D-Amino Acid Scan of γ-Melanocyte-Stimulating Hormone: Importance of Trp 8 on Human MC3 Receptor Selectivity Paolo Grieco, ‡,|| Preeti M. Balse, ‡,† David Weinberg, § Tanya MacNeil, § and Victor J. Hruby* ,‡ Department of Chemistry, University of Arizona, Tucson, Arizona 85721, and Department of Obesity Research, Merck Research Laboratories, Rahway, New Jersey 07065 Received May 18, 2000 In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide γ-melanocyte- stimulating hormone (γ-MSH). The native γ-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the γ-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp 8 analogue, there is an increase in binding affinity by about 1 order of magnitude (IC 50 ) 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe 6 analogue is about 10-fold more potent (IC 50 ) 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC 50 ) 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg 10 (EC 50 ) 35 nM) and DPhe 11 (EC 50 ) 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp 8 compound (EC 50 ) 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr 1 peptide is selective for the MC3R (EC 50 ) 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg 10 , but not Arg 7 , result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of γ-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg 10 (but not Arg 7 ), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far. Introduction The melanocortin receptors (MCRs) are members of the superfamily of G-protein coupled receptors (GPCRs). Until today, five melanocortin receptors (MC1R- MC5R) have been discovered and cloned. 1-9 The MC1R is expressed in melanocytes and leukocytes, and it is implicated in pigmentation and inflammation, respec- tively. 10,11 The MC2R is only expressed in the adrenal gland and mediates glucocorticoneogenesis. 12 The MC3R is mainly expressed in a few brain areas and some areas of the periphery, and its functional role is much less- defined. 5 The MC4R is exclusively localized in the brain where it is widely distributed. 13 Recently it has been demonstrated that this receptor plays an important role in weight homeostasis. 14,15 The MC5R is found in a variety of peripheral tissues and is believed to partici- pate in regulation of exocrine gland function. 16 The newly discovered MC3R, MC4R, and MC5R have comparatively lower affinity for the melanocortin pep- tides, and none of the natural hormones have enough selective properties which would be useful to distinguish between the physiological function of these subtypes of receptors. The natural melanocortin peptides (Table 1) are thus selective only for the MC1R, whereas ACTH is selective for the MC2R. 17-19 Consequently, potent and receptor-specific agonists and especially antagonists would be extremely valuable tools for determination of the physiological roles of MC3R, MC4R, and MC5R. To develop effective agonist and antagonist ligands of the melanocortin receptors, it is necessary to know the structure-activity relationships (SARs) of the natu- ral molecule in detail at each of the receptors. In the case of a short peptide like the γ-melanocyte-stimulating hormone (γ-melanotropin, γ-MSH), D-amino acid sub- stitution can be a useful strategy to increase our knowledge about the SARs for this natural peptide. As a result, we have studied the effects of D-amino acid substitution at each position of the γ-MSH peptide * To whom correspondence should be addressed. Phone: (520) 621- 6332. Fax: (520) 621-8407. E-mail: hruby@u.arizona.edu. ‡ University of Arizona. § Merck Research Laboratories. | Current address: Department of Pharmaceutical Chemistry and Toxicology, University of Napoli “Federico II”, Napoli 80131, Italy. † Current address: American Peptide Co., Inc., 777 E. Eveyln Ave., Sunnyvale, CA 94086. 4998 J. Med. Chem. 2000, 43, 4998-5002 10.1021/jm000211e CCC: $19.00 © 2000 American Chemical Society Published on Web 11/17/2000