From the Division de Pharmacologie Moléculaire
et Cellulaire, Institut de Recherches SERVIER, 78290-Croissy sur Seine,
§ Institut de Pharmacologie Moléculaire et Cellulaire,
CNRS UMR 6097 06100-Sophia-Antipolis, ¶ Division du
Métabolisme, Institut de Recherches SERVIER, 92150-Suresnes, and
Division des Peptides et de Chimie Combinatoire, Institut de
Recherches SERVIER, 92150-Suresnes, France
Received for publication, November 28, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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Melanin-concentrating hormone (MCH) is a
cyclic nonadecapeptide involved in the regulation of feeding behavior,
which acts through a G protein-coupled receptor (SLC-1) inhibiting
adenylcyclase activity. In this study, 57 analogues of MCH were
investigated on the recently cloned human MCH receptor stably expressed
in HEK293 cells, on both the inhibition of forskolin-stimulated
cAMP production and
guanosine-5'-O-(3-[35S]thiotriphosphate
([35S]- GTP Melanin-concentrating hormone
(MCH)1 has been initially
described in fish as a heptadecapeptide
(Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val (1)). Its structure was relatively conserved throughout evolution, although in mammals the sequence of MCH is a nonadecapeptide with differences mainly in the N terminus
(Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu- Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Gln-Val (2)). In rodents, there are now
several lines of evidence for the involvement of MCH in the central
regulation of feeding behavior as reviewed by Tritos and Maratos-Flier
(3). The MCH peptide and its receptor are expressed in the
hypothalamus, a region involved in energy balance and food intake
(4-7). In this particular brain area, MCH mRNA is overexpressed
and up-regulated during fasting in ob/ob mice as well
as in rats (8, 9). Intra-cerebroventricular injections of MCH promote
feeding in mice and rats (9-12). Finally, transgenic mice lacking the
MCH gene are lean and hypophagic (13). Interestingly, in peripheral
tissues, MCH also stimulates the release of leptin from isolated rat
adipocytes (14).
The lack of suitable binding conditions, mainly due to the hydrophobic
and sticky nature of MCH itself or derivatives (15, 16), was probably a
limitation for expression cloning of the receptor. The MCH receptor was
nevertheless recently identified by several groups using reverse
pharmacology (17-21). The MCH function was assigned to the previously
described orphan receptor SLC-1 (22, 23), using inhibition of
forskolin-stimulated cAMP production and induction of calcium rise.
Receptor cloning and association of functional tests open the way to
the search for pharmacological tools, especially receptor antagonists
that are needed to study receptor functions. One of the possible
strategies to this goal is the chemical modification of the natural
peptide including peptide shortening, amino acid substitution, and
conformation restriction with the help of structure-activity relationships and modeling studies toward optimized nonpeptide ligands.
Such a strategy has been successful for the design of subtype-specific
antagonists of neuropeptide Y receptors (24-27).
In the case of MCH, only two sets of data have been published on the
pharmacological action and binding affinity of MCH analogues in
vitro. A first series of experiments with fish MCH on fish, frog,
or other batrachian skin assays were reported (28-32), showing that
fish MCH could be shortened at both termini without major loss of the
biological activity (29, 31). It was also shown that the MCH ring was
essential (32) and that any modification (including amino acid
deletion) of this ring was deleterious for biological activity (30). A
second series of data have been published more recently using membranes
from mammalian cells that were tested for their capacity to bind the
current labeled derivative of MCH,
[Phe13,Tyr19]MCH (15, 16, 33-36). Among
other discrepancies, the reported affinities for salmon MCH in the cell
lines used (16, 34) were quite different from those reported at the rat
or human cloned receptor (17, 21). Furthermore, the mRNA for the
cloned receptor could not be found in some of those cell
lines.2 Thus, these results
cannot be attributed to the MCH receptor. More pharmacological data
should be gathered on the cloned human receptor. To date, a single
report described studies with MCH analogues (particularly
Arg11-modified ones) on human MCH receptor reported the
design of the weak MCH antagonist
[D-Arg11]MCH (37). It also showed, by
site-directed mutagenesis, that the residue Asp123 in the
third transmembrane domain of the receptor was critical in binding and
receptor activation.
In the present report, an extensive and detailed
structure-activity relationships study of MCH, including a panel
of 58 peptides, is presented using two different functional assays on
HEK293 cells stably expressing the human MCH receptor, the inhibition
of the intracellular cAMP production and the stimulation of
[35S]GTP Peptides--
Most of the natural and modified peptides were
purchased from NEOSYSTEM SA, Strasbourg, France, using the classical
methods of peptide chemistry. They were prepared on solid phase (see
Ref. 38 for example) using Fmoc for Cloning of the Human MCH Receptor (SLC-1)--
Human brain
poly(A)+ RNA (CLONTECH) was
reversed-transcribed with oligo(dT)12-18 using reverse
Transcriptase Superscript II (Life Technologies, Inc.). First strand
cDNA (corresponding to 1 µg of total RNA) was subjected to 35 cycles of amplification using the forward primer
5'-GAGACCCAAGCTTCTGGATGGACCTGGAAGCCT-3' and the reverse primer
5'-GATGACGCGGCCGCTCAGGTGCCTTTGCTTTCTG-3' (33). After an initial cycle
of denaturation at 94 °C for 1 min, polymerase chain reaction was
carried out for 35 cycles with the following cycle conditions:
94 °C, 1 min; 55 °C, 1 min; 72 °C, 3 min with a postincubation
of 72 °C for 7 min. The expected 1064-base pair fragment was
isolated and ligated into pcDNA3.1 (Invitrogen). The recombinant
plasmid, pcDNA3.1-SLC1, was sequenced on both strands by automated
sequencing (Applied Biosystems 377).
Establishment of a HEK293 Cell Line Stably Expressing the Human
MCH Receptor--
HEK293 cells grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 2 mM
glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin were
seeded at 5.106 cells in a T75 cm2 culture
flask. 24 h later, they were transfected with 10 µg of the
pCDNA3.1-SLC1 plasmid using LipofectAMINE as described by the
manufacturer (Life Technologies, Inc.). The day following transfection,
cells were trypsinized, resuspended in complete Dulbecco's modified
Eagle's medium containing 800 µg/ml of active geneticin, and seeded
at different dilutions in 96-well plates that were kept for 2-3 weeks
in an humidified CO2 incubator. At the end of this
selection period, isolated clones were picked, amplified, and further
characterized by cAMP experiments. One positive clone was subcloned in
limited dilution before being used for all the cAMP and
[35S]GTP Intracellular cAMP Assay--
Intracellular cAMP was determined
using the Flash plate technology (SMP004, PerkinElmer Life Sciences).
Briefly, forskolin (15 µM) and test peptides diluted in
0.1% bovine serum albumin were added into 96-well flash plates, and
incubation was started with the addition of cells (35,000 cells per
well). After 15 min at 37 °C, incubation was stopped by the addition
of the revelation mix, and 2 h later, plates were counted on a
TopCount (Packard Instrument Co.).
[35S]GTP Calcium Flux Measurements--
Stable HEK293 cells expressing
the human MCH receptor were seeded (40,000 cells per well) into 96-well
black-walled culture plates coated with poly-D-lysine
24 h before assay. Cells were loaded with a calcium kit assay
buffer (Molecular Devices) containing 2.5 mM probenecid and
incubated at 37 °C for 1 h in 6% CO2 atmosphere. After 10 s, the antagonist was added. For antagonist studies, tested substances were added 10 min before the addition of MCH. Increases of intracellular Ca2+ in the presence of peptides
were monitored using the fluorimetric imaging plate reader detection
system (Molecular Devices) at 488 nM for 120 s.
Peptides--
A total of 58 MCH analogues (including genuine MCH)
was obtained by synthesis on solid phase with a purity of Characterization of the HEK293 Cell Line Stably Expressing the
Human MCH Receptor--
The HEK293 cell line stably expressing the MCH
receptor was selected through MCH-induced inhibition of
forskolin-stimulated cAMP production. The density of the MCH receptors,
as determined in saturation binding experiments using
125I-labeled [3-iodo-Tyr13]MCH, was 759 ± 55 fmol/mg proteins, and the dissociation constant of the
radioligand was 0.46 ± 0.11 nM (n = 3) (data not shown).
Modifications of the MCH Peptide--
As shown in Fig.
1, human MCH (compound 42) strongly
inhibits the forskolin-induced intracellular cAMP level. Neither the substitutions of Tyr13 by Phe13 and
Val19 by Tyr19 (compounds 41) nor the
iodination of Tyr13 (compound 40) significantly affected
the potency of these peptides to inhibit forskolin-induced
intracellular cAMP level (Fig. 1 and Table
II). Salmon MCH (compound 45) was
slightly less potent than human MCH in this assay (Fig. 1 and Table
II). Replacement of the two cysteines by two serine residues, leading
to a linear MCH analogue (compound 44), dramatically decreased the
potency ~300-fold (Fig. 1 and Table II) indicating that the cyclic
part of MCH plays an essential role for activity. These MCH analogues were full agonists in the cAMP assay.
Shortening the MCH Peptide--
To establish the human MCH
structure-activity relationships, a number of analogues were designed
following the classical rules of peptide modification (review
Fauchère (43)). The last 2 and the first 5 amino acids of MCH
(compounds 43, 58, 39, 57, 56, and 31) were not essential since
deletions of these amino acids only decreased ~10-fold the potency to
inhibit forskolin-induced intracellular cAMP level, potency being still
in the nanomolar range (Table II). In contrast, the deletion of
Arg6 (compound 52) shifted the potency a further ~10-fold
in the cAMP assay. The minimal sequence for a strong activity thus
required 12 amino acids, from Arg6 to Trp17
(compound 31), i.e. the sequence MCH-(6-17).
Substitutions and Deletions in the Minimal Sequence of
MCH--
The influence of modifications of compound 31 was further
evaluated. First, analogue 31 was subjected to an Ala scan (Table III), which detected mandatory side
chains in positions 11 (Arg) and 13 (Tyr) and to a lesser extent in
position 8 (Met), since the substitution by Ala led to less active
(compound 9) or inactive peptides (compounds 24 and 27). In contrast,
the residues Leu9, Gly10, Val12,
Arg14, and Pro15 could be individually replaced
by Ala without abolishing the biological activity (compounds 17, 20, 25, 29, and 30). Furthermore, the presence of the ring was crucial for
activity as already shown for the entire human MCH peptide (see
compound 44), since the linear analogue of compound 31, compound 38, in
which both cysteines were replaced by serine residues, was inactive in
the cAMP assay. Other substitutions of the essential amino acid
Arg11 by other basic amino acids His or Lys (compounds 34 and 35) failed to restore potent agonist activity (~1000
nM, Table III). In contrast, the key amino acid
Tyr13 could be replaced by Phe in compound 31, the
resulting compound 28 keeping a similar biological activity. Finally,
inversion of the chirality of Tyr13 (compound 32) or of
Arg14 (compound 33) led to dramatically less active
peptides in the cAMP assay (Table III). Starting from compound 31, we
made several attempts to reduce the number of amino acids in the ring
and to change the type of cyclization. Linear peptides, cyclic
head-to-tail peptides, and compounds cyclized over an amide bond
between two amino acid side chains were found to be inactive, at least
in the cAMP assay (Tables IV and V).
Characterization of the [35S]GTP
Since the use of saponin was shown to increase GTP From Partial Agonists to Antagonists--
Beside compound 52, other peptides exhibited partial agonist activity in the
[35S]GTP
Starting from the information that compound 52 ([des-Arg]MCH-(6-17))
was a partial agonist in [35S]GTP Since the discovery that MCH is involved in many
physiological functions, especially in relation to food intake (5, 6, 9, 13, 45) and since the MCH receptor has been recently cloned
(17-21), the search for MCH receptor antagonists has become an
important pharmacological task to explore both the physiological role
of MCH and the therapeutic relevance of its receptor antagonists. In
fact several studies using lower vertebrates bioassays have been
performed to delineate the amino acids required to support MCH activity
(46). However, in view of the differences between the two MCH sequences
(fish and human), no extrapolation to mammals can be made without being
tested experimentally. Similarly, structure-activity relationships studies of the binding of MCH on various cell lines should be taken cautiously since they were not performed with the
genuine human MCH receptor as discussed in the Introduction (15, 16,
33-36). The purpose of the present work was therefore to find out the
structural requirements for human MCH derivatives to behave as agonists
or antagonists at the human MCH receptor stably expressed in HEK293
cells by using the cAMP and [35S]GTP Several studies at other receptors have shown that, due to
intracellular signal amplification, detection of partial agonist effects might not be seen with the cAMP assay and that the antagonist effect of new compounds might be difficult to detect (47, 48). Indeed
all the analogues tested in the cAMP assay, when active, were full
agonists as potent as MCH. Furthermore, when they were tested against
MCH, the inactive analogues were unable to reverse its effect. Thus,
the binding of [35S]GTP Our results obtained for MCH, salmon MCH (compound 45), and
[Phe13 Tyr19]MCH (compound 41) in the cAMP
assay were comparable to those of Chambers et al. (17) at
the human receptor. MCH could be shortened at both its C and N termini
without major loss of activity, leading to the dodecapeptide 31, MCH-(6-17). Similarly, in salmon MCH, the amino acid variations
outside the MCH ring had a limited impact on the agonistic effect of
the modified peptides (31). Interestingly, further shortening at the N
terminus of human MCH led to a weak agonist (compound 52) with partial
agonistic activity in the [35S]GTP Other modifications of MCH-(6-17) led to the discovery of pure
antagonists. Although the substitution of Tyr13 by its
D-counterpart led to the inactive compound 32, the conversion of
Arg14 to its D-counterpart (compound 33) led to an
antagonist with a submicromolar KB (0.75 µM). A similar substitution was reported for
Arg11 in the full MCH sequence (37) also leading to an
antagonist but of much weaker potency (15.8 µM in a
calcium flux assay). In attempts to shorten the dodecapeptide 31 within
the cystine loop, further deletions were tried, and 14 analogues were
generated, 3 of which were antagonists with KB in
the micromolar range (compounds 23, 26, and 22), whereas the others
were inactive. Interestingly, shorter analogues of the ring of fish MCH
have been described (using the scale melanophore bioassay) in which agonist activity was dependent on the composition of both the loop and
the N terminus (29). Inhibition of MCH activity was detected for some
of these compounds, which were shown in fact to counteract MCH activity
through an interaction with the MSH receptor (30).
Extensive substitutions with non-natural amino acids, in compound 31, led to the most potent antagonists described so far at the MCH
receptor, compounds 53, 54, and 55 (KB 0.1-0.2 µM). The antagonist potency of compound 53 was further
confirmed in a calcium flux assay. It should be noted that the calcium
assay may represent a complement to [35S]GTP In summary, the following key compounds 31, 3, and 53-55 have been
discovered in the present study. The dodecapeptide MCH-(6-17) (compound 31) with a conserved ring was shown to be the minimal sequence for full agonistic activity. These findings open also routes
for the discovery of new ligands with optimized biophysical characteristics, such as enhanced solubility, reduced hydrophobicity, and proteolytic stability, still retaining high receptor affinity. Furthermore the structural diversity of agonists, partial agonists, and
antagonists reported in the present work might be very informative in
the study of MCH and MCH receptor interactions. MCH-(6-17) will be
further used as a pharmacophore in future combinatorial approaches for
the generation of large numbers of peptide (54-56) and non-peptide
ligands (57). Potent small size antagonists made of non-natural amino
acids and cyclized over an amide bond, may represent useful tools for
the design of new radiolabeled ligands of the MCH receptor and for both
in vitro and in vivo studies of MCH functions.
S) binding. The dodecapeptide MCH-(6-17)
(MCH ring between Cys7 and Cys16, with a single
extra amino acid at the N terminus (Arg6) and at the C
terminus (Trp17)) was found to be the minimal sequence
required for a full and potent agonistic response on cAMP formation and
[35S]- GTP
S binding. We Ala-scanned this
dodecapeptide and found that only 3 of 8 amino acids of the ring,
namely Met8, Arg11, and Tyr13, were
essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range.
Cys7 and Cys16 were substituted by Asp and Lys
or one of their analogues, in an attempt to replace the disulfide
bridge by an amide bond. However, those modifications were
deleterious for agonistic activity. In [35S]- GTP
S
binding, these compounds behaved as weak antagonists (KB 1-4 µM). Finally, substitution
in MCH-(6-17) of 6 out of 12 amino acids by non-natural residues and
concomitant replacement of the disulfide bond by an amide bond led
to three compounds with potent antagonistic properties
(KB = 0.1-0.2 µM). Exploitation of
these structure-activity relationships should open the way to the
design of short and stable MCH peptide antagonists.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S binding on membrane preparations. The
minimal peptide sequence maintaining the agonistic activity was found
to be the dodecapeptide MCH-(6-17), which includes the cyclic part of
MCH. From this minimal structure, several antagonists with weak
(micromolar) to relatively high (0.1 µM) potency could be designed.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-protection (39),
tert-butyl type groups for side chain protection, and trityl
for cysteine. Arginine was used under its protected form,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl arginine (40). Final
deprotection and cleavage from the resin was achieved by treatment with
90% trifluoroacetic acid in the presence of thiol scavengers. Final
purification was obtained by preparative reverse phase chromatography
using a C18 Delta-Pak, 15 µM, 300 Å, in linear gradients
of acetonitrile/water (0.1% trifluoroacetic acid). Head-to-tail cyclic
compounds, such as compound 23, were synthesized on an
orthochloro-chlorotrityl resin, the N
-Fmoc
eliminated by the action of piperidine, the linear protected peptide
cleaved from the resin with 1% trifluoroacetic acid, and the cyclic
peptide obtained by bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP) activation (41) at high dilution. Compounds cyclized over a disulfide bridge, such as compounds 18-22,
were obtained via air oxidation under strong agitation for 16 h at
room temperature of the unprotected and cleaved linear peptide in
dilute solution. Finally, compounds cyclized over an amide bond, such
as compound 3, were assembled on a Merrifield resin using groups of the
benzyl type for side chain and final N
protection, and groups of the tert-butyl type for protection of the basic and acidic side chains were involved in the cyclization. After removal of the latter groups under mild acidic conditions (50%
trifluoroacetic acid in CH2Cl2), cyclization
was achieved under activation by PyBroP on the resin, and finally, the
cyclic peptide was both freed from its protecting groups and cleaved from the resin by treatment with fluohydric acid. The purity of each
peptide assessed by analytical reversed phase HPLC varied between
90 and 99%, and the molecular weight was confirmed by electrospray
mass spectroscopy. Analytical data are presented in Table I.
S experiments.
S Binding on Membrane
Preparations--
Cells grown at confluency were harvested in
phosphate-buffered saline containing 2 mM EDTA and
centrifuged at 1000 × g for 5 min (4 °C). The
resulting pellet was suspended in 20 mM HEPES (pH 7.5),
containing 5 mM EGTA and homogenized using a Kinematica Polytron. The homogenate was then centrifuged (95,000 × g, 30 min, 4 °C), and the resulting pellet was suspended
in 50 mM HEPES (pH 7.5), 10 mM
MgCl2, and 2 mM EGTA. Determination of protein content was performed according to the method of Lowry et
al. (42). Aliquots of membrane preparations were stored at
80 °C until use. Membranes and peptides were diluted in binding
buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 3 µM GDP, 5 mM MgCl2, 0.1% bovine
serum albumin, 10 µg/ml saponin). Incubation was started by the
addition of 0.2 nM [35S]GTP
S to membranes
(25 µg/ml) and drugs and further followed for 45 min at room
temperature. For experiments with antagonists, membranes were
preincubated with both the agonist and the antagonist for 30 min prior
the addition of [35S]GTP
S. Nonspecific binding was
defined using cold GTP
S (10 µM). Reaction was stopped
by rapid filtration through GF/B filters followed by three successive
washes with ice-cold buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
90%. They
included compounds cyclized over the side chain of two cysteines
(cystine analogues), compounds cyclized over an amide bond between two amino acid side chains, and head-to-tail cyclic analogues (Table I). All peptides were used as tools to
establish the ligand structure-activity relationships, both as agonists
or antagonists, at the human MCH receptor.
Analytical data of peptides
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Fig. 1.
Dose-dependent inhibition of
forskolin-induced cAMP accumulation in HEK293 cells stably expressing
the human MCH receptor. Effects of the human MCH (compound 42, ), human [Phe13,Tyr19]MCH (compound 41,
), salmon MCH (compound 45,
), or linear human
[Ser7,16]MCH (compound 44,
). Points shown are from
representative independent experiments performed in triplicate and
repeated at least three times.
Agonistic potency of shorter analogues of MCH to inhibit the
forskolin-induced cAMP production and to stimulate
[35S]GTPS binding at hSLC1 receptor
Agonistic potency of derivatives of the minimal sequence MCH-(6-17)
upon the forskolin-induced cAMP production and [35S]GTPS
binding at hSLC1 receptor
Agonistic potency of shortened derivatives of the minimal sequence
MCH-(6-17), and [35S]GTPS binding at hSLC1 receptor
S Binding Assay, a
Tool to Discover MCH Partial Agonists and Antagonists--
Strikingly,
all the tested peptides that were active in the cAMP assay behaved as
full agonists as compared with MCH. Furthermore, attempts to
characterize the antagonist properties of all those "inactive"
compounds against 1 or 10 nM of MCH failed to give significant results in the cAMP assay (Table III). This lack of sensitivity was probably due to intracellular signal amplification of
adenylyl cyclase activation. Therefore, another functional model
corresponding to the first step of receptor activation, [35S]GTP
S binding to G proteins, was set up on
membrane preparations. MCH induced ~1.5-fold stimulation of
[35S]GTP
S binding in membranes from HEK-SLC-1 cells
but not from native cells (data not shown).
S binding at the
adenosine A1 receptor (44), its effect was also evaluated in our assay. Indeed, saponin dose-dependently induced a
bell-shaped curve on [35S]GTP
S binding to membranes
expressing the SLC-1 receptor (Fig. 2).
The peak concentration of 10 µg/ml (for 15 µg of proteins/ml) corresponding to a 5-fold stimulation was further used. The
concentrations of NaCl, GDP, and MgCl2 were also optimized;
they were, respectively, 100 nM, 3 µM, and 5 mM (data not shown). The use of saponin did not modify the
potency of MCH to stimulate [35S]GTP
S binding
(EC50 = 4.51 ± 0.52 nM versus
6.05 ± 0.87 nM in the presence of saponin,
n = 3) (Fig. 3).
Comparing the two functional assays, there was a rightward shift
(~20-fold) in the potency of MCH to stimulate
[35S]GTP
S binding as compared with the cAMP assay
(Table II). This was also the case for the other agonists tested
(Tables II and III). However, the data obtained from
[35S]GTP
S binding were nicely correlated with those
gathered from the cAMP assay (r = 0.87, p < 0.0001, n = 19) (Fig.
4). Furthermore, the
[35S]GTP
S binding assay was able to discriminate
between partial and full agonists, as for instance for compound 52, which behaved as a partial agonist (half of the maximal effect of MCH)
in this assay, and which was able to reverse MCH-induced
[35S]GTP
S binding to the level of its partial agonist
intrinsic effect (Fig. 5 and Table
II).
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Fig. 2.
Dose-response effect of saponin upon specific
[35S]GTP S binding in basal (
) or MCH (1 µM)-stimulated (
) conditions on HEK293 cell membranes
stably expressing the human MCH receptor (25 µg/ml). The
stimulation ratio derived from data in A is represented in
B. Points shown are from representative experiments
performed in triplicate and repeated twice independently.
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Fig. 3.
Dose-response isotherm of MCH upon specific
[35S]GTP S binding in the absence
(
) or the presence of saponin 10 µg/ml (
)
on HEK293 cell membranes stably expressing the human MCH receptor.
Results are expressed as a percentage of effect versus basal
level. Points shown are from representative experiments performed in
triplicate and repeated three times independently.
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Fig. 4.
Correlation between agonist potencies
determined by forskolin-induced cAMP accumulation
(pIC50 = logIC50) and by
[35S]GTP
S binding
(pEC50 =
logEC50). The 19 compounds
that behaved as agonists in both tests were considered (data were
calculated from Tables I and II). The correlation coefficient was 0.877 (p < 0.0001).
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Fig. 5.
Concentration response isotherm of compound
52 upon specific [35S]GTP S
binding in the absence (
) or the presence of MCH 100 nM
(
) on HEK293 cell membranes stably expressing the human MCH
receptor. Results are expressed as a percentage of effect
versus the maximal effect of MCH. Points shown are from
representative experiments performed in triplicate, at least three
times independently.
S binding test, as compared with MCH (100%).
Examples are as follows: compounds 9 (51%) and 27 (36%) of the Ala
scan series (Table III) and compounds 34 and 35, the latter being
extremely weak (Table III). The linearization of MCH or of compound 31, via Ser substitutions at the two Cys residues, also led to weakly potent partial agonists with efficacies, respectively, of 64 (compound 44, Table II) and 41% (compound 38) (Table III). The "inactive peptides" in the cAMP assay were further tested in the
[35S]GTP
S binding test. They were inactive alone, but
when tested versus MCH (10 nM), some of them
showed full antagonist properties (Table III and IV). Indeed,
quite surprisingly, substitution of Arg14 (compound 33) by
D-Arg led to a mild antagonist (Table III). The single
deletion of Gly10 or of Val12 both in
Tyr13 and Phe13 analogues also led to
antagonists (compounds 23, 26, and 22, respectively, Fig.
6 and Table IV). The replacement of the
dipeptide Leu9-Gly10 by aminovaleryl, thus
keeping the backbone length constant but eliminating the side chain of
leucine, resulted in a completely inactive compound (compound 19, Table
IV). Similarly, an attempt to modify the peptide 31 by substituting the
Cys7 by Ala and by replacing the two dipeptide sequences,
Leu9-Gly10 and Pro15
-Cys16 by two aminohexanoic acids (compound 1, Table IV)
failed to produce antagonists. Furthermore, mimics of the ring of MCH
were generated, keeping the general shape of the compound 31 (length
and ring) but replacing the disulfide bridge between Cys7
and Cys16 by an amide bond between diaminobutyric acid
(Dab7) and glutamic acid (Glu16). This compound
3 was a partial agonist, with a potency in the 500 nM range
and an efficacy of 67% (Table V). All
the other cyclization attempts failed (Table V).
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Fig. 6.
Concentration response isotherm of compounds
53 ( ) and 26 (
) upon specific
[35S]GTP
S binding in the
presence of MCH 10 nM on HEK293 cell membranes stably
expressing the human MCH receptor. Results are expressed as a
percentage of effect versus the maximal effect of MCH.
Points shown are from representative experiments performed in
triplicate, at least three times independently.
Attempts to find active small cyclic analogues of MCH
S binding (Fig. 5 and
Table II) and that the residues Leu9, Gly10,
Val11, Arg14, and Pro15 could be
individually replaced by Ala without abolishing the biological activity
in both assays (Table III), we designed compound 53 in which not less
than five residues were substituted by non-natural residues. This
compound was a potent antagonist (KB = 148 nM, Fig. 6 and Table VI). A
second compound was synthesized in which the disulfide bridge of
compound 53 was replaced by an amide bond between Dab and Asp (compound
54) or
,
-diaminobutyric acid and Asp (compound 55). These
compounds were also antagonists of good potency (KB = 158 and 180 nM, respectively, Table VI) and should
display enhanced stability in biological fluids. Activity of these
antagonists was further confirmed by calcium flux measurement. MCH was
able to increase Ca2+ in HEK cells stably expressing the
MCH receptor (Fig. 7A). The MCH activation curve was rightward shifted in the presence of compound
53 at 10 and 30 µM (Fig. 7A). Furthermore,
this functional test was able to show the partial agonistic nature of
compound 52 as compared with MCH-(6-17) (compound 31) (Fig.
7B).
Structural and antagonistic activity of highly substituted
undecapeptides derived from MCH-(6-17)
View larger version (18K):
[in a new window]
Fig. 7.
Intracellular Ca2+ signaling in
HEK293 cells stably expressing the human MCH receptor after treatment
with MCH and analogues. A, concentration response
isotherms of MCH alone ( ) or in the presence of compound 53 at 10 (
) and 30 µM (
). The MCH EC50 (6 nM) was decreased to 34 and 57 nM,
respectively. B, concentration response isotherms of
compounds 31 (
) and 52 (
); potencies were 22 and 394 nM, respectively. Results are expressed as the mean
percentage of the calcium peak height with the peak height of 1 µM MCH taken as 100%. Points shown are from
representative experiments performed in triplicate, at least three
times independently.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S binding assays.
S on membrane preparations,
corresponding to the first step of agonist/receptor activation
(49-52), was established. In this test briefly described by Lembo
et al. (21) at the human MCH receptor, a stimulation ratio
of 1.5 over basal [35S]GTP
S binding was confirmed in
our hands. Cohen et al. (44) demonstrated that the use of
saponin greatly enhanced the level of [35S]GTP
S
binding at the adenosine receptor in the presence of the agonist. The
use of saponin was also successfully applied to our assay, leading to a
5-fold stimulation ratio, without modifying the potency of MCH itself.
The effect of saponin is probably linked to a higher recruitment of G
proteins as attested by the number of G proteins measured through
homologous inhibition of [35S]GTP
S binding (51, 52), 1 versus 4.5 pmol/mg in the presence of saponin (data not
shown). There was a highly significant correlation between agonist
potencies obtained in the cAMP assay and affinities in the
[35S]GTP
S binding test (Fig. 4). As expected,
[35S]GTP
S binding was more sensitive since it also
allowed the detection of partial agonists as well as the detection of
weak antagonists. Thus, in the following discussion about MCH
analogues, when partial agonists and antagonists are described, we
refer to results from the [35S]GTP
S binding test.
S binding assay.
Compound 31 was thus considered the minimal MCH sequence required for
agonistic activity and on which an Ala scan and other structural
variations had to be performed. Incidentally, the equivalent fish
minimal sequence, MCH-(5-15), was demonstrated as being more
proteolytically stable than the sMCH (53). Ala scan studies showed that
Met8, Tyr13, and Arg14 residues
were essential for agonistic activity. Whereas substitution of the
Met8 residue diminished agonistic activity, substitutions
of Tyr13 and Arg14 were very
destructive. In fact, the key role of the Arg14
residue was also reported by Macdonald et al. (37) and shown to interact with the residue Asp123 of the MCH receptor. In
contrast, the Tyr13 residue could be replaced by a Phe in
several analogues without loss of activity (compounds 41 and 28).
Substitutions in the dodecapeptide 31 also provided keys for the
conversion of full agonists into partial agonists since the single
substitution of Met8 (compound 9) and Tyr13
(compound 27) by Ala or Arg11 by His or Lys (compounds 34 and 35) afforded partial agonists of low to extremely low potency and
efficacy. The lack of agonistic activity of the compounds in which
Arg11 was replaced by His or Lys strongly suggests that the
negative charge of the chain is not key in the residue interaction with the receptor but rather the chemical nature of this side chain (i.e. guanidinium).
S binding
since both partial agonists (compound 52) and antagonists (compound 53)
were easily detectable. In two of these analogues, 54 and 55, the
cystine bridge was successfully modified into an amide bond without
loss of the antagonistic potency. A similar result was observed for
compound 3. Conversely replacing the Cys by Ser in MCH (compound 44) or
in MCH-(6-17) (compound 38) induced a great loss of potency, and these
compounds then behaved as partial agonists. The importance of
cyclization to keep biological activity has also been documented for
the fish MCH (32).
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ACKNOWLEDGEMENT |
---|
We thank Nelly Fabry for help with the manuscript preparation.
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FOOTNOTES |
---|
* This work was supported by a a Convention CIFRE between the Association Nationale de la Recherche Technique, the Institut de Recherches SERVIER and the Centre National de la Recherche Scientifique (to T. S.).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: Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78 290 Croissy-sur-Seine, France. Tel.: 33 1 55 72 27 48; Fax: 33 1 55 72 28 10; E-mail: jean.boutin@fr.netgrs.com.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M010727200
2 M. Rodriguez and L. Maulon, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
MCH, melanin-concentrating hormone (human, rat, mouse);
sMCH, salmon MCH;
[35S]GTPS, guanosine-5'-O-(3-[35S]thiotriphosphate);
Fmoc, N-(9-fluorenyl)methoxy-carbonyl;
SLC-1, somatostatin-like receptor 1;
HEK, human embryonic kidney;
PyBrop, bromo-tris-pyrrolidinophosphonium hexafluorophosphate;
TEAP, triethylamine phosphate;
HPLC, high pressure liquid chromatography;
Dab, diaminobutyric acid.
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REFERENCES |
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