglinide bound to mutant chan- this drug in tissues such as heart and smooth muscles, in nels. This is consistent with the idea that binding of which K channels are composed of SUR2A and SUR2B, nateglinide and tolbutamide, but not repaglinide, is ATP abolished by the SUR1[S1237Y] mutation and that the respectively (10,11). binding site for repaglinide is not identical to that of In addition to the classic sulphonylureas, KATP channels nateglinde/tolbutamide. These results are discussed in are inhibited by benzamido compounds and their deriva- terms of a conformational analysis of the drug mole- tives (e.g., meglitinide) (12). This suggests that like other cules. Diabetes 51:2789–2795, 2002 ATP-binding cassette transporters, SUR possesses a large multifaceted drug-binding pocket that can accommodate several structurally distinct compounds. Individual compounds differ in the residues within this pocket, with ulphonylureas such as tolbutamide and gliben- which they interact. Studies of recombinant K channels clamide are widely used to treat type 2 diabetes ATP suggest that drugs containing a sulphonylurea moiety (e.g., because they stimulate insulin secretion. Their primary mode of action is to bind to ATP-sensi- tolbutamide, glibenclamide) interact with residues in the Sϩ TM15-16 linker of SUR1 and that a single serine residue tive K (K ) channels in the pancreatic ␤-cell and induce ATP (S1237) within this region is critical for drug binding their closure (1–4). KATP channels serve a critical role in glucose-stimulated insulin secretion. At low glucose levels, and/or transduction (8,13). Mutagenesis and affinity-label- these channels are open, permitting an efflux of Kϩ ions ing studies also suggest that residues within the cytosolic that hyperpolarizes the ␤-cell membrane. Elevation of loop linking transmembranes (TMs) 5 and 6 may be essential for [3H]glibenclamide binding (14,15). Thus, amino acid residues derived from two distinct regions of From 1Discovery, Novo Nordisk A/S, Bagsvaerd, Denmark; and the 2University SUR1 appear to contribute to the glibenclamide binding Laboratory of Physiology, Oxford University, Oxford, U.K. Address correspondence and reprint requests to Dr. Philip Wahl, Depart- site. Because glibenclamide contains both a sulphonylurea ment of Islet Discovery Research, Discovery, Novo Alle´, Novo Nordisk A/S, group and a benzamido moiety, these moieties may inter- DK-2880 Bagsvaerd, Denmark. E-mail: [email protected]. Received for publication 21 December 2001 and accepted in revised form 7 act with different parts of SUR1, with the sulphonylurea June 2002. moiety interacting with the TM15-16 linker. F.M.A. is a paid consultant for Novo Nordisk. The extent to which this model can be applied to drugs GFP, green fluorescent protein; IC50, half-maximal inhibitory concentration; ϩ that do not contain a sulphonylurea group remains un- KATP, ATP-sensitive K ;KD, equilibrium dissociation constant; SUR, sulphonylurea receptor; TM, transmembrane. clear. Recent studies have shown that inhibition of Kir6.2/ DIABETES, VOL. 51, SEPTEMBER 2002 2789 INTERACTION OF REPAGLINIDE AND NATEGLINIDE WITH SUR1 SUR1 currents by the nonsulphonylurea mitiglinide, as time course of drug inhibition. In this case, currents were sampled at 25 Hz with tolbutamide, is abolished by the S1237Y mutation and filtered at 6.25 Hz. Molecular modeling. Conformational analyses were performed using Macro- (16). This suggests that this residue is not specific for the Model version 6.0 (20). The compounds were subjected to a Monte Carlo sulphonylurea moiety but can interact with other com- search using the MMFF force field (21), with a solvation model for water (22). pounds. We therefore explored whether this part of the For each molecule, 5,000 conformations were generated, with an energy drug-binding pocket of SUR1 can also accommodate repa- cut-off of 25 kJ/mol. The resulting low-energy conformations were imported into Sybyl version 6.6 (Tripos, St. Louis, MO), and all further manipulations glinide and nateglinide, two members of a new class of were performed using this program. Initially, a number of alternative super- insulin secretagogues termed “prandial glucose regula- impositions were generated using the GASP algorithm (23), as implemented in tors.” These drugs differ structurally from the sulphonyl- Sybyl. The final pharmacophore models were generated by manually adjusting ureas in that they do not possess a sulphonylurea moiety. the superimpositions generated using GASP. In all cases, the resulting Chemically, they are based on benzoic acid (in the case of conformations were compared with the minimum-energy conformations found in the conformational search. repaglinide [17]) and phenylalanine (nateglinide). Both Data analysis. Data are presented as mean Ϯ 1 SD unless otherwise stated. drugs are known to inhibit KATP channel activity. The Concentration-response curves for drug-induced KATP current inhibition were half-maximal inhibitory concentration (IC50) for nateglin- constructed by expressing the current in the presence of the drug as a fraction ␤ ␮ of the current before the drug was added. Data were analyzed in Prism ide inhibition of native rat -cell KATP channels (7 mol/l) (18) lies, as with tolbutamide, in the low micromolar (GraphPad) using the four-parameter logistic equation: range. In contrast, repaglinide resembles glibenclamide in 100 Ϫ a y ϭ a ϩ ͑ Ϫ͓ ͔ ⅐ producing high-affinity block of both native and recombi- 1 ϩ 10 log IC50 L nH ␤ ϭ nant -cell KATP channels (IC50 0.9–7 nmol/l) (18,19). Our results suggest that nateglinide, but not repaglinide, where y is the current expressed as a percentage of that recorded before drug was added, a is the percentage of current remaining after maximal inhibition interacts with serine 1237 of SUR1 to mediate inhibition of by the drug, IC50 is the drug concentration that results in half-maximal the KATP channel. inhibition, [L] is the concentration of drug, and nH is the Hill coefficient. The same equation was used to fit the dose-inhibition curves for displacement of [3H]repaglinide binding, but in this case, y was specific binding and a RESEARCH DESIGN AND METHODS was nonspecific binding. The experimentally measured IC50 values for the Molecular biology. Human SUR1 cDNA (GenBank L78207) and human Kir6.2 competitive ligands were converted into Ki values using the Cheng-Prusoff (GenBank D50582) were cloned into pcDNA3.1(Ϫ) (Invitrogen). The point equation: mutation SUR1[S1237Y] was constructed by standard molecular biology techniques and confirmed by DNA sequencing. IC ϭ 50 Cell culture and transfection. HEK293 cells were cultured at 37°Cina Ki 1 ϩ ͓L͔/KD humidified atmosphere of 95% air and 5% CO2 in Dulbecco’s modified Eagles 3 medium with 4.5 g/l glucose (BioWhittaker) supplemented with 10% FCS, where [L] is the concentration of [ H]repaglinide, and KD is the equilibrium penicillin (100 units/ml), and streptomycin (0.1 mg/ml). dissociation constant for [3H]repaglinide. Transient transfections were performed using FuGene 6 Transfection Chemicals. Tolbutamide was purchased from Sigma and glibenclamide from Reagent (Roche), according to the manufacturer’s instruction. Cells were Research Biochemicals International. Nateglinide was synthesized at Novo seeded at 50% confluency and transfected with Kir6.2 and SUR1[S1237Y] at a Nordisk A/S and repaglinide at Boehringer Ingelheim (Biberach, Riss, Germa- plasmid ratio of 1:3 on the next day.

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