(AGRP)âMelanocortin Chimeric Tyr-c[Î²-Asp-H - American Chemical

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J. Med. Chem. 2005, 48, 3060-3075

Structure-Activity Relationships of the Unique and Potent Agouti-Related Protein (AGRP)-Melanocortin Chimeric Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Peptide Template Andrzej Wilczynski,† Krista R. Wilson,† Joseph W. Scott,† Arthur S. Edison,‡ and Carrie Haskell-Luevano*,† University of Florida, Departments of Medicinal Chemistry and Biochemistry and Molecular Biology, Gainesville, Florida 32610 Received December 5, 2004

The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structureactivity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for 1H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[β-Asp-His-DPhe-Ala-Trp-AsnAla-Phe-Dpr]-Tyr-NH2 and Tyr-c[β-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a γ-turn secondary structure involving the DPhe4 in the central position of the Tyr-c[β-Asp-Phe-DPheArg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology. Introduction The melanocortin pathway has been implicated in mediating a variety of physiological responses including pigmentation,1 obesity, energy homeostasis,2-4 and sexual function.5 The melanocortin system consists of endogenous agonists derived from posttranslational modification of the proopiomelanocortin (POMC) gene transcript,6,7 five G-protein coupled receptors (MC15R),8-14 and the endogenous antagonists agouti15 and agouti-related protein (AGRP).16 The endogenous agonists R-, β-, γ-melanocyte-stimulating hormones (MSH) and adrenocorticotropin (ACTH) all contain a core “HisPhe-Arg-Trp” tetrapeptide sequence that has been hypothesized to be important for melanocortin receptor molecular recognition and stimulation.17,18 Interestingly, the endogenous melanocortin receptor antagonists agouti and AGRP have a core “Arg-Phe-Phe” tripeptide sequence that has been demonstrated to be important for agouti and AGRP to bind and antagonize the melanocortin receptors.19,20 It has been postulated that the antagonist hAGRP Arg-Phe-Phe amino acids may be mimicking the agonist Phe-Arg-Trp residues. Chimeric peptide ligands that utilize the melanocortin agonist NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe* Reprint requests should be addressed to Carrie Haskell-Luevano, University of Florida, Department of Medicinal Chemistry, P.O. Box 100485, Gainesville, FL 32610-0485. Phone (352) 846-2722, Fax (352) 392-8182, e-mail: [email protected]. † Department of Medicinal Chemistry. ‡ Department of Biochemistry.

Arg-Trp-Gly-Lys-Pro-Val-NH2)21 and MTII (Ac-Nlec[Asp-His-DPhe-Arg-Trp-Lys]-NH2)22,23 or the antagonist hAGRP(109-118) Tyr-c[Cys-Arg-Phe-Phe-Asn-AlaPhe-Cys]-Tyr20,24 peptide templates have been examined where the agonist Phe-Arg-Trp or antagonist Arg-PhePhe were substituted for the corresponding residues in the normal peptide template.25,26 These latter studies resulted in the identification of the novel melanocortin receptor peptide template Tyr-c[β-Asp-His-DPhe-ArgTrp-Asn-Ala-Phe-Dpr]-Tyr-NH2 that is equipotent to R-MSH at the mouse MC1, MC3, and MC5 receptors, but is more potent than R-MSH at the mMC4R.25 Upon the basis of the discovery of this novel melanocortin receptor template25 and previous structure-activity studies on the agonist Ac-His-DPhe-Arg-Trp-NH2 tetrapeptide template,27-30 where incorporation of unusual amino acids at various positions of the tetrapeptide template resulted in unique pharmacology, we designed the study reported herein to determine if this is a “novel” peptide template or if instead it resembles the “classical” melanocortin peptide agonist ligand pharmacology and structure-activity relationships (SAR). This study utilizes the Tyr-c[β-Asp-His-DPhe-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2 melanocortin agonist template that incorporates substitution of the His, DPhe, Arg, and Trp amino acids with natural and unnatural amino acids that have been “classically” demonstrated to result in significant and consistent changes in melanocortin receptor pharmacology.

10.1021/jm049010r CCC: $30.25 © 2005 American Chemical Society Published on Web 03/26/2005

AGRP-Melanocortin Chimeric Peptide Template

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3061

Table 1. Summary of the Peptide Modifications Performed Using the Chimeric AGRP-Melanocortin Templatea Tyr

c[β-Asp

His

DPhe

Arg

Trp

Ala Pro Phe Atc (rac)

Ala Pro (pI)DPhe DNal(2′) DNal(1′) DBip

Ala Pro Lys

Ala Pro Nal(2′) DNal(2′) Bip Tic

Asn

Ala

Phe

Dpr]

Tyr-NH2

a The template peptide is indicated on the first line. Individual amino acid modifications are listed below the template amino acid with the new amino acid substitution indicated (all other residues remain the same as the template peptide). The lactam cyclization is formed using the CR carboxylic acid of Asp and the side chain amine of Dpr resulting in a 29-membered ring.

Figure 1. Structure of the chimeric antagonist hAGRPmelanocortin peptide template utilized in this study. The bold bonds and circle focuses upon the Asp amino acid where the lactam bridge involves the Asp backbone domain and the side chain is incorporated into the peptide backbone. This configuration is different from the typical incorporation of the Asp residue that generally results in the residue side chain participating in the formation of the lactam bridge cycle, versus the peptide backbone, in melanocortin ligands.

Results Chemical Design, Synthesis, and Characterization. The chimeric peptide template examined in this study is derived from the melanocortin receptor antagonist hAGRP(109-118) sequence with the antagonist Arg-Phe-Phe amino acids replaced with the melanocortin agonist His-DPhe-Arg-Trp residues and has the disulfide bridge of hAGRP(109-118) replaced by a lactam bridge between Asp and Dpr resides. This template has been previously discovered by our laboratory to result in full agonist pharmacology at the melanocortin receptors examined herein, and is as potent as the endogenous melanocortin agonist R-MSH.25 Contrary to the latter studies where the template utilized a lactam bridge cyclization between the side chains of the Asp and diaminopropionic (Dpr) amino acids, for this study, the lactam bridge is formed using the CR carboxylic acid of Asp instead of the typical side chain carboxylic acid moiety (Figure 1). This difference in lactam cyclization has been previously identified as resulted in unique structural changes and pharmacology for AGRP decapeptides.31 Substitution and placement of the natural and unnatural amino acids used herein (Table 1, Figure 2) was based upon tetrapeptide studies incorporating these amino acids and resulting in unique and unexpected melanocortin receptor pharmacology.27-30

Figure 2. Summary of the amino acid abbreviations and structures used in this study.

These chimeric hAGRP-melanocortin peptides were synthesized using standard tert-butyloxycarbonyl (Boc) methodology.32,33 The peptides were purified to homogeneity using semipreparative reversed-phase highpressure liquid chromatography (RP-HPLC). The peptides possessed the correct molecular weights as determined by mass spectrometry. The purity of these peptides (>95%) were assessed by analytical RP-HPLC in two diverse solvent systems. Biological Evaluation. Table 2 summarizes the pharmacology at the mouse melanocortin receptors, mMC1R, mMC3R, mMC4R, and mMC5R, of the 21 peptides synthesized in this study. Additionally, the control melanocortin agonists and antagonist listed in Table 2 were assayed in parallel with the compounds reported herein, unless otherwise noted. The melanocortin receptor pharmacology of lead template, peptide 1, is consistent with our previous results,25 with the ligand possessing full sub nM agonist potencies at the mMC1 and mMC3-5 receptors and is equipotent with R-MSH at these receptors (within the inherent experimental error). Alanine Scanning of the His-DPhe-Arg-Trp Amino Acids. Since this is the first structure-activity study being performed using this potentially “novel” melanocortin receptor chimeric template, we performed an Ala scan of the His-DPhe-Arg-Trp sequence. Peptide 2, with the His replaced with Ala, resulted in full

Tyr-c[β-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DBip-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Pro-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Lys-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Pro-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Nal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-DNal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Bip-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Tic-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-DPhe-Dpr]-Tyr-NH2

12 13 14 15 16 17 18 19 20 21

46800 1 25 42 18 4500 3100 4500 2 1 53 98 12

10300 ( 3200 0.30 ( 0.05 5.56 ( 3.40 9.20 ( 0.97 3.87 ( 0.85 990 ( 290 680 ( 82 1000 ( 170 0.53 ( 0.13 0.20 ( 0.05 11.6 ( 3.5 21.6 ( 10.3 2.61 ( 0.14

1 30 60% @ 100 µM 24000 14000

320 ( 250 33.0 ( 21.3 17300 ( 290 30800 ( 4200 39200 ( 13200 5.56 ( 2.72 6.50 ( 1.87 450 ( 130 3400 ( 1200 14.0 ( 1.8

pA2 ) 8.2 ( 0.2 330 34 18000 32000 40000 6 7 460 3500 14

16700 ( 1300 partial agonist pA2 ) 9.4 ( 0.3 partial agonist pA2 ) 9.1 ( 0.1 0.57 ( 0.08 0.57 ( 0.07 4300 ( 1900 8600 ( 2200 7200 ( 1800 0.27 ( 0.12 0.67 ( 0.25 42.6 ( 9.7 260 ( 99 0.66 ( 0.20

115 ( 26

0.64 ( 0.17

32.5 ( 16.7 710 ( 170 0.63 ( 0.20

0.13 ( 0.04 0.36 ( 0.07 14700 ( 1000

pA2 ) 6.8 ( 0.24

1.93 ( 0.39 0.16 ( 0.03 0.053 ( 0.011 pA2 ) 10.4

EC50 (nM)

mMC4R

4 4 33000 66000 55000 2 5 330 2000 5

128000

880

5

1000 5400 5

1 3 113000

fold difference

3.13 ( 1.89 5.27 ( 2.12 3200 ( 2400 800 ( 120 700 ( 110 1.93 ( 1.10 0.95 ( 0.40 3.43 ( 0.90 42.7 ( 10.0 4.87 ( 2.29

22.3 ( 10.6

7900 ( 4000 2.33 ( 0.96

440 ( 230

3.15 ( 1.38

2.06 ( 0.67 50.1 ( 9.6 7.16 ( 0.22

0.37 ( 0.26 0.46 ( 0.18 690 ( 98

>100000

0.32 ( 0.09 0.095 ( 0.02 0.068 ( 0.021 2.31 ( 0.45

EC50 (nM)

8 14 8600 2100 1900 5 3 9 115 13

60

21000 6

1200

9

6 135 19

1 1 1800

fold difference

mMC5R

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8

a The indicated errors represent the standard error of the mean determined from at least three independent experiments. The antagonistic pA values were determined using the Schild 2 analysis and the agonist MTII (note that Ki ) -log pA2). SA denotes that some stimulatory activity was observed at 100 µM concentrations, but not enough to determine an EC50 value since the curve did not plateau. The percentage value associated with an SA value indicates the relative percentage observed stimulation at 100 µM of the ligand, as compared to the maximal response generated by R-MSH. Partial agonist denotes that some stimulatory activity was observed in addition to the antagonist pharmacology. *Peptides SHU9119 and hAGRP(109-118) values were previously reported in refs 24, 25, 49, and 69.

Tyr-c[β-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

11

Tyr-c[β-Asp-(rac)Atc-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 690 ( 150

8

Tyr-c[β-Asp-His-Pro-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-(pI)DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

6.04 ( 1.36

Tyr-c[β-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

7

9 10

27

66.5 ( 26.8 350 ( 160 22.1 ( 18

Tyr-c[β-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Ala-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-Pro-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

4 5 6

3100

300 1590 100

0.22 ( 0.14 7.20 ( 2.89 14000 ( 1700

Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-Ala-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 23800 ( 18900 13500 ( 3700 partial agonist pA2 ) 7.2 ( 0.2 partial agonist pA2 ) 7.2 ( 0.6 SA 40% @ pA2 ) 6.4 ( 0.2 100 µM 13200 ( 3400 14000 pA2 ) 8.8 ( 0.1

0.97 ( 0.49 29.0 ( 7.5 SA

1 33 64000

5120 ( 3040

Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr-NH2

hAGRP* (109-118) 1 2 3

EC50 (nM) 0.57 ( 0.08 0.089 ( 0.013 0.13 ( 0.027 partial agonist pA2 ) 9.5 >100000

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2

R-MSH NDP-MSH MTII SHU9119*

EC50 (nM)

fold difference

mMC3R

0.50 ( 0.11 0.018 ( 0.005 0.022 ( 0.006 0.64 ( 0.26

structure

peptide

fold difference

mMC1R

Table 2. Functional Activity of the Chimeric Antagonist hAGRP-Melanocortin Agonist Peptides at the Mouse Melanocortin Receptorsa

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Figure 3. Antagonist mouse melanocortin-3 receptor pharmacology of peptides 6 and 7, modified at the His position in the chimeric hAGRP-melanocortin peptide template. Both these ligands result in mMC3R antagonists that possess partial agonist activity.

Figure 4. Antagonist mouse melanocortin-3 and -4 receptor pharmacology of peptides 10 and 11, modified at the DPhe position in the chimeric hAGRP-melanocortin peptide template. Both these ligands result in mMC3R and mMC4R antagonists that possess partial agonist activity.

agonist activity at the melanocortin receptors, with potency ranging from 29 to 0.4 nM, and was equipotent or possessed up to 33-fold decreased potency, as compared to 1. Peptide 3, with the DPhe substituted with Ala resulted in >1800-fold decreased agonist potency, and was only able to generate a 60% maximal response (compared to R-MSH) at the mMC3R (100 µM concentrations). Peptide 4, with Ala substituting for the Arg residue, resulted in full nM agonist potency at the mMC1 and mMC4-5 receptors, however at the mMC3R 24000-fold decreased potency resulted, as compared to 1. Replacement of Trp by Ala (peptide 5), resulted in 130- to 14000-fold decreased potency at the melanocortin receptors, as compared with the lead peptide 1. Substitutions at the His Position. Modification at the His position with the Pro (6), Phe (7), and racemic 2-aminotetraline-2-carboxylic acid (Atc, 8) resulted in full mMC1, mMC4, and mMC5 receptor agonists, but was a mMC3R antagonist, with some stimulatory agonist activity. Figure 3 shows the mMC3R antagonist pharmacology curves for peptides 6 and 7 possessing

equipotent antagonist Ki (Ki) -Log pA2) values of ca. 63 nM. Peptide 8 possesses an antagonist Ki value ca. 400 nM which is 6-fold less potent than peptides 6 and 7, presumably due to the compound being tested as a diastereomeric mixture. Substitutions at the Phe Position. Substitutions of the Phe position by Pro (9), p-iodo-D-phenylalanine [(pI)DPhe, 10], D-2-naphthylalanine [DNal(2′), 11], D-1naphthylalanine [DNal(1′), 12], and 4-phenyl-D-phenylalanine (DBip, 13) resulted in mixed melanocortin receptor pharmacology. Peptides 10 and 11 resulted in mMC3R and mMC4R antagonists (Figure 4) with some stimulatory activity. The Pro containing peptide 9 possessed µM melanocortin agonist potency that was >14000-fold less potent than 1. Peptide 12 containing the DNal(1′) substitution at the DPhe position resulted in low nM agonist potency at the mMC1R, mMC4R, and mMC5R but possessed a mMC3R agonist EC50 value of 320 nM. The DBip-containing peptide 13 resulted in potent nM agonist activity at the melanocortin receptors examined in this study.

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Figure 5. Summary of the NOE intensities from 400 ms NOESY data observed for peptides 1, 2, 6, 7, 11, and 12. The height of the bars indicates the strength of the NOE.

Substitutions at the Arg Position. Substitution of the Arg amino acid of peptide 1 with Pro (14) or Lys (15) resulted in significantly reduced agonist melanocortin receptor potency at the mMC1 and mMC3-5 receptors. Substitutions at the Trp Position. Modification of the Trp indole side chain by the insertion of different amino acids [Pro, Nal(2′), DNal(2′), Bip, or 1,2,3,4tetrahydroisoquinoline-3-carboxylic acid (Tic)] resulted in a diverse range of melanocortin receptor agonist pharmacological profiles. The Pro (16) and Tic (20) peptides resulted in the least potent melanocortin receptor ligands ranging from 98- to 55000-fold de-

creased potency compared with the lead peptide 1. The Nal(2′) containing peptide 17 and DNal(2′)-containing peptide 18, possessed nearly identical pharmacological profiles and possessed less than 10-fold decreased potency, as compared with 1. Peptide 19, containing the Bip substitution for the Trp amino acid, resulted in a range of agonist EC50 values from 3 to 450 nM at the mouse melanocortin receptors examined in this study. Finally, peptide 21 which has a stereochemical inversion of the Phe at the 9 position to DPhe was designed to test a previous hypothesis that the differences between the hAGRP decapeptides that were MC1R agonists versus an MC1R antagonist is a reverse turn



Table 3. 1H NMR Chemical Shifts Assignment for Peptides 1, 2, 6, 7, 11, and 12

putatively in the Ala-Phe-Dpr sequence.31 Precedent regarding the use of stereochemical conversion of L-Phe to D-Phe to stabilize a putative reverse turn in the melanocortin peptides has been previously documented with great success.21-23 Herein using this peptide template and DPhe at the 9 position, full nM melanocortin receptor agonists resulted that possessed 5- to 14fold decreased potency, as compared to 1. Structural Analysis by 1H NMR and ComputerAssisted Molecular Modeling. Figure 5 summarizes the NOE intensities observed for peptides 1, 2, 6, 7, 11, and 12. Table 3 provides the NMR-derived proton assignments for peptides 1, 2, 6, 7, 11, and 12. The β-turn secondary structural motif consists of four consecutive residues defined by positions i, i + 1, i + 2, i + 3 that are not within an a-helix and the distance between CR (i) and CR (i + 3) is less than 7 Å.34 Based upon this definition, a reverse turn structure was identified in almost all of the cyclic peptides studied. Another common feature that was observed in these peptides examined by NMR was a significant upfield shift of β and γ methylene protons of the Arg5 residue side chain (especially distinct in peptides containing DNal residues peptides 11 and 12), suggesting that these protons may be shielded by aromatic rings. These data are consistent with previous reports of NMR-based structural studies of the cyclic synthetic melanocortin peptides MTII and SHU9119 in addition to linear R-MSH analogues that observed similar findings.35,36 Peptide 1 Tyr-c[β-Asp-His-DPhe-Arg-Trp-AsnAla-Phe-Dpr]-Tyr-NH2. The representative structure of the most highly populated conformation family of peptide 1 (62% of total number of conformers) has β-hairpin-like structure (Figure 6), which may be stabilized by hydrophobic interactions between side chains of the His3, DPhe4 and Phe9 aromatic residues. The His3 side chain has a perpendicular alignment with the phenyl ring of Phe9, whereas hydrophilic Arg5 and Asn7 residues are pointed to the other direction, suggesting that this molecule possesses amphiphilic character. A reverse β-turn structure involving Trp6 and Asn7 residues has been observed for this peptide. Examination of the backbone phi and psi angles at these residues showed that this turn can be defined as type VIII, which is the most common nonclassical β-turn occurring in peptides and proteins.37 The i+2 position of this type VIII β-turn is occupied by Asn7, which has been identified as a “preferred” amino acid at this position. The key ligand residues DPhe5, Arg6 and Trp7 are positioned on one of the strands forming the β-hairpin structure and their side chains are located at opposite sides of plane formed by β-hairpin. The core melanocortin-based sequence, composed of His-DPhe-Arg-Trp amino acids, examined in this peptide has similar orientations as previously observed in previous NMR-based structural studies of MTII and other linear analogues of R-MSH.38,39 Peptide 2, Tyr-c[β-Asp-Ala-DPhe-Arg-Trp-AsnAla-Phe-Dpr]-Tyr-NH2. The overall NMR structure of the peptide 2, which is a full agonist at melanocortin receptors 1, 3, 4 and 5, is similar to that of peptide 1 described above (Figure 6). Peptide 2 also possesses a type VIII β-turn with Trp6 and Asn7 residues at i+1 and i+2 positions, respectively. The His3, DPhe4 and Phe9 aromatic residues do not form a distinct hydrophobic

amino acid

HN

HR

Hβ

other

Peptide 1, Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr1 4.16 3.06 7.07 6.38 Asp2 8.67 4.55 2.59 2.72 His3 8.45 4.64 3.03 3.11 6.99 DPhe4 8.64 4.57 2.89 7.34 7.22 Arg5 8.40 4.02 1.5 1.32 1.04 0.92 Trp6 8.43 4.50 3.29 7.24 Asn7 7.76 4.30 2.1 2.53 7.46 6.78 Ala8 7.83 3.99 1.16 Phe9 8.18 4.38 2.90 7.09 Dpr10 R-7.93 β-7.97 4.42 3.52 3.14 Tyr11 8.17 4.44 2.87 3.08 7.16 7.64 Peptide 2, Tyr-c[β-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr1 4.15 3.06 7.08 Asp2 8.68 4.57 2.61 Ala3 8.32 4.29 1.25 DPhe4 8.58 4.59 3.02 7.23 7.34 Arg5 8.38 4.00 1.5 1.3 1.05 0.92 Trp6 8.40 4.54 3.33 7.58 7.27 Asn7 7.62 4.30 1.98 2.52 7.48 Ala8 7.84 3.98 1.16 Phe9 8.18 4.42 2.93 7.1 Dpr10 R-7.88 β-8.13 4.43 3.54 3.11 Tyr11 8.26 4.43 2.88 3.1 7.18 Peptide 6, Tyr-c[β-Asp-Pro-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr1 4.47 2.92 3.13 Asp2 8.73 4.43 2.78 Pro3 4.30 2.17 1.63 3.52 DPhe4 8.52 4.66 3.00 Arg5 8.41 4.07 1.57 1.39 1.17 1.04 Trp6 8.56 4.48 3.29 7.55 7.25 Asn7 7.60 4.33 2.05 2.50 7.5 Ala8 7.87 3.93 1.19 Phe9 8.22 4.38 2.93 Dpr10 R-7.79 β-7.95 4.39 3.51 3.10 Tyr11 8.78 4.48 3.09 2.84 Peptide 7, Tyr-c[β-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr1 3.99 3.05 Asp2 8.58 4.60 2.61 Phe3 8.43 4.60 3.00 2.93 DPhe4 8.40 4.36 2.59 2.67 7.32 7.15 Arg5 8.28 3.93 1.42 1.2 0.92 0.75 Trp6 8.38 4.53 3.33 7.26 Asn7 7.58 4.26 1.92 2.54 7.58 Ala8 7.82 3.98 1.14 Phe9 8.17 4.41 2.93 Dpr10 R-7.87 β-8.17 4.42 3.54 3.09 Tyr11 8.28 4.39 2.86 3.07 Peptide 11, Tyr-c[β-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr1 4.15 3.04 Asp2 8.67 4.54 2.58 2.7 His3 8.42 4.66 3.00 3.13 6.95 DNal2′ 8.73 4.68 3.07 Arg5 8.30 3.95 1.34 1.14 0.66 0.55 Trp6 8.41 4.49 3.29 7.23 Asn7 7.73 4.30 2.16 2.51 7.48 Ala8 7.81 3.98 1.16 Phe9 Dpr10 R-7.92 β-8.00 4.00 3.13 3.51 Tyr11 Peptide 12, Tyr-c[β-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr1 4.16 Asp2 8.68 4.54 2.6 2.7 His3 8.47 4.68 3.02 3.14 DNal1′ 8.73 4.73 3.36 Arg5 8.03 3.78 1.27 1.00 0.57 0.35 Trp6 8.38 4.45 3.27 7.21 Asn7 7.65 4.26 1.95 2.48 7.43 Ala8 7.75 3.95 1.13 Phe9 Dpr10 R-7.90 β-8.00 4.40 3.1 3.5 Tyr11 -

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Figure 6. Different conformers of chimeric AGRP-melanocortin peptides 1, 2, 6, 7, 11, and 12. (A) Sausage representations of backbone superposition of members of major families of peptide 1 (RMSD ) 0.99 ( 0.62 Å), peptide 2 (RMSD ) 0.63 ( 0.38 Å), peptide 6 (RMSD ) 0.77 ( 0.38 Å), peptide 7 (RMSD ) 0.39 ( 0.19 Å), peptide 11 (RMSDfamily 1 ) 0.38 ( 0.18 Å, RMSDfamily 2 ) 0.57 ( 0.21 Å), and peptide 12 (RMSD ) 0.37 ( 0.16 Å). (B) XCluster derived representative structures. Side chains of Arg are shown in blue, DPhe (DNal1′ and DNal2′) in yellow, and Trp in red. Residues involved in lactam bridge (Asp and Dpr) and both N- and C-terminal Tyr amino acids are omitted.

cluster as observed for peptide 1. The putative disruption of the β-hairpin structure observed in peptide 1, but absent in peptide 2, is postulated to be a result of the His substitution by Ala, that resulted in 30-fold decreased agonist potency at MC3R as compared to the analogue 1. Peptide 6, Tyr-c[β-Asp-Pro-DPhe-Arg-Trp-AsnAla-Phe-Dpr]-Tyr-NH2. Cluster analysis of the mo-

lecular dynamics trajectory of peptide 6 identified two major conformational families. Both representative conformational family structures are characterized by a reverse β-turn with Arg6 and Trp7 residues at i+1 and i+2 positions, respectively. Analysis of the amino acid phi and psi backbone dihedral angles revealed that this turn most likely resembles a type I β-turn (Figure 6). It was previously demonstrated that the cyclic melano-



cortin MTII analogue containing Pro6 instead of His6 (R-MSH numbering)40,41 lacks a clearly defined classical turn structure, however this analogue displays a different pharmacological profile at melanocortin receptors as compared to peptide 6 (peptide 6 is a partial agonist at MC3R whereas MTII analogue is a full agonist). Peptide 7, Tyr-c[β-Asp-Phe-DPhe-Arg-Trp-AsnAla-Phe-Dpr]-Tyr-NH2. The largest conformational family (60% of total) identified for peptide 7 possessed a well-defined structure with Phe, DPhe and Arg residues participating in a γ-turn secondary structural motif (Figure 6). Based on values of phi and psi angles of DPhe residue, this type of turn can be classified as a classic γ-turn.34 The very close proximity of Phe3 and Trp6 may suggest that the structure is additionally stabilized by aromatic interactions involving side chains of these residues. The examination of dynamics trajectory revealed that the distance between centroids of the aromatic rings of Phe3 and Trp6 was conserved and ranged between 4 and 7 Å which is in good agreement with previous reported data.42 Additionally, the orientation of the side chains indicates that there is a parallel arrangement of aromatic rings.43 Peptide 11, Tyr-c[β-Asp-His-DNal(2′)-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2. XCluster analysis of peptide 11 which possesses message sequence His-DNal(2′)Arg-Trp resulted in two distinct families of structures (approximately 40% and 50% of total population of structures derived from dynamics trajectory). Within the representative structure of family 1 we can identify the cluster of aromatic residues His3, DNal(2′),4 Trp6 and Phe9 and a hydrophilic patch involving Arg5 and Asn7 amino acids (Figure 6). Both patches are located at the opposite sides of backbone plane. This arrangement to some extent resembles that in peptide 1. Additionally a β-turn of type I spanning Trp6 and Asn7 residues was identified. In the representative structure of family 2 the reverse turn is located at Arg5 and Trp6 position (Figure 6). Values of the phi and psi backbone angles allow us to categorize this as a type II turn. Generally, the conformation of family 2 of the peptide 11 is similar to the peptide 6 which contains Pro instead of His3. Peptide 12, Tyr-c[β-Asp-His-DNal(1′)-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2. Clustering analysis of peptide 12 resulted in one main family which contains 80% of the structures (Figure 6). Two consecutive turns were identified within the representative structure of this family. The first turn belongs to the type II′ with DNal(1′)4 and Arg5 amino acids, whereas the second turn involves residues Arg5 and Trp6 and belongs to the type I.

His imidazole side chain with the Ala (2) or Phe (7) side chains resulted in ca. 30-fold decreased mMC1R potency, compared with 1, consistent with previous melanocortin agonist pharmacology at the MC1R.27,45,46 Incorporation of Pro (6) and racemic Atc (8) at the His position in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 peptide template resulted in 100- to 3100-fold decreased mMC1R potency, as compared with 1, respectively. Modification of the His residue in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-TyrNH2 template is consistent with previous MC1R data upon incorporation of the racemic Atc amino acid at this position in the cyclic c[β-Asp-His-DPhe-Arg-Trp-Lys]NH2 template,47 but resulted in a notable difference in melanocortin receptor pharmacology, based upon incorporation of these modifications into the linear tetra- and pentapeptide templates.27,46 Replacement of the DPhe residue in Tyr-c[β-Asp-HisDPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 with Ala, peptide 3, resulted in 64000-fold decreased MC1R potency, compared to 1. When Phe was replaced by Ala in R-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-ProVal-NH2) there was a 509-fold decrease in binding affinity at the mouse B16 melanoma cells (putative MC1R), and this analogue was 178-fold less potent than the native hormone in functional activity.45 Comparison of these data suggest that the DPhe residue may serve a more crucial functional role of agonism at the MC1R in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]Tyr-NH2 cyclic template versus the linear R-MSH-based template. Substitution of the DPhe benzyl side chain with (pI)DPhe (10), DNal(2′) (11), DNal(1′) (12) and DBip (13) resulted in up to a 40-fold decreased mMC1R potency in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 peptide template. Modification of the DPhe in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 peptide template (peptides 10-13) resulted in different MC1R potency as compared with the linear melanocortin-based agonist templates previously reported,28,48 but is consistent with studies using the cyclic MTII or SHU9119 templates.22,23,49,50 Replacement of the Arg side chain in the Tyr-c[β-AspHis-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template with Ala (4), Pro (14) or Lys (15) resulted in 300-. 4500-, and 3100-fold decreased mMC1R agonist potency, respectively, compared to 1. Interestingly, the methyl side chain of Ala (4) resulted in a compound that was 10-fold more potent than the basic amine moiety of Lys (15). This latter observation is in contrast to similar substitutions in the Ac-His-DPhe-Arg-Trp-NH2 template where substitution of the Arg side chain with the Lys moiety was slightly more potent than the Ala substitution.30 At the MC1R, modification at the Arg position in the chimeric AGRP-melanocortin template presented herein results in different structure-activity relationships (SARs) than those previously reported for the melanocortin-based peptides at the MC1R.30,45 Substitution of the Trp indole side chain with Nal(2′) (17), DNal(2′) (18), Bip (19), and Tic (20) in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-TyrNH2 peptide template resulted in equipotent or up to ca. 100-fold decreased mMC1R potency, compared to 1 (Table 2). When the Ala methyl side chain (5) replaced the Trp indole side chain (1), a 1500-fold decreased

Discussion Comparative Ligand-Melanocortin Receptor Structure-Activity Relationships. Melanocortin-1 Receptor. The peripheral skin melanocortin receptor, MC1R, is involved in human skin pigmentation1,8,14 and animal coat coloration.44 Substitution with (pI)DPhe (10) at the DPhe position, and Nal(2′) (17) or DNal(2′) (18) at the Trp position in the Tyr-c[β-Asp-His-DPheArg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in equipotent agonists at the mMC1R, as compared with the lead peptide 1. Substitution of the

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mMC1R potency resulted, significantly different compared to the functional activity of the homologous substitutions in the R-MSH and Ac-His-DPhe-Arg-TrpNH2 linear templates at the MC1R.30,45 SAR comparisons demonstrate that substitution at the Trp position in the chimeric template with amino acid substitutions that have been previously reported to possess equipotent MC1R agonists results in an overall different pharmacological MC1R profile.29,48 Melanocortin-3 Receptor. The MC3R is expressed both peripherally and centrally and appears to be involved in metabolism and energy homeostasis.9,10,51,52 Alanine scanning of the His, DPhe, Arg, and Trp residues in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 peptide template were not well tolerated at this receptor. Analogue 2, where the His is replaced by the Ala amino acid, resulted in 30-fold decreased mMC3R potency, compared to 1. Whereas substitution of Ala at the DPhe (3), Arg (4), and Trp (5) positions resulted in µM agonist EC50 values, or in the case of peptide 3, was only able to stimulate the mMC3R to 60% at 100 µM concentrations (Table 2). The MC3R pharmacological profile, even within the various melanocortin peptide-based templates, appears to vary significantly at the His position.27-29,53-56 Remarkably however, replacement of His by Pro (6), Phe (7), and racemic Atc (8) in the Tyr-c[β-Asp-His-DPhe-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2 template all resulted in mMC3R antagonists, with or without (8) partial agonist activity (Figures 3 and 4). These results were unexpected, as it has been well demonstrated in the literature that modification of the melanocortin agonist His position results in MC4R versus MC3R agonist ligand selectivity.27,41,56-59 Peptide 10. Tyr-c[β-Asp-His-(pI)DPhe-Arg-Trp-AsnAla-Phe-Dpr]-Tyr-NH2 resulted in an MC3R antagonist, similar to this substitution in both the NDP-MSH (SHU9005) and MTII templates and the Ac-His-DPheArg-Trp-NH2 template.28,50 Surprisingly, peptide 9, Tyrc[β-Asp-His-Pro-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2, in which the Pro was substituted for the DPhe, retained full agonist activity at the MC3R. This is surprising, as it is well documented in the melanocortin-based peptide templates that the Phe seven position is critical for biological activity and the substitution of this key residue by amino acids other than aromatic or “bulky” result in a loss of ligand activity. Modification of the Phe seven position with DBip and DNal(1′) resulted in 13-and 29-fold decreased mMC3R potency in the AcHis-DPhe-Arg-Trp-NH2 template,28 whereas in the chimeric template Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 (1), these substitutions resulted in 34- and 330-fold decreased mMC3R potency. Characteristically, the DNal(2′) substitution for DPhe in the template used herein (11) resulted in MC3R antagonism, typical for this substitution in melanocortin-based peptide templates.50,60 Generally, as observed above, modifications of the chimeric template used herein result in different MC3R pharmacological profiles than previously observed for the melanocortin peptides, with the specific exception of the DNal(2′) substitution that will be discussed further in the MC4R section below. Substitution of Arg in the Tyr-c[β-Asp-His-DPhe-ArgTrp-Asn-Ala-Phe-Dpr]-Tyr-NH2 template with Ala (4),

Pro (14), and Lys (15) resulted in 24000-, 18000-, and 32000-fold decreased mMC3R potency, respectively. Significant differences in MC3R pharmacology result for the same amino acid substitutions of the Arg residue in different melanocortin-based templates,30,41,53,54,61 therefore it is difficult to compare the chimeric template (1) results with published melanocortin-based peptides. Substitution of Trp in the Tyr-c[β-Asp-His-DPhe-ArgTrp-Asn-Ala-Phe-Dpr]-Tyr-NH2 template with Ala (5), Pro (16), Nal(2′) (17), DNal(2′) (18), Bip (19), and Tic (20) resulted in 14000-, 40000-, 6-, 7-, 460-, and 3400fold decreased mMC3R potency, respectively. Both differences and similarities exist between the chimeric template 1 versus the melanocortin peptide templates at the MC3R, depending upon the specific amino acid modification examined.29,41,53-55 Melanocortin-4 Receptor. The central MC4R has been identified as physiologically participating in food consumption3 and obesity in mice2 with several polymorphisms of the MC4R observed in obese humans.62-67 Substitution with Ala (2), Pro (6), or Phe (7) at the His position, DNal(1′) (12) or DBip (13) at the DPhe position, and Nal(2′) (17) or DNal(2′) (18) at the Trp position in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]Tyr-NH2 peptide template resulted in equipotent agonist potency at the mMC4R, as compared with the lead peptide 1 (within experimental error). These results were unexpected, as it has been well demonstrated in the literature that modification of the melanocortin agonist His position results in MC4R versus MC3R selectivity.27,41,56-59 Specifically, modification of the His position by the Pro side chain in the MTII template resulted in the identification of modifications that might lead to increased MC4R selectivity versus the MC3R.41,58,59 Peptide 8, containing racemic Atc at the His position in the Tyr-c[β-Asp-His-DPhe-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2 peptide template, resulted in 880-fold decreased mMC4R potency, compared with 1, and was a ca. 400 nM potent MC3R antagonist, versus a MC4R selective agonist as demonstrated previously in different melanocortin peptide templates.27,46,47,57 Consistent with previous reports of homologous substitutions in the cyclic MTII template,41,54 peptides 6 (Pro) and 7 (Phe) modifications at the His position in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-TyrNH2 peptide template resulted in nearly equipotent (5fold) mMC4R potency as the control compound 1. However, the mMC4R pharmacology of peptides 6 (Pro) and 7 (Phe) was significantly different from the linear tetrapeptide template27 and cyclic (c[CO-CH2CH2COHis-DPhe-Arg-Trp-Lys]-NH2)55 templates containing identical substitutions at the His position. Substitution of DPhe with Ala (3) in the Tyr-c[β-AspHis-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in 113000-fold decreased mMC4R agonist potency. Replacement of Phe by Ala in the endogenous melanocortin agonist γ2-MSH (Tyr-Val-MetGly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly) template resulted in a loss of full hMC4R agonist activity at up to 10µM concentrations.53 Replacement of the DPhe residue of MTII (Ac-Nle-[Asp-His-DPhe-Arg-Trp-Lys]-NH2) with Ala resulted in a modification that possessed only slight agonist activity at the hMC4R.54 Thus, while all the templates resulted in a significant loss of agonist



activity at the MC4R when Phe is substituted with Ala, uniquely, peptide 3 presented herein was still able to fully stimulate the mMC4R at high concentrations. Similarly, in peptide 9 the DPhe substitution with Pro in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]Tyr-NH2 peptide template resulted in 16700-fold decreased mMC4R agonist potency, but still maintain full agonist stimulation at high concentrations. Surprisingly, substitution with DNal(2′), peptide 11, at the DPhe position in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 peptide template, resulted in partial agonist activity at the mMC4R (Figure 3), in addition to the expected potent competitive antagonist pharmacology. In all the various melanocortin templates, both linear and cyclic, substitution of L/DPhe with DNal(2′) resulted in potent MC4R competitive antagonists devoid of any partial agonist activity, reviewed by Irani et al.,68 since its original discovery in the SHU9119 compound.50 Peptide 10, with the (pI)DPhe replacing DPhe in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-TyrNH2 peptide template, resulted in partial agonist activity at the mMC4R (Figure 4) in addition to the expected potent competitive antagonist pharmacology. These data are consistent with previous reports that (pI)DPhe substitution in the linear tridecapeptide NDP-MSH and cyclic MTII templates result in mixed partial agonist and antagonist MC4R pharmacology.50,69 However, the tetrapeptide Ac-His-(pI)DPhe-Arg-Trp-NH2 is a potent mMC4R agonist.28 Thus, at the MC4R, peptide 10 is consistent with previous melanocortin templates NDP-MSH and MTII that incorporate the (pI)DPhe at the seven position (R-MSH numbering). Peptides 12 [DNal(1′)] and 13 (DBip), modified at the DPhe position in the chimeric template, resulted in equipotent (4-fold) mMC4R agonist potency as compared with the control (1), similar to homologous substitutions in linear tetra- and pentapeptide melanocortin-based templates.28,48 Modification of the Arg position in the Tyr-c[β-AspHis-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template with Ala (4), Pro (14), and Lys (15) resulted in 1000- to 66000-fold decreased mMC4R potency (Table 2). Modification at the Arg position in the Tyr-c[β-AspHis-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template results in MC4R pharmacology different than previously reported homologous substitutions in other melanocortin peptide templates.30,41,53,54,61 The functional role of the Trp indole side chain in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-TyrNH2 peptide template at the mMC4R was explored by substitution with the following amino acids: Ala (5, 5400-fold decreased mMC4R potency), Pro (16, 55000fold decreased mMC4R potency), Nal(2′) (17, equipotent), DNal(2′) (18, equipotent), Bip (19, 330-fold decreased mMC4R potency), and Tic (20, 2000-fold decreased mMC4R potency). In the chimeric peptide template examined herein (Table 1), Trp modifications with Nal(2′), peptide 17, and DNal(2′), peptide 18 resulted in consistent MC4R pharmacology as previously reported.29,48,55 However, the Ala (5), Pro (16), Bip (19), and Tic (20) amino acid substitutions of the Trp amino acid in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-AsnAla-Phe-Dpr]-Tyr-NH2 peptide template resulted in MC4R pharmacology different from that previously

reported for homologous substitutions in other melanocortin peptide templates.29,41,53,54 Melanocortin-5 Receptor. The peripheral MC5R is expressed in a variety of tissues and has been implicated as physiologically participating in the role of exocrine gland function.12,70,71 Substitution with Ala (2), at the His position, (pI)DPhe (10) at the DPhe position, Nal(2′) (17) or DNal(2′) (18) at the Trp position, and Ala (4) at the Arg position in the Tyr-c[β-Asp-His-DPhe-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in equipotent agonist potency at the mMC5R, as compared with the lead peptide 1 (within experimental error). Due to the fact that substitution with identical amino acids at homologous positions in different melanocortin receptor peptide templates result in drastically different MC5R pharmacology,27,41,53-55 from equipotent to no activity for the same modification in different templates, comparisons of His modifications in the Tyr-c[β-Asp-HisDPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template with homologous substitutions in other melanocortin templates becomes confounding. Modification of the DPhe residue in the Tyr-c[β-AspHis-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template with Ala (3) resulted in a 690 nM full agonist with 1800-fold decreased mMC5R potency. Thus, Ala substituted for DPhe in the chimeric peptide 3 retained high nM MC5R agonist potency, while other melanocortin receptor templates with the identical modification resulted in compounds that were unable to stimulate the MC5R at up to 100 µM concentrations.28,53,54 Peptides 10 [(pI)DPhe], 12, [DNal(1′)], and 13 (DBip) resulted in 6- to 14-fold decreased mMC5R potency, compared to 1 (Table 2), consistent with previously reported results.28 However, the Tyr-c[β-Asp-HisDNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide 12, resulted in 60-fold decreased mMC5R potency which is inconsistent with previously published DNal(2′) substitutions at the DPhe position in the tetrapeptide template.28 The Tyr-c[β-Asp-His-Pro-Arg-Trp-Asn-AlaPhe-Dpr]-Tyr-NH2 peptide 9, resulted in µM MC5R agonist potency but was 21000-fold less active than the control peptide 1. Modification of the Arg side chain with Ala (4), Pro (14), and Lys (15) in the Tyr-c[β-Asp-His-DPhe-Arg-TrpAsn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in 6-, 8600-, and 2100-fold decreased MC5R potency. The Tyr-c[β-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-TyrNH2 peptide 4 possesses unique MC5R pharmacology as compared with homologous modifications in other melanocortin peptide templates.30,53,54 Peptide 14, containing the Pro amino acid modification for the Arg, resulted in 8600-fold decreased MC5R potency, consistent with the same substitution in the tetrapeptide template,30 but different in the MTII template.41 Peptide 15, containing the Lys residue substitution for the Arg, resulted in 2100-fold decreased MC5R potency, which is different than the homologous modifications in the tetrapeptide and MTII templates.30,61 In the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-PheDpr]-Tyr-NH2 peptide template, the Trp amino acid was substituted with Ala (5), Pro (16), Nal(2′) (17), DNal(2′) (18), Bip (19), and Tic (20) residues. The Ala-containing peptide 5 resulted in 135-fold decreased mMC5R potency. Peptide 16, Tyr-c[β-Asp-His-DPhe-Arg-Pro-Asn-

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Table 4. Summary of mMC4R versus mMC3R Selective Peptides Identified in the Current Study

peptide

structure

mMC3R EC50 (nM)

4 12 2 13 17 21 5 20 19 18

Tyr-c[β-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DBip-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Nal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-DPhe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Ala-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Tic-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-Bip-Asn-Ala-Phe-Dpr]-Tyr-NH2 Tyr-c[β-Asp-His-DPhe-Arg-DNal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2

23800 320 29.0 33.0 5.56 14.0 13500 3400 450 6.50

Ala-Phe-Dpr]-Tyr-NH2, possessed 700 nM mMC5R agonist potency, but was 1900-fold less potent than 1. Comparison of the chimeric versus the MTII templates41 with Pro substituted for Trp resulted in dramatically different MC5R pharmacology. The Trp amino acid substitution by Nal(2′) (17), DNal(2′) (18), and Bip (19) resulted in nearly equipotent MC5R pharmacology as control (1) and is consistent with identical substitutions in the tetrapeptide template,29 but the Nal(2′) substitution in the (c[CO-CH2CH2CO-His-DPhe-Arg-Nal(2′)Lys]-NH2) peptide resulted in significantly different MC5R pharmacology.55 In summary, these structure-function substitution comparisons of the chimeric template examined herein, versus other various linear and cyclic melanocortinbased peptide agonist templates, demonstrate that the chimeric Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-PheDpr]-Tyr-NH2 peptide is a novel melanocortin agonist template possessing distinct receptor pharmacological profiles. Melanocortin Receptor Selectivity. The melanocortin pathway consists of five known receptor isoforms, located in a variety of tissues, and is involved in a variety of physiological functions.72 The melanocortin receptor knock out mice have provided valuable information attributing a melanocortin receptor to a particular phenotype, but not all the physiological functions attributed to the melanocortin pathway have been linked to a specific melanocortin receptor(s). Therefore, the search for novel melanocortin receptor selective ligands with unique pharmacology is being pursued by both academic and industrial research teams.27,28,41,55-57,73-75 Current emphasis regarding melanocortin receptor selectivity focuses upon distinction between the MC3 and MC4 receptors that are both expressed in the hypothalamus of the brain and are involved in the regulation of energy homeostasis. Herein, peptide 4, Tyr-c[β-Asp-His-DPhe-Ala-Trp-Asn-Ala-PheDpr]-Tyr-NH2, resulted in a ca. 24 µM MC3R agonist but possessed 2 to 70 nM agonist potencies at the mMC1R, and mMC4-5Rs, and is 730-fold mMC4R versus mMC3R selective. Table 4 summarizes the peptides examined in this study that possess greater than 10-fold selectivity for the MC4R versus the MC3R. Modifications at each of the His-DPhe-Arg-Trp positions in the Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]Tyr-NH2 chimeric peptide template resulted in some MC4R versus MC3R selectivity. Interestingly, with exception of the Pro and Ala substitutions, modification at the Trp position resulted in MC4 versus MC3 receptor selectivity not previously identified by identical amino

mMC4R EC50 (nM)

selectivity ratio: mMC3R EC50/ mMC4R EC50

32.5 0.57 0.36 0.57 0.27 0.66 710 260 42.6 0.67

730 560 80 58 21 21 19 13 11 10

acid substitutions in other melanocortin receptor templates.29,41,48,53-55 Additionally, previous reports identifying that modification at the His position with Pro and other residues in cyclic melanocortin peptides results in MC4R versus MC3R selective agonists.27,41,56-59 However, in the chimeric peptide template, substitution of His with Pro (6), Phe (7), and racemic Atc (8) all resulted in ligands that were mMC3R antagonists but mMC4R agonists, clearly possessing different pharmacological profiles compared with other published melanocortin receptor templates. 1H NMR and Computer-Assisted Molecular Modeling-Based Structural Studies. In attempts to correlate the melanocortin receptor functional studies with peptide structure, we performed 2D 1H NMR and CAMM experiments. Six peptides were selected from the 21 peptides pharmacologically evaluated in Table 2, based upon controls (peptides 1 and 2), unique and unanticipated MC3R versus MC4R differences in function (peptides 6, 7, and 11), and comparisons with similar substitutions in other NMR-based structural studies of melanocortin ligands MTII and SHU911936,38,76 (peptides 11 and 12). For all the peptides examined by NMR in this study, a reverse turn conformation within the putative His-DPhe-Arg-Trp message sequence was found. Our results are consistent with previous studies that identified the presence of a reverse turn in this HisDPhe-Arg-Trp domain of various melanocortin agonist ligands.36,38,77-81 NMR-Based Structural Features of Chimeric Agonist Peptides. Peptides 1 and 2 possessing agonist activity at all the mouse melanocortin receptor examined in this study and are generally characterized to possess a β-turn involving the Trp6 and Asn7 residues. The 30-fold decreased potency of peptide 2 at the mMC1R and mMC3 may suggest that stacking interaction between side chains of His3 and DPhe4 observed in peptide 1 (Figure 6) may be important for the agonist potency at these receptors. NMR studies of melanocortin ligands has shown that this type of β-turn secondary structure may also exist in MTII, which contains the same message sequence His-DPhe-Arg-Trp, as peptide 1.38 Another interesting structural feature of peptide 1 is the positioning of the side chain of the Arg5 residue which is directed in an opposite orientation from the DPhe4 and Trp6 residues (Figure 6). This side chain orientation may explain how peptide 1 possesses the amphiphilic characteristic similar to structures observed in other melanocortin ligands.38,78,82 Peptide 12, containing a bulkier DNal(1′) residue instead of the DPhe amino acid, is a full agonist at mMC1R and mMC3-



with the DPhe4 in the central position, possibly explaining the difference between mMC3R antagonist and mMC4R agonist pharmacology.

Figure 7. Comparison of the DPhe (yellow), Arg (blue), and Trp (red) side chain orientations in peptide 6 (A) and 7 (B).

5Rs, albeit with 40-fold decreased potency at the mMC1 and 330-fold decreased agonist potency at the mMC3, as compared to peptide 1. Interestingly, this analogue contains two reverse turns involving His-DNal(1′)-ArgTrp-Asn residues, which may be defined as consecutive turns of a 310-helix.37 As a result of this putative 310helical secondary structure for peptide 12, the side chains of the message His-DNal(1′)-Arg-Trp sequence are oriented differently than observed in peptide 1 (Figure 6). Both the differences in the peptide backbone and side chain orientations may account for the differences observed in melanocortin receptor potency. NMR-Based Structural Features of Chimeric mMC3 and mMC4 Receptor Partial Agonist and Antagonist Peptides. The common feature of this group of peptides possessing partial agonist or antagonist melanocortin receptor pharmacology is a reverse turn including the Arg5 and Trp6 residues (Figure 6). Additionally, these peptides are generally more flexible, as compared with the full agonist peptides discussed above. Peptides 6 and 7, with Pro and Phe substitutions at the His position respectively, possess strikingly identical melanocortin receptor pharmacological profiles in which both peptides are partial agonist/antagonist at the mMC3R and full agonists at the mMC1R, mMC4R, and mMC5R (Table 2). These peptides possess different overall tertiary structures with peptide 7 possessing a more ordered conformation as compared with peptide 6. However, comparison of the peptide fragments containing the melanocortin “key” DPhe-ArgTrp residues (Figure 7) identified that both peptides 6 and 7 possess similar side chain orientations in this local domain, supporting the hypothesis that both these peptides may be interacting with the melanocortin receptors in a similar molecular recognition motif. Peptide 11 is a potent competitive antagonist at mMC3R, is a partial agonist and antagonist at mMC4R (Figure 4) and possesses full nM agonist potency at the mMC1 and mMC5 receptors (data not shown). This “mixedagonist/antagonist ligand pharmacology” of peptide 11 may be attributed to the conformational equilibrium between two identified conformational families (Figure 6). Family 1 conformer contains a reverse turn involving the Trp6-Asn7 residues, which is similar to the analogous turn existing in the full agonist peptide 1. Additionally however, a highly populated family 2 conformer possesses a reverse turn involving the Arg5-Trp6 amino acids that resembles that major structural family observed for peptide 6, which also possesses partial agonist activity at MC3R (Figure 3). Peptide 7, which is a partial agonist and antagonist at the MC3R (Figure 3) but is a full nM mMC4R agonist, possesses a γ-turn

Conclusions This study reports incorporation of selected amino acid substitutions into a chimeric antagonist AGRPmelanocortin agonist peptide template Tyr-c[β-Asp-HisDPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 to determine if the pharmacological profiles of these chimeric molecules resemble the published “classical” melanocortin receptor antagonists or antagonists. Detailed melanocortin receptor comparison of amino acid substitutions incorporated into homologous positions in the chimeric template versus other published melanocortin peptide template structure-activity studies has resulted in the conclusion that the melanocortin chimeric Tyr-c[β-AspHis-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 template results in novel receptor pharmacology profile and is a new lead template for further structure-activity studies, both from the in vitro ligand-receptor standpoint as well as providing novel tools to study the in vivo melanocortin pathways. The conformational studies presented herein suggest that single amino acid substitutions can induce a broad range of structural conformations and as a consequence may cause different pharmacological function at the melanocortin receptors, and that perhaps specifically a γ-turn secondary structure involving the DPhe4 in the central position can differentiate mMC3R antagonist and mMC4R agonist functions. Experimental Section Peptide Synthesis. The chimeric hAGRP-melanocortin peptides were synthesized using standard Boc methodology32,33 on an automated synthesizer (Advanced ChemTech 440MOS, Louisville, KY). The amino acids Boc-Asp(NR carboxyl-OFm), Boc-Tyr(2,6-dichloro-Bzl), Boc-His(3-Bom), Boc-DPhe, BocTrp(For), Boc-Asn, Boc-Ala, Boc-Phe, Boc-Pro, Boc-(2-Naphthyl)-alanine [Nal(2′)], Boc-(1-naphthyl)-D-alanine [DNal(1′)], Boc-Lys(2-chloro-Z), and Boc-(2-naphthyl)-D-alanine [DNal(2′)] were purchased from Bachem (Torrance, CA). The BocArg(Tos) and Boc-diaminopropionic (Dpr) amino acids were purchased from Peptides International (Louisville, KY). The amino acids Boc-p-iodo-D-phenylalanine [(pI)DPhe], Boc-4phenyl-D-phenylalanine (DBip), Boc-4-phenyl-phenylalanine (Bip), and Boc-1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid (Tic) were purchased from Synthetech (Albany, OR). The racemic amino acid 2-N-Boc-amino-tetrahydro-2-naphthyl carboxylic acid (Atc) was purchased from Pharmacore (High Point, NC). p-Methylbenzhydrylamine Resin (p-MBHA Resin, 0.28 mequiv/g substitution) was purchased from Peptides International. The coupling reagents: benzotriazol-1-yl-N-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-hydroxybenzotriazole (HOBt) were obtained from Peptides International. Glacial acetic acid (HOAc), dichloromethane (DCM), methanol (MeOH), acetonitrile (ACN), and anhydrous ethyl ether were purchased from Fisher (Fair Lawn, NJ). N,NDimethylformamide (DMF) was purchased from Burdick and Jackson (McGaw Park, IL). Trifluoroacetic acid (TFA), 1,3diisopropylcarbodiimide (DIC), and piperidine were purchased from Sigma (St. Louis, MO). N,N-Diisopropylethylamine (DIEA) was purchased from Aldrich (Milwaukee, WI). All reagents and chemicals were ACS grade or better and were used without further purification. The syntheses were performed using a 40-well Teflon reaction block with a coarse Teflon frit. Approximately 200 mg of resin (0.08 mmol) was added to each reaction block well. The resin was allowed to swell for 2 h in 5 mL of DMF and deprotected using 4 mL of 50% TFA and 2% anisole in DCM

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Table 5. Analytical Data for the Peptides Synthesized in This Studya

peptide

HPLC k′ (system 1)

structure

1 2 3 4 5 6

Y-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-Ala-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-Ala-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-Pro-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

7 8

Y-c[β-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-(rac)Atc-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

9 10 11 12 13 14 15 16 17 18 19 20 21

Y-c[β-Asp-His-Pro-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-(pI) DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DBip-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Pro-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Lys-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-Pro-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-Nal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-DNal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-Bip-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-Tic-Asn-Ala-Phe-Dpr]-Tyr-NH2 Y-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-DPhe-Dpr]-Tyr-NH2

4.8 5.2 4.9 5.5 3.9 peak 1: peak 2: 6.8 peak 1: peak 2: 5.0 5.0 5.0 4.9 5.3 3.2 4.0 3.9 5.3 4.9 5.5 5.6 4.7

5.5 5.8 6.4 6.6

HPLC k′ (system 2) 8.1 8.7 8.9 9.2 7.0 peak 1: peak 2: 9.5 peak 1: peak 2: 8.8 9.2 9.2 8.6 9.1 11.2 7.3 7.4 8.9 8.7 9.4 9.6 8.1

8.7 9.2 11.3 12.7

M +1 (calcd)

mass spectral analysis (M + 1)

purity, %

1486.61 1420.54 1410.51 1401.50 1371.47 1446.58

1486.06 1419.60 1408.53 1399.94 1370.59 1445.83

>96 >96 >96 >98 >96 >96

1496.64 1521.68

1495.62 1523.31

>95 >95

1436.55 1612.50 1536.66 1536.66 1562.70 1427.54 1458.59 1397.51 1497.63 1497.63 1523.67 1459.58 1486.61

1435.62 1610.89 1535.24 1535.33 1561.34 1448.91 1457.83 1397.46 1497.82 1498.43 1524.64 1461.00 1485.86

>95 >95 >95 >98 >96 >95 >96 >98 >96 >98 >96 >95 >96

a HPLC k′ ) [(peptide retention time - solvent retention time)/solvent retention time] in solvent system 1 (10% acetonitrile in 0.1% trifluoroacetic acid/water and a gradient to 90% acetonitrile over 35 min) or solvent system 2 (10% methanol in 0.1% trifluoroacetic acid/water and a gradient to 90% methanol over 35 min). An analytical Vydac C18 column (Vydac 218TP104) was used with a flow rate of 1.5 mL/min. The peptide purity was determined by HPLC at a wavelength of 214 nm. Multiple peaks indicate a racemic mixture (both peaks having the same mass spectral M + 1).

for 3 min followed by a 20 min incubation at 500 rpm and washed with DCM (4.5 mL, 2 min, 500 rpm three times). The peptide-resin salt was neutralized by the addition of 4 mL of 10% DIEA in DCM (3 min, 500 rpms, two times) followed by a DCM wash (4.5 mL, 2 min, 500 rpm four times). A positive Kaiser83 test resulted indicated free amine groups on the resin. The growing peptide chain was added to the amide-resin using the general amino acid cycle as follows: 500 µL of DMF was added to each reaction well to “wet the frit,” 3-fold excess amino acid starting from the C-terminus is added [400 µM of 0.5 M solution in 0.5 M HOBt and BOP in DMF] followed by the addition of 400 µL of 0.5 M DIC in DMF and the reaction well volume is brought up to 3 mL using DMF. The coupling reaction was mixed for 1 h at 500 rpm, followed by emptying of the reaction block by positive nitrogen gas pressure. A second coupling reaction was performed by the addition of 500 µL of DMF to each reaction vessel, followed by the addition of 400 µL of the respective amino acid (3-fold excess), 400 µL of 0.5 M HOBt and BOP, 300 µL of 1 M DIEA, the reaction well volume was brought up to 3 mL with DMF, and mixed at 500 rpm for 1 h. After the second coupling cycle, the reaction block was emptied and the resin-NR-protected peptide was washed with DCM (4.5 mL 4 times). NR-Boc deprotection was performed by the addition of 4 mL of 50% TFA, 2% anisole in DCM and mixed for 5 min at 500 rpm followed by a 20 min deprotection at 500 rpm. The reaction well was washed with 4.5 mL of DCM (4 times), neutralized with 10% DIEA (3 min, 500 rpms, 2 times) followed by a DCM wash (4.5 mL, 2 min, 500 rpm 4 times), and the next coupling cycle is performed as described above. The Fmoc and OFm protecting groups were removed from Dpr and Asp, respectively by treatment with 4.5 mL of 25% piperidine in DMF (20 min at 500 rpm) with a positive Kaiser test resulting. The lactam bridge between the Asp and Dpr amino acids was formed using 5-fold excess BOP and 6-fold excess DIEA as coupling agents and mixing at 500 rpm and monitored for cyclization completion by a negative Kaiser test. Deprotection of the remaining amino acid side chains and cleavage of the amide-peptide from the resin was performed by incubation the peptide-resin with anhydrous hydrogen fluoride (HF, 5 mL, 0°C, 1 h) and 5% m-cresol, 5% thioanisole as scavengers. After the reaction was complete and the HF was distilled off, the peptide was ether precipitated

(50 mL × 1) and washed with 50 mL of cold (4°) anhydrous ethyl ether. The peptide was filtered off using a coarse frit glass filter, dissolved in glacial acetic acid, frozen and lyophilized. The crude peptide yields ranged from 60% to 90% of the theoretical yields. A 40 mg sample of crude peptide was purified by RP-HPLC using a Shimadzu chromatography system with a photodiode array detector and a semipreparative reversed phase high performance liquid chromatography (RPHPLC) C18 bonded silica column (Vydac 218TP1010, 1.0 × 25 cm) and lyophilized. The purified peptide was >95% pure as determined by analytical RP-HPLC and had the correct molecular mass (University of Florida protein core facility) Table 5. Cell Culture and Transfection. HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum and seeded 1 day prior to transfection at 1 to 2 × 106 cell/100-mm dish. Melanocortin receptor DNA in the pCDNA3 expression vector (20 µg) were transfected using the calcium phosphate method. Stable receptor populations were generated using G418 selection (1 mg/mL) for subsequent bioassay analysis. Functional Bioassay. HEK-293 cells stably expressing the melanocortin receptors were transfected with 4µg CRE/βgalactosidase reporter gene as previously described.18,69,84 Briefly, 5000 to 15000 posttransfection cells were plated into 96-well Primera plates (Falcon) and incubated overnight. Forty-eight hours posttransfection the cells were stimulated with 100 µL of peptide (10-4 to 10-12 M) or forskolin (10-4 M) control in assay medium (DMEM containing 0.1 mg/mL BSA and 0.1 mM isobutylmethylxanthine) for 6 h. The assay media was aspirated and 50 µL of lysis buffer (250 mM Tris-HCl pH ) 8.0 and 0.1% Triton X-100) was added. The plates were stored at -80 °C overnight. The plates containing the cell lysates were thawed the following day. Aliquots of 10 µL were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysate plates, 40 µL of phosphate-buffered saline with 0.5% BSA was added to each well. Subsequently, 150 µL of substrate buffer (60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM β-mercaptoethanol, 200 mg/100 mL ONPG) was added to each well and the plates were incubated at 37°. The sample absorbance, OD405, was measured using a 96-well plate reader (Molecular



Devices). The relative protein was determined by adding 200 µL of 1:5 dilution Bio Rad G250 protein dye:water to the 10 µL cell lysate sample taken previously, and the OD595 was measured on a 96-well plate reader (Molecular Devices). Data points were normalized both to the relative protein content and nonreceptor dependent forskolin stimulation. The antagonistic properties of these compounds were evaluated by the ability of these ligands to competitively displace the MTII agonist (Bachem) in a dose-dependent manner, at up to 10 µM concentrations.69 The pA2 values were generated using the Schild analysis method.85 Data Analysis. EC50 values represent the mean of duplicate experiments performed in quadruplet or more independent experiments. EC50 value estimates, and their associated standard errors, were determined by fitting the data to a nonlinear least-squares analysis using the PRISM program (v3.0, GraphPad Inc.). The results are not corrected for peptide content. NMR Spectroscopy. Peptide NMR samples were prepared by dissolving 1.0-1.3 mg of peptide in 660 µL of 95% H2O/5% D2O, adjusting the pH to 3.6 and adding DSS as an internal standard (0.0 ppm). NMR data were collected using Bruker Avance spectrometer operating at 600 MHz at the Advanced Magnetic Resonance and Imaging and Spectroscopy (AMRIS) Facility in the McKnight Brain Institute at the University of Florida. All NMR data were collected at 13 °C. Standard proton TOCSY and NOESY 2D NMR datasets were collected, processed, and analyzed as described previously.31 NOESY data were collected at both 250 and 400 ms mixing times, and proton-proton distances were obtained from the 400 ms dataset. Computer-Assisted Molecular Modeling (CAMM). Proton-proton distances were calibrated using the wellresolved Arg or Dpr amino acid methylene protons based on the relationship r ) rref *(ηref/η)1/6, where r is the distance between atoms, η is the NOESY cross-peak volume, rref is the known distance, and ηref is the corresponding volume of the NOESY calibration cross-peak. The NOE volumes were categorized as strong (1.8-3.0 Å), medium (1.8-3.5 Å), or weak (1.8-5.0 Å) NOEs (Figure 5). All conformational modeling was performed using SYBYL v6.9 software from Tripos Inc. (St. Louis, MO) on a Silicon Graphics workstation. Restrained molecular dynamics (RMD) simulations were run in vacuo with a dielectric constant of 4.0 and a temperature of 500 K using the Tripos force field and the Ga¨steiger-Hu¨ckel method of calculation of partial atomic charges. The peptides were built in fully extended linear conformations. In the first step of modeling, only sequential constraints were used and the RMD simulations were run for 1 ns. The resulting dynamics trajectories were evenly sampled and energy minimized, and structures consistent with medium range NOESY restraints were used for the next simulation. The next step was a 1 ns RMD simulation using medium range NOE restraints, structures were identified in which the side chains of aspartic acid and diaminopropionic acid were close in space, and lactam bridges were then formed. These steps ensured that the cyclization steps did not trap the molecule in local energy minima. The resulting cyclic peptides were then energy minimized without restraints. Finally, all the NOE restraints were included, and 20 ns RMD trajectories were collected. Following the RMD simulations, structures from 100 equally spaced points along the dynamics trajectory were energy minimized, analyzed, and grouped into conformational families using the XCluster program.86 The clustering was done by comparison of backbone phi and psi dihedral angles in the loop region corresponding to residues 3-9.

Appendix

Acknowledgment. This work has been supported by NIH Grants DK57080 and DK64250. Andrzej Wilczynski is a recipient of an American Heart Association Postdoctoral Fellowship.

Abbreviations. Tyr-c[β-Asp-Xaa-Yaa-Zaa-Baa-AsnAla-Phe-Dpr]-Tyr-NH2 (cyclo Asp RCOOH-Dpr βNH): Xaa ) His, Ala, Pro, Phe, and (rac)Atc; Yaa ) DPhe, Ala, Pro, (pI)DPhe, DNal(2′), DNal(1′), and DBip; Zaa ) Arg, Ala, Pro, and Lys; Baa ) Trp, Ala, Pro, Nal(2′), DNal(2′), Bip, and Tic. References (1) Lerner, A. B.; McGuire, J. S. Effect of Alpha- and BetaMelanocyte Stimulating Hormones on the Skin Colour of Man. Nature 1961, 189, 176-179. (2) Huszar, D.; Lynch, C. A.; Fairchild-Huntress, V.; Dunmore, J. H.; Smith, F. J. et al. Targeted Disruption of the Melanocortin-4 Receptor Results in Obesity in Mice. Cell 1997, 88, 131-141. (3) Fan, W.; Boston, B. A.; Kesterson, R. A.; Hruby, V. J.; Cone, R. D. Role of Melanocortinergic Neurons in Feeding and the agouti Obesity Syndrome. Nature 1997, 385, 165-168. (4) Yaswen, L.; Diehl, N.; Brennan, M. B.; Hochgeschwender, U. Obesity in the Mouse Model of Pro-opiomelanocortin Deficiency Responds to Peripheral Melanocortin. Nat. Med. 1999, 5, 10661070. (5) Van Der Ploeg, L. H.; Martin, W. J.; Howard, A. D.; Nargund, R. P.; Austin, C. P. et al. A Role for the Melanocortin 4 Receptor in Sexual Function. Proc. Natl. Acad. Sci. U S A 2002, 99, 11381-11386. (6) Eipper, B. A.; Mains, R. E. Structure and Biosynthesis of ProACTH/Endorphin and Related Peptides. Endocr. Rev. 1980, 1, 1-26. (7) Smith, A. I.; Funder, J. W. Proopiomelanocortin Processing in the Pituitary, Central Nervous System and Peripheral Tissues. Endocr. Rev. 1988, 9, 159-179. (8) Mountjoy, K. G.; Robbins, L. S.; Mortrud, M. T.; Cone, R. D. The Cloning of a Family of Genes that Encode the Melanocortin Receptors. Science 1992, 257, 1248-1251. (9) Roselli-Rehfuss, L.; Mountjoy, K. G.; Robbins, L. S.; Mortrud, M. T.; Low, M. J. et al. Identification of a Receptor for γ Melanotropin and Other Proopiomelanocortin Peptides in the Hypothalamus and Limbic System. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8856-8860. (10) Gantz, I.; Konda, Y.; Tashiro, T.; Shimoto, Y.; Miwa, H. et al. Molecular Cloning of a Novel Melanocortin Receptor. J. Biol. Chem. 1993, 268, 8246-8250. (11) Gantz, I.; Miwa, H.; Konda, Y.; Shimoto, Y.; Tashiro, T. et al. Molecular Cloning, Expression, and Gene Localization of a Fourth Melanocortin Receptor. J. Biol. Chem. 1993, 268, 1517415179. (12) Gantz, I.; Shimoto, Y.; Konda, Y.; Miwa, H.; Dickinson, C. J. et al. Molecular Cloning, Expression, and Characterization of a Fifth Melanocortin Receptor. Biochem. Biophys. Res. Commun. 1994, 200, 1214-1220. (13) Chhajlani, V.; Muceniece, R.; Wikberg, J. E. S. Molecular Cloning of a Novel Human Melanocortin Receptor. Biochem. Biophys. Res. Commun. 1993, 195, 866-873. (14) Chhajlani, V.; Wikberg, J. E. S. Molecular Cloning and Expression of the Human Melanocyte Stimulating Hormone Receptor cDNA. FEBS Lett. 1992, 309, 417-420. (15) Lu, D.; Willard, D.; Patel, I. R.; Kadwell, S.; Overton, L. et al. Agouti Protein is an Antagonist of the Melanocyte-StimulatingHormone Receptor. Nature 1994, 371, 799-802. (16) Ollmann, M. M.; Wilson, B. D.; Yang, Y.-K.; Kerns, J. A.; Chen, Y. et al. Antagonism of Central Melanocortin Receptors in Vitro and in Vivo by Agouti-Related Protein. Science 1997, 278, 135138. (17) Hruby, V. J.; Wilkes, B. C.; Cody, W. L.; Sawyer, T. K.; Hadley, M. E. Melanotropins: Structural, Conformational and Biological Considerations in the Development of Superpotent and Superprolonged Analogs. Pept. Protein Rev. 1984, 3, 1-64. (18) Haskell-Luevano, C.; Holder, J. R.; Monck, E. K.; Bauzo, R. M. Characterization of Melanocortin NDP-MSH Agonist Peptide Fragments at the Mouse Central and Peripheral Melanocortin Receptors. J. Med. Chem. 2001, 44, 2247-2252. (19) Kiefer, L. L.; Veal, J. M.; Mountjoy, K. G.; Wilkison, W. O. Melanocortin Receptor Binding Determinants in the Agouti Protein. Biochemistry 1998, 37, 991-997. (20) Tota, M. R.; Smith, T. S.; Mao, C.; MacNeil, T.; Mosley, R. T. et al. Molecular Interaction of Agouti Protein and Agouti-Related Protein with Human Melanocortin Receptors. Biochemistry 1999, 38, 897-904. (21) Sawyer, T. K.; Sanfillippo, P. J.; Hruby, V. J.; Engel, M. H.; Heward, C. B. et al. 4-Norleucine, 7-D-Phenylalanine-RMelanocyte-Stimulating Hormone: A Highly Potent R-Melanotropin with Ultra Long Biological Activity. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 5754-5758.

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