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

Abstract

Melanin-concentrating hormone (MCH) is a cyclic nona- Melanin-concentrating hormone (MCH)1 has been initially described in fish as a heptadecapeptide (Asp-Thr-Met-Arg-CysMet-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-MetLeu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-GlnVal (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 * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.*

Synthesis and Biological Evaluation in Vitro of a Selective, High Potency Peptide Agonist of Human Melanin-concentrating Hormone Action at Human Melanin-concentrating Hormone Receptor 1

“…Aequorin Bioluminescence Functional Assays (24,26,27)-For the functional receptor activation assays, stable cell lines expressing either the MCH-1R or the MCH-2R and the aequorin reporter protein were used. The assays were performed on a Luminoskan RT luminometer (Labsystems Inc., Gaithersburg, MD) controlled by custom software written for a PC-compatible computer.…”

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini

“…The Sphyrna MCH peptide is composed of 19 AA and is identical to the mammalian MCH, except for a C-terminal residue. A ring structure formed by a disulfide bond, an Arg residue in the N-terminal side chain, and a Trp residue in the C-terminal tail of human MCH are essential for the binding of the protein to its receptor [4]. This structure is well conserved in Sphyrna MCH, suggesting that this peptide might be functional in the shark.…”

Appetite-Boosting Property of Pro-Melanin-Concentrating Hormone131–165 (Neuropeptide-Glutamic Acid-Isoleucine) Is Associated with Proteolytic Resistance

“…This suggests that shortened cyclic peptides on the N-or C-terminal parts could be still active in the rat brain. In contrast, linear compound or deletion in the ring structure led to complete inactivity in binding/functional assays using MCH 1 -transfected cells and in fish or rat bioassays (Audinot et al, 2001a;Bednarek et al, 2001;). This allows us to consider endopeptidase 24.11 and related enzymes as overt MCH-inactivating peptidases.…”

“…Because NEI-MCH acts as a superagonist, we hypothesized that it may be a better ligand or activator of signaling pathways than MCH at either of the MCH receptors. Previous studies have identified the structural domains of MCH responsible for binding and affinity to MCH1 (MacDonald et al, 2000;Audinot et al, 2001a) and MCH2 (Bednarek et al, 2001;Rodriguez et al, 2001). These and Arg 14 as a potential site of interaction with MCH1 and MCH 6 -17 (ring between Cys 7 and Cys 16 ) as a minimal sequence required for an agonistic response.…”

“…Are endopeptidase 24.11 and aminopeptidases/carboxypeptidases truly physiological MCH-inactivating peptidases? Structure-activity studies performed with a number of cyclic fragments and MCH analogs demonstrated that the last two and the first five amino acids of the cyclic MCH [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] are not essential for full potency in rat/human MCH 1 -transfected cellular models (MacDonald et al, 2000;Audinot et al, 2001a;Bednarek et al, 2001;Suply et al, 2001) and in vivo ). This suggests that shortened cyclic peptides on the N-or C-terminal parts could be still active in the rat brain.…”