Inhibition of ATP-sensitive K+-channels by a sulfonylurea analogue with a phosphate group

Article abstract of DOI:10.1016/S0006-2952(02)01566-6

Hypoglycaemic sulfonylureas initiate insulin secretion by direct inhibition of ATP-sensitive K⁺-channels in the pancreatic β-cells. These channels are composed of two proteins, a pore-forming subunit (K_IR6.2 in the case of β-cells) a

Full text of DOI:10.1016/S0006-2952(02)01566-6

Biochemical Pharmacology 65 (2003) 599±602
Short communication
‡
Inhibition of ATP-sensitive K -channels by a sulfonylurea
analogue with a phosphate group
Katja Hastedt, Uwe Panten*
Institute of Pharmacology and Toxicology, Technical University of Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany
Received 11 September 2002; accepted 6 November 2002
Abstract
‡
Hypoglycaemic sulfonylureas initiate insulin secretion by direct inhibition of ATP-sensitive K -channels in the pancreatic b-cells.
These channels are composed of two proteins, a pore-forming subunit (KIR6.2 in the case of b-cells) and a regulatory subunit, the
sulfonylurea receptor (SUR). In the present study we characterised the interaction with SURs of the new sulfonylurea analogues 5-chloro-
N-[2-(4-hydroxyphenyl)ethyl]-2-methoxybenzamide (compound I) and {4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenyl}phosphate
(compound II). Compounds I and II differ from the sulfonylurea analogue meglitinide only in so far as the carboxylic group of meglitinide
is replaced by a hydroxyl group or a phosphate group, respectively. The binding af®nities of compound II for the SUR subtypes SUR1
(identi®ed in b-cells) and SUR2A (identi®ed in heart and skeletal muscle) were higher by 55 or 21-fold, respectively, than the
‡
corresponding af®nities for compound I. In inside-out patch±clamp experiments compound II inhibited ATP-sensitive K -channels of the
SUR1/KIR6.2-type (characteristic of b-cells) with an IC value of 0.16 mM which is 6-fold lower than the corresponding value for
50
meglitinide. These ®ndings strongly support the conclusion that the interaction of sulfonylureas and acidic analogues with SURs is
favoured by the anionic group of these drugs and that a phosphate group allows more ef®cient ligand interaction with SUR1 than a
carboxylic group.
# 2002 Elsevier Science Inc. All rights reserved.
Keywords: KATP-channel; Sulfonylurea receptor subtypes; Sulfonylureas; Sulfonylurea analogue with a phosphate group; Structure±activity relationship;
Pancreatic b-cell
1. Introduction
While SUR1 acts as the regulatory subunit of the KATP-
channels in b-cells and many neurons, SUR2A has been
suggested to represent the SUR in heart and skeletal
muscle and SUR2B in smooth muscle [3].
‡
K -channels inhibited by cytosolic ATP (KATP-chan-
nels) have been identi®ed in the plasma membranes of
pancreatic b-cells, many neurons, cardiac myocytes, ske-
letal muscle and vascular and non-vascular smooth muscle
[1]. Hypoglycaemic sulfonylureas (e.g. glibenclamide),
too, inhibit KATP-channels and thereby initiate insulin
release in the pancreatic b-cells [2]. KATP-channels are
Several derivatives of 3-phenylpropionic acid (e.g. nate-
glinide) or benzoic acid (e.g. meglitinide and the more
lipophilic repaglinide) inhibit KATP-channels by binding to
the same receptor site that mediates the responses to
sulfonylureas [2,4±6]. Two of these sulfonylurea analogues
(repaglinide and nateglinide) have recently been intro-
duced into the therapy of type 2 diabetes mellitus [7].
The receptor site for sulfonylureas and their analogues is
located at the cytoplasmic face of the plasma membrane
[8]. Interaction with this site is favoured by the anionic
group (sulfonamide or carboxylic group) characteristic of
the sulfonylureas and analogues which are used in the
antidiabetic therapy [5]. However, it is unknown whether
the ligand interaction with the receptor site can be
improved by an anionic group containing more than one
negative charge. To test this possibility the effects of a
composed of two proteins, an inwardly rectifying KATP
-
channel subunit (KIR6.1 or KIR6.2) and the SUR, a member
of the ATP-binding cassette protein superfamily [3]. Three
SUR isoforms have been cloned, SUR1, and two splice
products of a single gene differing only in their carbox-
yterminal 42±45 amino acids, SUR2A and SUR2B [3].
* Corresponding author. Tel.: ‡49-531-3915669; fax: ‡49-531-3918182.
E-mail address: u.panten@tu-bs.de (U. Panten).
Abbreviations: KATP-channel, ATP-sensitive K -channel; KIR
‡
,
inwardly-rectifying KATP-channel subunit; SUR, sulfonylurea receptor.
0006-2952/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 0 6 - 2 9 5 2 ( 0 2 ) 0 1 5 6 6 - 6

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K. Hastedt, U. Panten / Biochemical Pharmacology 65 (2003) 599±602
zamide) was prepared by the addition of 5-chloro-2-meth-
oxybenzoyl chloride to tyramine and puri®ed by
recrystallisation from ethanol to water. Using a minor
modi®cation of a previously described method [10], com-
pound II ({4-[2-(5-chloro-2-methoxybenzamido)ethyl]-
phenyl}phosphate) was prepared by the addition of POCl3
to compound I and puri®ed by column chromatography on
silica gel 60 (Merck). Melting points (uncorrected values)
were 1278 for compound I and 1978 for compound II.
Elementary analyses of compounds I and II were within
‡0.1, Æ0.7 and À5% of the theoretical values (100%) for C,
H and N, respectively. The structures of compounds I and II
(Fig. 1A) were con®rmed by 1H-NMR (DMSO, 400 MHz),
13C-NMR (DMSO, 100 MHz) and 31P-NMR (DMSO,
81 MHz) spectroscopy and infrared spectroscopy, sup-
ported by electron-impact and fast atom bombardment mass
spectrometry.
A solution (100 mM) of compound I was prepared daily
in DMSO and diluted 1/100 in 50 mM KOH to give a 1 mM
stock solution. A 10 mM stock solution of compound II
was prepared daily in KOH (50 mM). Appropriate amounts
of the stock solutions were added slowly, while stirring, to
Tris buffer (binding experiments) or intracellular solution
supplemented with 0.3 mM ADP (patch±clamp experi-
ments). The pH of all test solutions was determined after
addition of test substances and was readjusted if necessary.
Compounds I and II were completely dissolved at the
applied concentrations.
Fig. 1. Structures (A) and SUR binding (B) of compounds I and II. (A)
The anionic form of compound II is shown. (B) In membranes from COS-1
cells transiently expressing hamster SUR1 or rat SUR2A, the effects of
compounds I (squares) and II (circles) on specific [3H]-ligand binding
were measured. For binding to recombinant SUR1 (filled symbols) the
[3H]-ligand was [3H]-glibenclamide (0.3 nM), for binding to recombinant
SUR2A (open symbols) the [3H]-ligand was [3H]-P1075 (3 nM).
Nonspecific binding was defined by incubations in the additional presence
of 100 nM glibenclamide or 100 mM pinacidil, respectively. Results are
expressed as percentage of control (absence of displacing drug). Symbols
are means (with SEM shown when larger than symbols) from three to six
separate binding experiments.
2.2. Binding experiments
Culture and transient transfections of COS-1 cells
(obtained from the German Collection of Microorganisms
and Cell Cultures), membrane preparation and measure-
ment of ligand binding to the membranes were performed
as described previously [5].
sulfonylurea analogue with a phosphate group (compound
II, Fig. 1A) on SUR binding and KATP-channel activity
were examined. Compound II differs from meglitinide
only in so far as the carboxylic group of meglitinide is
replaced by a phosphate group.
2.3. Electrophysiological experiments
Transient cotransfection of COS-1 cells with pECE-
SUR1 and pECE-KIR6.2 complementary DNA and
patch±clamp experiments using inside-out patches of these
È
cells were performed as previously described by Dorschner
2. Materials and methods
et al. [11] and Meyer et al. [5], respectively. The membrane
potential was clamped at À50 mV, and inward membrane
currents ¯owing through KATP-channels were measured at
room temperature. The solution at the cytoplasmic side of
the inside-out membrane (intracellular solution) contained
(in mM): KCl 140, CaCl2 2, MgCl2 1, EGTA 10 and HEPES
5 (titrated to pH 7.3 with KOH) (free ‰Ca2‡Š ˆ 0:05 mM).
The bath was perfused continuously at 2 mL/min with
intracellular solution containing 1 mM ATP, 0.3 mM ADP
or 0.3 mM ADP ‡ compound II (0.03±1 mM). The free
Mg2‡ concentration was held close to 0.7 mM by adding
appropriate amounts of MgCl2 to nucleotide-containing
solutions. The pipette solution contained (in mM): KCl
2.1. Chemicals and solutions
Chemicals for organic syntheses were purchased from
Aldrich Chemical Co. and Fluka Chemie AG. All other
chemicals and radioactively labelled compounds were
obtained from sources described elsewhere [5].
Unless stated otherwise, chemicals for syntheses (pure
grade) were used as received. Solvents were dried and
distilled before use. Using a minor modi®cation of the
method described by Brown and Foubister [9], compound
I (5-chloro-N-[2-(4-hydroxyphenyl)ethyl]-2-methoxyben-

K. Hastedt, U. Panten / Biochemical Pharmacology 65 (2003) 599±602
601
146, CaCl2 2.6, MgCl2 1.2, HEPES 10 (titrated to pH 7.4
with KOH). The mean KATP-channel current during the last
minute of application of 0.3 mM ADP (control solution) or
0.3 mM ADP ‡ compound II (test solution) was measured
and used to express the KATP-channel activity in the presence
of compound II as percentage of channel activity in control
solution before and after application of compound II. The
single channel current amplitudes of the KATP-channels were
not changed by the applied concentrations of nucleotides and
compound II.
2.4. Treatment of results
Values are presented as mean Æ SEM. Relations
between drug concentration and speci®c binding or chan-
nel activity and calculation of Kd values were performed as
described [5]. Signi®cances were calculated by the two-
tailed U-test of Wilcoxon and of Mann and Whitney.
P < 0:05 was considered signi®cant.
3. Results and discussion
Competitive inhibition assays (Fig. 1B) revealed that
compounds I and II inhibited speci®c [3H]-glibenclamide
binding to SUR1 half-maximally at 89:6 Æ 5:7 mM (Hill
coefficientˆÀ1:11) and 1:63Æ0:07 mM (Hill coefficientˆ
Fig. 2. Effect of compound II on KATP-channel activity in inside-out
patches of COS-1 cells transiently expressing SUR1 and KIR6.2. (A)
Current trace obtained from an inside-out patch. Free Mg2‡ (0.7 mM) was
always present in the solutions applied at the cytoplasmic membrane side.
The dotted line denotes the current level when all KATP-channels are closed
by 1 mM ATP (90 sec periods of ATP application 4 min before and 12 min
after the indicated current trace). The horizontal bars below the current
trace indicate application of intracellular solution containing 0.3 mM ADP
(with or without 0.03±1 mM compound II) by the bath. (B) Relationship
between normalised KATP-channel activity and concentration of compound
II in the presence of ADP. By use of the experimental design shown in (A),
KATP-channel activity was normalised to KATP-channel activity during the
control periods (presence of ADP, absence of drug) before and after
application of each drug concentration. Symbols are means (with SEM
shown when larger than symbols) from six experiments.
À1:06), respectively. From these IC values dissociation
50
constants (Kd) of 58:0 Æ 3:7 mM for compound
I
and 1:06 Æ 0:04 mM for compound II were calculated.
Compounds I and II inhibited speci®c [3H]-P1075
binding to SUR2A half-maximally at 148:7 Æ 2:9 mM
(Hill coefficient ˆ À0:82) and 7:01 Æ 0:54 mM (Hill
coefficient ˆ À0:87), respectively. These IC50 values corre-
sponded to Kd values of 139:6 Æ 2:7 mM for compound I
and 6:58 Æ 0:51 mM for compound II.
As shown in Fig. 2A, compound II applied at the
cytoplasmic membrane face rapidly inhibited KATP-chan-
nels of the SUR1/KIR6.2-type which is characteristic of b-
cells and many neurons. The periods of current recording
in the sole presence of 0.3 mM ADP revealed run-down of
channel activity. Inhibition by 0.03 or 1 mM of compound
II amounted to 9.5 or 83.5%, respectively (Fig. 2A).
Inhibition was rapidly and completely reversible. Analysis
of the dependence of channel inhibition on the concentra-
that the interaction of sulfonylureas and acidic analogues
with SURs is favoured by the anionic group of these drugs
[5].
The structure of compound II (Fig. 1A) differs from the
structure of the sulfonylurea analogue meglitinide only in
so far as the carboxylic group of meglitinide is replaced by
a phosphate group. The binding af®nity (1/Kd) for SUR1
and the KATP-channel-inhibitory potency (1/IC ) of com-
50
tion of compound II (Fig. 2B) yielded an IC value of
50
pound II were higher by 6.2 and 7.5-fold, respectively, than
the corresponding values for meglitinide [5,8]. Thus, as
compared with a carboxylic group a phosphate group
allows more ef®cient ligand interaction with SUR1. The
reason could be the greater negative charge of the phos-
phate group and/or the different position of the negative
charge relative to the neighbouring benzene ring of the
ligand.
0.16 mM (Hill coefficient ˆ À1:33). This IC value was
50
6.6-fold lower than the corresponding Kd value for binding
of compound II to SUR1. Similar differences (2±10-fold)
have been regularly observed for sulfonylureas and their
analogues [4,5,8]. These differences re¯ect the fact that
occupation of one of the four SUR binding sites per channel
complex is suf®cient for KATP-channel closure [11].
The ®nding that the Kd values for binding of compound
I were 21±55-fold higher than the Kd values for binding
of compound II strongly supports the previous conclusion
The binding af®nities of meglitinide for SUR1 and
SUR2A did not differ signi®cantly [5], in contrast to the
observation that the binding af®nity of compound II for

602
K. Hastedt, U. Panten / Biochemical Pharmacology 65 (2003) 599±602
References
SUR1 was 6.2-fold higher (P < 0:01) than the binding
af®nity of compound II for SUR2A. Thus, replacement of
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produces a SUR1-selective ligand. This information might
help to characterise the biochemical properties of the
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The insulin releasing property of compound II has
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the present inside-out patch±clamp experiments (Fig. 2).
It is unknown whether endogenous ligands for the SUR
sites exist. The possibility that endogenous ligands might
be found among intracellular phosphorylated peptides and
proteins is stressed by the structure of compound II which
includes the tyrosine phosphate side chain occurring in
peptides and proteins.
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We thank Haide FuÈrstenberg, Carolin Rattunde and Ines
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È
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