Design, Synthesis, and Biological Evaluation of New Cyclic Melanotropin Peptide Analogues Selective for the Human Melanocortin-4 Receptor
2007

Abstract
Intensive efforts have been made to develop potent and selective ligands for certain human melanocortin receptors as possible treatments for obesity and sexual dysfunction due to the role of these receptors in feeding behavior, energy homeostasis, sexual function, etc. A number of novel ?-MSH analogues were designed and synthesized primarily on the basis of our previous MTII NMR structure. In these peptide analogues, a disulfide or lactam bridge between residues at positions 5 and 8 was used as a conformational constraint to enhance the ?-turn spanning His6 and d-Phe7, while the pharmacophore group in Arg8 was mimicked via N?-alkylation of residues 8 or 9 with the guanidinylbutyl group. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues have good binding affinity for the hMC4R (0.7–4.1 nM), but have no binding affinity up to 10 ?M at the other three melanocortin receptors. Interestingly, the three hMC4R selective analogues display only 50% binding efficiency, suggesting there is allosteric modulation of the melanocortin-4 receptor. These analogues were found to act as antagonists of the hMC4R. This result represents a discovery of very selective peptide-based antagonists for the hMC4R. The high selectivity may be due to the strong conformational constraint via ring contraction as compared to MTII, and the rigid conformation preferred by these new ligands allows them to recognize only the hMC4R, but not to activate the second messenger. The MTII NMR structure-based design thus not only examined the structural model of melanocortin ligands, but also yielded new biologically unique ?-MSH analogues.

Intro
?-, ?-, and ?-Melanocyte stimulating hormone (MSHa) and adrenocorticotropin (ACTH) are posttranslational products of the proopiomelanocortin (POMC) prohormone. As natural ligands interacting with five melanocortin receptors (MC1R-MC5R), these endogenous neuropeptides and hormones play an important role in regulating a large number of diverse physiological functions, including skin pigmentation, adrenal cortical function, energy homeostasis, feeding behavior, sexual function, exocrine gland function, pain, and many others. These physiological functions mediated in the melanocortin systems have made melanotropins useful lead peptides to develop drugs for treating obesity, sexual dysfunction, pigmentary problems, etc. To this end, earlier studies have focused on elucidation of the structure–activity relationships of ?-MSH and identified His-Phe-Arg-Trp as the message sequence of melanotropin peptides. These studies also have led to early discoveries of several ?-MSH analogues with biological profiles significantly different from those of ?-MSH itself, thus providing diverse molecular tools for better understanding the biological roles of melanocortin pathways. More recently, much effort has been made to develop receptor selective analogues, peptidomimetics, and small molecule mimics of ?-MSH. Selectivity for the MC4R is a particularly desirable goal due to the role of this receptor in feeding regulation and weight control. Suitable ligands highly selective for the human MC4R (hMC4R) may be developed as specific molecular probes to better study the receptor functions and as possible treatments for obesity.

One approach to development of receptor selective ligands is to apply conformational constraints to the existing lead peptides. In this regard, the structural insights obtained in our previous NMR study of MTII and other melanotropin analogues provide useful information on the design of potentially selective ligands with new conformational constraints. Despite the conformational differences observed, most of these ?-MSH analogues were found to contain a type II ?-turn structure spanning residues 6 and 7 distorted to different extents. In this study, we designed, synthesized, and biologically analyzed a number of new conformationally restricted analogues on the basis of the NMR-obtained conformation. The design was aimed to enhance the type II ?-turn at positions 6 and 7 of ?-MSH with a linker between residues 5 and 8. It turned out that several of these analogues selectively bind to the human MC4R only and act as antagonists of this receptor. The NMR structure-based design and synthesis of the new analogues thus not only served as a test of our previous structural model of melanocortin ligands, but also resulted in new biologically unique and receptor selective ?-MSH analogues.

Results
The binding affinity and adenylate cyclase activity of the peptides developed in this study were evaluated for various human melanocortin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) using MTII as a standard control. Peptide 1 was found to have good binding affinity toward the hMC4R (IC50 = 1.8 nM) but exhibited no receptor–ligand binding for the other melanocortin receptor subtypes up to 10?5 M. Interestingly, this analogue was an antagonist with a binding efficiency of about 50%. This may suggest the existence of an allosteric function for 1 on the hMC4R. Such an allosterically linked binding site prohibits the binding of a ligand to the other site of the receptor protein. Furthermore, the allosteric modulator changes the shape of the receptor, probably through conformational selection and stabilization of one or some of the receptor's ensemble of states. This results in a completely new set of reactivity toward the ligand and the cellular components. To verify the existence of allosteric function of the melanocortin receptors, we did the experiments where dose–response curves of MTII were obtained in the presence of antagonists. No significant shift in dose–response curves were observed which supports the existence of an allosteric binding site for antagonist peptide 1. In contrast to peptide 1, peptide 2 was obtained by using the d-enantiomer of Cys in position 8. The resulting peptide was found to be entirely inactive at all the melanocortin receptor subtypes, which suggests that such substitution drastically affects the secondary structure of the corresponding peptide. A shift of the N?-guanidylbutyl moiety to the Trp9 position resulted in peptide 3, which demonstrated a potent binding affinity (IC50 = 4.1 nM), and weak partial agonist activity (18% maximal stimulation) at the hMC4R. Peptide 4 was obtained by substituting Trp9 in the peptide 3 sequence with d-Trp9, which led to a further improvement in binding potency to hMC4R (IC50 = 0.7 nM). In the cAMP accumulation assay, this compound was determined to be a weak partial agonist (19% maximal stimulation) at the hMC4R, similar to peptide 3. As was observed for peptide 1, peptides 3 and 4 also exhibited remarkable receptor selectivity for the hMC4R and once again the binding efficiency could reach to the level of about 50% only. This evidence provides further support for the existence of an allosteric binding site at the hMC4R. These results also suggest that the C? stereochemistry of Trp9 does not appear to be as critical to biological activity as that of the residue at position 8. Presumably, this is because Trp9 is located outside the ring structure, which affords much more flexibility of its side chain group for a favorable orientation for interaction with the receptor.

Conclusion
A number of new cyclic ?-MSH analogues was designed on the basis of the MTII structure. In these peptide analogues, novel disulfide or lactam bridges with macrocyclization were used as conformational constraints, while the pharmacophore group in Arg8 was mimicked via N?-alkylation of certain amino acid residues with the guanidinylbutyl group. All eight cyclic peptides were successfully synthesized on a solid-phase support. The Mitsunobu reaction was used to achieve the N?-alkylation for mimicking the Arg8 pharmacophore group in these syntheses. The binding affinity and adenylate cyclase activity assays of these peptides at human melanocortin receptors showed that three of the new ?-MSH analogues act as antagonists and exhibited high selectivity toward the human melanocortin-4 receptor. The selectivity may be due to the strong conformational constraint via ring contraction as compared to MTII. The MTII NMR structure-based design thus not only examined the structural model of melanocortin ligands, but also yielded new biologically unique ?-MSH analogues. As observed in this study, allosterically modulated GPCRs have been recently recognized as an alternative approach to gain selectivity in drug action. It is highly important to further modify the majority of assays currently being used to study such novel compounds that could be a great lead in the drug discovery process. A number of peptide analogues are in the works in our laboratory that are based on this unique design concept.