From the Departments of Molecular Pharmacology & Biochemistry and ¶ Medicinal Chemistry, Merck Research
Laboratories, Rahway, New Jersey 07065
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ABSTRACT |
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We have identified a series of potent,
orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole
glucagon receptor antagonists.
2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrrole (L-168,049), a prototypical member of this series, inhibits
binding of labeled glucagon to the human glucagon receptor with an
IC50 = 3.7 ± 3.4 nM
(n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 µM. The
binding affinity of L-168,049 for the human glucagon receptor is
decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent
EC50 for glucagon stimulation of adenylyl cyclase in
Chinese hamster ovary cells expressing the human glucagon receptor and
decreases the maximal glucagon stimulation observed, with a
Kb (concentration of antagonist that shifts the
agonist dose-response 2-fold) of 25 nM. These data suggest
that L-168,049 is a noncompetitive antagonist of glucagon action.
Inclusion of L-168,049 increases the rate of dissociation of labeled
glucagon from the receptor 4-fold, confirming that the compound is a
noncompetitive glucagon antagonist. In addition, we have identified two
putative transmembrane domain residues, phenylalanine 184 in
transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for
which substitution by alanine reduces the affinity of L-168,049 46- and
4.5-fold, respectively. These mutations do not alter the binding of
labeled glucagon, suggesting that the binding sites for glucagon and
L-168,049 are distinct.
Glucagon is a 29-amino acid peptide that is an important
counter-regulatory hormone in the control of glucose homeostasis (1).
Glucagon secretion from the endocrine pancreas induces an increase in
hepatic glycogenolysis and gluconeogenesis, and it attenuates the
ability of insulin to inhibit these processes. As such, the overall
rates of hepatic glucose synthesis and glycogen metabolism are
controlled by the systemic ratio of insulin and glucagon (2, 3).
Therefore, glucagon antagonists have the potential to improve hepatic
insulin sensitivity and to be effective hypoglycemic agents.
Peptidyl glucagon antagonists and their hypoglycemic activity were
first described over 15 years ago, and an extensive exploration of the
structure/activity relationships of these glucagon analogs has been
reported (4-6). The hepatic receptor for glucagon was cloned recently
(7, 8), confirming that it is a member of the seven-transmembrane
domain, G-protein-coupled receptor superfamily. This receptor
superfamily has a binding pocket for small-molecule ligands within the
transmembrane domain that has made it possible to identify non-peptidyl
antagonists for many receptor families in which the endogenous ligands
are small peptides or proteins (9). Thus, we initiated an effort to
identify non-peptidyl, orally active antagonists for the human glucagon receptor.
Collins et al. (10) have described a dichloroquinoxaline
glucagon antagonist with weak affinity (IC50 = 4 µM) for the rat glucagon receptor. However, there have
been no subsequent reports in the patent or scientific literature
describing the development of potent antagonists from this series. Our
initial screening efforts identified a series of triarylimidazole and
triarylpyrrole compounds with significant binding affinity for the
human glucagon receptor, and efforts to evaluate the structure-activity
relationships of this series have lead to the identification of potent
glucagon antagonists (11). In the present article, we describe the
identification and characterization of a potent glucagon antagonist
from this series.
Characterization of Binding Affinity and Functional
Activity--
Stable CHO1
cell lines or COS cells transiently expressing the human glucagon
receptor were prepared as described previously (8, 12). Antagonist
binding affinity was assessed by measuring inhibition of radiolabeled
glucagon binding to CHO cell membranes. Briefly,
125I-glucagon (58 pM) binding to the membrane
preparation was measured in 20 mM Tris, pH 7.4, containing
1 mM dithiothreitol, 5 µg/ml leupeptin, 10 µg/ml
benzamidine, 40 µg/ml bacitracin, 5 µg/ml soybean trypsin
inhibitor, and 3 µM o-phenanthroline ± 1 µM glucagon for 1 h at room temperature. Bound cpm
were recovered by filtration using a Tomtec harvester and quantified in
a
The ability of compound to inhibit glucagon-stimulated adenylyl cyclase
was assessed as described previously (12). Briefly, cells were
harvested from monolayers with enzyme-free cell dissociation solution
(Specialty Media, Inc.) and were pelleted at 500 × g. The cells were resuspended at 100,000 cells/100 µl in 75 mM Tris-HCl, pH 7.4, containing 250 mM sucrose,
12.5 mM MgCl2, 1.5 mM EDTA, 0.1 mM Ro-20-1724 (Biomol, Inc.), leupeptin (5 µg/ml),
benzamidine (10 µg/ml), bacitracin (40 µg/ml), soybean trypsin
inhibitor (5 µg/ml), and 0.02% bovine serum albumin. Cells were
incubated for 30 min at 22 °C with increasing peptide concentrations
in the presence or absence of antagonist followed by lysis by boiling. Lysates were analyzed for cAMP content versus a
nonacetylated cAMP standard curve using the Amersham cAMP
radioimmunoassay scintillation proximity assay kit. Data were analyzed
using Packard TopCount with RIASmart and GraphPad Prism software.
Preparation and Assessment of Mutant Glucagon Receptors--
The
wild-type glucagon receptor was cloned into pCI-neo (Promega Corp.,
Madison, WI) at the NheI and XbaI sites.
Mutations at amino acid residues 184 (TM2, F184A) and 239 (TM3, Y239A)
of the wild-type glucagon receptor were prepared using recombinant polymerase chain reaction. The sense primers for the F184A and the
Y239A mutants were 5'-TTTGCGTCCGCCGTGCTGAA-3' and
5-GTGGCCAACGCCTGCTGGCTGCT-3', respectively. The resulting mutant
fragments were excised with either ApaI/SacII
(F184A) or SacII/BclI (Y239A) and ligated into the pCI-neo wild-type glucagon receptor. Mutations were confirmed by
automated sequencing. All other mutants were prepared in a similar
manner using oligonucleotides specific to the production of an alanine
at the indicated position with the wild-type receptor, and mutations
were confirmed by sequencing. The restriction sites for the other
mutations were as follows: TM1 (Tyr-145, Tyr-149, and Ser-152, digested
with ApaI/SacII), TM2 (Phe-184 and Ser-190, digested with ApaI/SacII), TM3 (Gln-232, Tyr-233,
Asn-238, and Tyr-239, digested with SacII/BclI),
TM4 (Phe-278, digested with SstII/BclI), TM5
(Phe-312, Phe-319, and Phe-320, digested with KspI/PflM 1), and TM7 (Lys-381, Phe-383, Phe-384,
Phe-387, Ser-390, and Phe-391, digested with PflM
1/XbaI).
The transfection of COS cells and the preparation of membranes for
binding assays were performed as described previously (12). Compound
titrations using wild-type receptor transiently expressed in COS cells
were performed as controls in all experiments utilizing mutant receptors.
Several triarylimidazole compounds were identified from compound
collection screening that inhibit labeled glucagon binding to the human
glucagon receptor with potencies ranging from 300 nM to 1 µM (11). Optimization of this activity by structural modification of these lead compounds led to the identification of the
triarylpyrrole glucagon antagonist, L-168,049 (Fig.
1). This compound inhibits binding of
labeled glucagon with an IC50 of 3.7 ± 3.4 nM (n = 7). Surprisingly, the affinity of
L-168,049 is decreased 48-fold by the inclusion of 5 mM
MgCl2 in the assay (Fig. 2).
In the presence of 5 mM MgCl2, the compound
inhibits binding of labeled glucagon with an IC50 of
179 ± 86 nM (n = 3). An equivalent
decrease in affinity is also observed with MnCl2 and
CaCl2, but not with NaCl, indicating that the loss in
affinity is divalent cation-dependent (data not shown).
L-168,049 does not inhibit binding of labeled
glucagon-like peptide-1 (GLP-1) to the highly homologous GLP-1 receptor at concentrations as high as 10 µM.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of the glucagon antagonist
L-168,049.
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Fig. 2.
Inhibition of the binding of
125I-glucagon to the human glucagon receptor by L-168,049
in the absence (closed circles) or presence
(open circles) of 5 mM
MgCl2. Assays were performed using CHO cell
membranes as described under "Materials and Methods." Data are the
average of three separate experiments.
Inclusion of increasing doses of L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase and decreases the maximal stimulation observed (Fig. 3). A competitive antagonist should not alter maximal agonist-induced activation of the receptor, suggesting that L-168,049 is a noncompetitive antagonist of glucagon. Schild transformation of these data is linear with a slope of 0.6, which is consistent with a noncompetitive mechanism. The affinity of L-168,049, as measured by the concentration of antagonist that shifts the agonist dose-response 2-fold (Kb), is 25 nM. In the absence of exogenously added glucagon, L-168,049 does not alter intracellular cAMP levels, indicating that it has no agonist activity.
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125I-Glucagon dissociates from the human glucagon receptor
in the presence of MgCl2 with
k1 = 0.013 ± 0.001 min
1 (n = 2). Inclusion of L-168,049 (1 µM) increases the rate of dissociation 4-fold (Fig.
4). These data confirm that
L-168,049 is a noncompetitive antagonist of glucagon. The
nonhydrolyzable guanine nucleotide analog, guanosine
5'-(
,
-imido)-triphosphate, which reduces glucagon affinity by
uncoupling the receptor from G-protein, increases the rate of
dissociation 29-fold. These data suggest that L-168,049 does not
inhibit glucagon binding by this mechanism. Although the data shown are
in the presence of divalent cation, L-168,049 also increases the rate
of dissociation of 125I-glucagon 4-fold in the absence of
divalent cation.
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To confirm that L-168,049 binds within the transmembrane domain of the glucagon receptor, 19 residues predicted to be within this region (8) were separately mutated to alanine, and the binding affinities of these mutants for glucagon and L-168,049 were determined following transient transfection into COS cells. These include Tyr-145, Tyr-149, Ser-152, Phe-184, Ser-190, Gln-232, Tyr-233, Asn-238, Tyr-239, Phe-278, Phe-312, Phe-319, Phe-320, Lys-381, Phe-383, Phe-384, Phe-387, Ser-390, and Phe-391. Alanine substitution of these individual residues does not alter affinity for labeled glucagon. Whereas most of these substitutions also do not alter affinity for L-168,049, the substitution of alanine for Phe-184 and Tyr-239 results in a 46- and a 4.5-fold reduction in the affinity for L-168,049, respectively (Table I). Inclusion of divalent cation reduced the affinity of the compound for both the wild-type and mutant receptors, indicating that these residues were not involved in mediating the effect of divalent cations on compound affinity (data not shown).
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The triarylpyrrole glucagon antagonists have poor affinity for the rat,
guinea pig, and rabbit glucagon receptors (>1 µM). However, L-168,049 inhibits labeled glucagon binding to the murine and
canine glucagon receptors with IC50 = 63 ± 44 nM (n = 3) and 60 nM,
respectively. As with the human receptor, these affinities are reduced
20-fold in the presence of 5 mM MgCl2
(IC50 = 331 ± 122 nM (n = 3)) and >1 µM for the murine and dog receptors, respectively.
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DISCUSSION |
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We have identified a structural class of non-peptidyl glucagon antagonists. Preliminary data indicate that these compounds are absorbed systemically after oral administration (11), suggesting that they have the potential to be useful, orally active hypoglycemic agents. The prototype of this class, L-168,049, is a high affinity, noncompetitive antagonist of the human glucagon receptor, which inhibits glucagon-stimulated adenylyl cyclase with a Kb of 25 nM. This compound has poor affinity for the highly homologous GLP-1 receptor and for a panel of other G-protein-coupled receptors, indicating that it is a selective glucagon antagonist.
The observation that inclusion of L-168,049 alters both the apparent affinity and maximal receptor activation of glucagon suggests that these compounds are noncompetitive glucagon antagonists. The demonstration that L-168,049 increases the dissociation rate of labeled glucagon confirms this hypothesis, because these data suggest that both glucagon and L-168,049 are able to bind to the receptor at the same time.
Whereas mutagenesis data suggest that the binding site for glucagon is within the amino-terminal domain and other extracellular domains of the glucagon receptor (13-15), our data suggest that the L-168,049 binding site is within the transmembrane domain. The binding of glucagon to the 19 transmembrane domain mutant receptors is unaltered, suggesting that these mutations did not dramatically effect overall receptor conformation. Phenylalanine 184 and tyrosine 239, the two mutations with altered affinity for L-168,049, are predicted to be within the second and third transmembrane domains, respectively. Thus, these compounds appear to bind in the same common binding pocket that has been postulated for other G-protein-coupled receptors (9). These data, and the observation that L-168,049 is a noncompetitive antagonist of glucagon, suggest that the compound allosterically modulates the binding of glucagon to the receptor.
Divalent cations have been shown to allosterically enhance the binding of agonists to many types of G-protein-coupled receptors, including the binding of glucagon to the human glucagon receptor (12), but divalent cation effects on antagonist binding affinity are less well documented. However, inclusion of 5 mM divalent cation decreases the affinity of L-168,049 48-fold. The activity of antagonists in the cellular functional assays that are performed under more physiologically relevant buffer conditions correlates more closely with the binding affinity measured in the presence of divalent cation. More importantly, because circulating levels of MgCl2 are in the low mM range, the decrease in affinity observed in the presence of divalent cation would be expected to be important for the activity of antagonists in vivo.
We previously showed that the deletion of residues 252-259 in the second intracellular domain of the glucagon receptor attenuates the effects of divalent cation on the binding affinity for glucagon (12). However, this mutation does not significantly alter the affinity for L-168,049, and inclusion of divalent cation reduces affinity of L-168,049 for this mutant protein 30-fold. Thus, the effects of divalent cation on agonist and antagonist binding appear to be distinct.
Because L-168,049 has poor affinity for all of the species orthologs of the glucagon receptor tested to date, it has been difficult to test its in vivo efficacy. However, L-168,049 has the highest affinity for the murine receptor of any compound in this series identified to date, and high levels of compound (3 µM) increase the apparent EC50 for glucagon-stimulated adenylyl cyclase activation in murine liver membranes by >10-fold.
These data suggest that the compound is an antagonist of glucagon
activity in a physiological tissue preparation. However, the compound
(50 mg/kg, per os) does not inhibit glucagon-stimulated increases in the blood glucose in mice, suggesting that its activity at
the murine receptor is insufficient to be a useful tool in this
species. Thus, identification of a suitable animal model is required
for the analysis of the effects of this class of orally active glucagon
antagonists on glucose homeostasis in vivo. Recently we have
generated a murine line in which the native murine glucagon receptor
has been replaced with the human receptor by homologous recombination,
and preliminary data suggest that the activity of compounds in liver
membranes from these mice correlates with their affinity as measured
using the cloned human
receptor.2 Our current
efforts are focused on expanding this murine line to extend these
observations and to determine the effect of this class of compounds on
glucose homeostasis.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Merck Research Laboratories, 80M-213, P. O. Box 2000, Rahway, NJ 07065. Tel.: 732-594-4609; Fax: 732-594-3337; E-mail: peggy_cascieri{at}merck.com.
2 L.-L. Shiao, K. A. Sullivan, and M. A. Cascieri, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CHO, Chinese hamster ovary; GLP-1, glucagon-like peptide-1; TM, transmembrane (domain); L-168, 049, 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2- propyloxyphenyl)pyrrole.
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