Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but non-selective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4 and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation which resembles other a-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH 2 ) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2′)-Arg-Trp-Lys]-NH 2 ) by replacing the His-D-Phe and His-D-Nal(2′) fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx = D-Phe/pCl-D-Phe/D-Nal(2′)). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists. KeywordsHuman melanocortin receptors; 4-Amino-1,2,4,5-tetrahydro-2-benzazepin-3-ones; Cyclic lactam analogues; Conformational restrictions; hMC3R/hMC5R antagonists The α-melanocyte stimulating hormone (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-TrpGly-Lys-Pro-Val-NH 2 ) plays a role in a wide range of biological responses like feeding behavior, pain modulation, learning behavior, pigmentation, sexual function, energy homeostasis, and thermoregulation. 1 In particular, the human melanocortin 4 subtype receptor is an attractive drug target because of its role in regulation of feeding behavior. 2,3 Design of selective hMC4R antagonists is considered to have great potential for the treatment of anorexia. 4,5 The hMC3R on the other hand has been shown to play a role in the physiological process of energy partitioning and body weight. 6 Controlled modulation of these receptors could lead to promising results in the field of feeding disorders.The principal pharmacophore groups of α-MSH were found to be the side chains of the central tetrapeptide His 6 -Phe 7 -Arg 8 -Trp 9 . 7,8 Molecular modeling as well as conformational analysis of a variety of cyclic analogues led to the discovery of a superpotent lactam analogue MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH 2 ). 9 This cyclic peptide analogue, first introduced by Al-Obeidi et al. 9 was a very potent, but non-selective, agonist for the human melanocortin receptor subtypes MC1R, MC3R, MC4R, and MC5R. It also showed a very high stability against all proteolytic enzymes and tissue homogenates. 9 Because of the lack in selectivity, finding selective ligands for each of the four human melanocortin receptors * Corresponding author. Tel.: +32 26293298; fax: +32 26293 304; e-mail: sballet@vub.ac.be. The 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold (Fig. 1) has proven to be an excellent tool for the design of novel peptide mimetics. 17-22 Its qualities can be described as two folded: (1) the scaffold can be considered as a conformationally restricted Phe analogue where the aromatic side chain is anchored to the α-amine of the next residue by means of a methylene bridge (dotted line); 17 (2) on the other hand, one can see the Aba template as a 'privileged template'. 23 NIH Public AccessIn scaffold 1 only the g(+) (χ 1 = +60°) and trans (χ 1 = 180°) staggered conformations are allowed for the C α -C β bon...