Structure-Activity Relationship Studies of Melanin-concentrating Hormone (MCH)-related Peptide Ligands at SLC-1, the Human MCH Receptor*

Valérie AudinotDagger , Philippe BeauvergerDagger , Chantal LahayeDagger , Thomas SuplyDagger §, Marianne RodriguezDagger , Christine OuvryDagger , Véronique LamamyDagger , Jérôme ImbertDagger , Hervé RiqueDagger , Jean-Louis Nahon§, Jean-Pierre GalizziDagger , Emmanuel CanetDagger , Nigel Levens, Jean-Luc Fauchère||, and Jean A. BoutinDagger **

From the Dagger  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




    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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]- GTPgamma 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]- GTPgamma 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]- GTPgamma 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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]GTPgamma 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 Nalpha -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 Nalpha 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.

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]GTPgamma S experiments.

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]GTPgamma 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]GTPgamma 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]GTPgamma S. Nonspecific binding was defined using cold GTPgamma S (10 µM). Reaction was stopped by rapid filtration through GF/B filters followed by three successive washes with ice-cold buffer.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peptides-- A total of 58 MCH analogues (including genuine MCH) was obtained by synthesis on solid phase with a purity of >= 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.


                              
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Table I
Analytical data of peptides
All the peptides were solid-phase synthesized, purified, and analyzed by reverse phase HPLC and characterized by mass spectrometry.

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.



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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, black-diamond ), salmon MCH (compound 45, black-triangle), or linear human [Ser7,16]MCH (compound 44, black-down-triangle ). Points shown are from representative independent experiments performed in triplicate and repeated at least three times.


                              
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Table II
Agonistic potency of shorter analogues of MCH to inhibit the forskolin-induced cAMP production and to stimulate [35S]GTPgamma S binding at hSLC1 receptor
All experiments were performed independently at least three times in triplicate determinations.

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).


                              
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Table III
Agonistic potency of derivatives of the minimal sequence MCH-(6-17) upon the forskolin-induced cAMP production and [35S]GTPgamma S binding at hSLC1 receptor
All experiments were performed independently at least three times in triplicate determinations.


                              
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Table IV
Agonistic potency of shortened derivatives of the minimal sequence MCH-(6-17), and [35S]GTPgamma S binding at hSLC1 receptor
All experiments were performed independently at least three times in triplicate determinations.

Characterization of the [35S]GTPgamma 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]GTPgamma S binding to G proteins, was set up on membrane preparations. MCH induced ~1.5-fold stimulation of [35S]GTPgamma S binding in membranes from HEK-SLC-1 cells but not from native cells (data not shown).

Since the use of saponin was shown to increase GTPgamma 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]GTPgamma 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]GTPgamma 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]GTPgamma 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]GTPgamma 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]GTPgamma 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]GTPgamma 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]GTPgamma S binding in basal (open circle ) 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]GTPgamma S binding in the absence (open circle ) 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]GTPgamma 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]GTPgamma S binding in the absence (open circle ) 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.

From Partial Agonists to Antagonists-- Beside compound 52, other peptides exhibited partial agonist activity in the [35S]GTPgamma 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]GTPgamma 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 (open circle ) upon specific [35S]GTPgamma 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.


                              
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Table V
Attempts to find active small cyclic analogues of MCH
All experiments were performed independently at least three times in triplicate determinations.

Starting from the information that compound 52 ([des-Arg]MCH-(6-17)) was a partial agonist in [35S]GTPgamma 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 alpha ,gamma -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).


                              
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Table VI
Structural and antagonistic activity of highly substituted undecapeptides derived from MCH-(6-17)
All experiments were performed independently at least three times in triplicate determinations.



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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 (down-triangle) and 30 µM (triangle ). The MCH EC50 (6 nM) was decreased to 34 and 57 nM, respectively. B, concentration response isotherms of compounds 31 (black-triangle) and 52 (black-square); 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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]GTPgamma S binding assays.

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]GTPgamma 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]GTPgamma S binding was confirmed in our hands. Cohen et al. (44) demonstrated that the use of saponin greatly enhanced the level of [35S]GTPgamma 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]GTPgamma 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]GTPgamma S binding test (Fig. 4). As expected, [35S]GTPgamma 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]GTPgamma S binding test.

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]GTPgamma 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).

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]GTPgamma 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).

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.


    ACKNOWLEDGEMENT

We thank Nelly Fabry for help with the manuscript preparation.


    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.


    ABBREVIATIONS

The abbreviations used are: MCH, melanin-concentrating hormone (human, rat, mouse); sMCH, salmon MCH; [35S]GTPgamma S, 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Kawauchi, H., Kawazoe, I., Tsubokawa, M., Kishida, M., and Baker, B. I. (1983) Nature 305, 321-323[Medline] [Order article via Infotrieve]
2. Vaughan, J. M., Fischer, W. H., Hoeger, C., Rivier, J., and Vale, W. (1989) Endocrinology 125, 1660-1665[Abstract]
3. Tritos, N. A., and Maratos-Flier, E. (1999) Neuropeptides 33, 339-349[CrossRef][Medline] [Order article via Infotrieve]
4. Bittencourt, J. C., Presse, F., Arias, C., Peto, C., Vaughan, J., Nahon, J. L., Vale, W., and Sawchenko, P. E. (1992) J. Comp. Neurol. 319, 218-245[Medline] [Order article via Infotrieve]
5. Viale, A., Zhixing, Y., Breton, C., Pedeutor, F., Coquerel, A., Jordan, D., and Nahon, J. L. (1997) Mol. Brain Res. 46, 243-255[CrossRef][Medline] [Order article via Infotrieve]
6. Elmquist, J. K., Elias, C. F., and Saper, C. B. (1999) Neuron 22, 221-232[Medline] [Order article via Infotrieve]
7. Hervieu, G. J., Cluderay, J. E., Harrison, D., Meakin, J., Maycox, P., Nasir, S., and Leslie, R. A. (2000) Eur. J. Neurosci. 12, 1194-1216[CrossRef][Medline] [Order article via Infotrieve]
8. Presse, F., Sorokovsky, I., Max, J. P., Nicolaidis, S., and Nahon, J. L. (1996) Neuroscience 71, 735-745[CrossRef][Medline] [Order article via Infotrieve]
9. Qu, D., Ludwig, D. S., Gammeltoft, S., Piper, M., Pelleymounter, M. A., Cullen, M. J., Mathes, W. F., Przupek, J., Kanarek, R., and Maratos-Flier, E. (1996) Nature 380, 243-247[CrossRef][Medline] [Order article via Infotrieve]
10. Rossi, M., Choi, S. J., O'Shea, D., Miyoshi, T., Ghatei, M. A., and Bloom, S. R. (1997) Endocrinology 138, 351-356[Abstract/Free Full Text]
11. Tritos, N. A., Vincent, D., Gillette, J., Ludwig, D. S., Flier, E. S., and Maratos-Flier, E. (1998) Diabetes 47, 1687-1692[Abstract]
12. Kokkotou, E., Mastaitis, J. W., Qu, D., Hoersch, D., Slieker, L., Bonter, K., Tritos, N. A., and Maratos-Flier, E. (2000) Neuropeptides 34, 240-247[CrossRef][Medline] [Order article via Infotrieve]
13. Shimada, M., Tritos, N. A., Lowell, B. B., Flier, J. S., and Maratos-Flier, E. (1998) Nature 396, 670-674[CrossRef][Medline] [Order article via Infotrieve]
14. Bradley, R. L., Kokkotou, E. G., Maratos-Flier, E., and Cheatham, B. (2000) Diabetes 49, 1073-1077[Abstract]
15. Drozdz, R., and Eberle, A. N. (1995) J. Receptor Signal Transduc. Res. 15, 487-502[Medline] [Order article via Infotrieve]
16. Burgaud, J. L., Poosti, R., Fehrentz, J. A., Martinez, J., and Nahon, J. L. (1997) Biochem. Biophys. Res. Commun. 241, 622-629[CrossRef][Medline] [Order article via Infotrieve]
17. Chambers, J., Ames, R. S., Bergsma, D., Muir, A., Fitzgerald, L. R., Hervieu, G., Dytko, G. M., Foley, J. J., Martin, J., Liu, W. S., Park, J., Ellis, C., Ganguly, S., Konchar, S., Cluderay, J., Leslie, R., Wilson, S., and Sarau, H. M. (1999) Nature 400, 261-265[CrossRef][Medline] [Order article via Infotrieve]
18. Saito, Y., Nothacker, H. P., Wang, Z., Lin, S. H. S., Leslie, F., and Civelli, O. (1999) Nature 400, 265-269[CrossRef][Medline] [Order article via Infotrieve]
19. Shimomura, Y., Mori, M., Sugo, T., Ishibashi, Y., Abe, M., Kurokawa, T., Onda, H., Nishimura, O., Sumino, Y., and Fujino, M. (1999) Biochem. Biophys. Res. Commun. 261, 622-626[CrossRef][Medline] [Order article via Infotrieve]
20. Bachner, D., Kreienkamp, H. J., Weise, C., Buck, F., and Richter, D. (1999) FEBS Lett. 457, 522-524[CrossRef][Medline] [Order article via Infotrieve]
21. Lembo, P. M., Grazzini, E., Cao, J., Hubatsch, D. A., Pelletier, M., Hoffert, C., St-Onge, S., Pou, C., Labrecque, J., Groblewski, T., O'Donnell, D., Payza, K., Ahmad, S., and Walker, P. (1999) Nat. Cell Biol. 1, 267-271[CrossRef][Medline] [Order article via Infotrieve]
22. Kolakowski, J. L. K., Jung, B. P., Nguyen, T., Johnson, M. P., Lynch, K. R., Cheng, R., Heng, H. Q., George, S. R., and O'Dowd, B. F. (1996) FEBS Lett. 398, 253-258[CrossRef][Medline] [Order article via Infotrieve]
23. Lakaye, B., Minet, A., Zorzi, W., and Grisar, T. (1998) Biochim. Biophys. Acta 1401, 216-220[Medline] [Order article via Infotrieve]
24. Beck-Sickinger, A. G., Wieland, H. A., Wittneben, H., Willim, K. D., Rudolf, K., and Jung, G. (1994) Eur. J. Biochem. 225, 947-958[Abstract]
25. Doods, H. N., Wieland, H. A., Engel, W., Eberlein, W., Willim, K. D., Eutzeroth, M., Wienen, W., and Rudolf, K. (1996) Regul. Pept. 65, 71-77[CrossRef][Medline] [Order article via Infotrieve]
26. Keire, D. A., Mannon, P., Kobayashi, M., Walsh, J. H., Solomon, T. E., and Reeve, J. R. (2000) Am. J. Physiol. 279, G126-G131[Abstract/Free Full Text]
27. Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem. 275, 36043-36048[Abstract/Free Full Text]
28. Kawazoe, I., Kawauchi, H., Hirano, T., and Naito, N. (1987) Int. J. Pept. Protein Res. 29, 714-721[Medline] [Order article via Infotrieve]
29. Lebl, M., Hruby, V., Castrucci, A. M., Visconti, M. A., and Hadley, M. E. (1988) J. Med. Chem. 31, 949-954[Medline] [Order article via Infotrieve]
30. Lebl, M., Hruby, V., Castrucci, A. M., and Hadley, M. E. (1989) Life Sci. 44, 451-457[Medline] [Order article via Infotrieve]
31. Matsunaga, T. O., Hruby, V. J., Lebl, M., Castrucci, A. M. L., and Hadley, M. E. (1989) Peptides (Elmsford) 10, 773-778[CrossRef][Medline] [Order article via Infotrieve]
32. Matsunaga, T. O., Hruby, V. J., Lebl, M., Castrucci, A. M. L., and Hadley, M. E. (1992) Life Sci. 51, 679-685[Medline] [Order article via Infotrieve]
33. Drozdz, R., and Eberle, A. N. (1995) J. Pept. Sci. 1, 58-65[Medline] [Order article via Infotrieve]
34. Drozdz, R., Siegrist, W., Baker, B. I., Chluba-de Trapia, J., and Eberle, A. N. (1995) FEBS Lett. 359, 199-202[CrossRef][Medline] [Order article via Infotrieve]
35. Drozdz, R., Hintermann, E., and Eberle, A. N. (1998) Ann. N. Y. Acad. Sci. 839, 210-213[Free Full Text]
36. Hintermann, E., Drozdz, R., Tanner, H., and Eberle, A. N. (1999) J. Recept. Signal Transduct. Res. 19, 411-422[Medline] [Order article via Infotrieve]
37. Macdonald, D., Murgolo, N., Zhang, R., Durkin, J. P., Yao, X., Strader, C. D., and Graziano, M. P. (2000) Mol. Pharmacol. 58, 217-225[Abstract/Free Full Text]
38. Atherton, E., and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis: A Practical Approach , pp. 1-203, IRL Press at Oxford University Press, Oxford
39. Fields, G. B., and Noble, R. L. (1990) Int. J. Pept. Protein Res. 35, 161-214[Medline] [Order article via Infotrieve]
40. Carpino, L. A., Shroff, H., Triolo, S. A., Mansour, E. S., Wenschuh, H., and Albericio, F. (1993) Tetrahedron Lett. 34, 7829-7832[CrossRef]
41. Coste, J., Frérot, E., Jouin, P., and Castro, B. (1991) Tetrahedron Lett. 32, 1967-1970[CrossRef]
42. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
43. Fauchère, J. L. (1995) in Computer-aided Drug Design in Industrial Research (Hermann, E. C. , and Franke, R., eds) , pp. 129-161, Springer-Verlag, Berlin
44. Cohen, F. R., Lazareno, S., and Birdsall, N. J. M. (1996) Br. J. Pharmacol. 117, 1521-1529[Abstract]
45. Nahon, J. L. (1994) Crit. Rev. Neurobiol. 8, 221-262[Medline] [Order article via Infotrieve]
46. Baker, B. I. (1991) Int. Rev. Endocrinol. 126, 1-47
47. Adham, N., Ellerbrock, B., Hartig, P., Weinshank, R. L., and Branchek, T. (1993) Mol. Pharmacol. 43, 427-433[Abstract]
48. Stanton, J. A., and Berr, M. S. (1997) Eur. J. Pharmacol. 320, 267-275[CrossRef][Medline] [Order article via Infotrieve]
49. Hilf, G., Gieschi, K. P., and Jakobs, K. H. (1989) Eur. J. Pharmacol. 186, 725-731
50. Lazareno, S., Farries, T., and Birdsall, N. J. M. (1993) Life Sci. 52, 449-456[CrossRef][Medline] [Order article via Infotrieve]
51. Newman-Tancredi, A., Conte, C., Chaput, C., Verrièke, L., and Millan, M. J. (1997) Neuropharmacology 36, 451-459[CrossRef][Medline] [Order article via Infotrieve]
52. Audinot, V., Newman-Tancredi, A., and Millan, M. J. (2000) Neuropharmacology 40, 57-64[CrossRef]
53. Castrucci, A. M., Visconti, M. A., Matsunaga, T. O., Hadley, M. E., and Hruby, V. J. (1992) Comp. Biochem. Physiol. B 103, 317-320[Medline] [Order article via Infotrieve]
54. Boutin, J. A., Hennig, P., Lambert, P. H., Bertin, S., Petit, L., Mahieu, J. P., Serkiz, B., Volland, J. P., and Fauchère, J. L. (1996) Anal. Biochem. 234, 126-141[CrossRef][Medline] [Order article via Infotrieve]
55. Boutin, J. A., Lambert, P. H., Bertin, S., Volland, J. P., and Fauchère, J. L. (1999) J. Chromatogr. 725, 17-37
56. Fauchère, J. L., Boutin, J. A., Henlin, J. M., Kucharczyk, N., and Ortuno, J. C. (1998) Chemometrics Intelligent Lab. Syst. 43, 43-68[CrossRef]
57. Boutin, J. A., Lahaye, C., Pegurier, C., Nicolas, J. P., Fauchère, J. L., Langlois, M., Rebard, P., Delagrange, P., and Canet, E. (2000) J. Recept. Signal Transduct. Res. 20, 105-118[Medline] [Order article via Infotrieve]


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