Structure−Activity Relationships of the Unique and Potent Agouti-Related Protein...

Structure-Activity Relationships of the Unique and Potent Agouti-RelatedProtein (AGRP)-Melanocortin ChimericTyr-c[â-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-proteincoupled receptors, and auxiliary proteins that are involved in the regulation of complexphysiological functions such as energy and weight homeostasis, feeding behavior, inflammation,sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide templateTyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 that was characterized using aminoacids previously reported in other melanocortin agonist templates. Twenty peptides wereexamined in this study, and six peptides were selected for 1H NMR and computer-assistedmolecular modeling structural analysis. The most notable results include the identificationthat modification of the chimeric template at the His position with Pro and Phe resulted inligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mousemelanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[â-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 and Tyr-c[â-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 re-sulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that alsopossessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified areverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed thatmay account for the differences in melanocortin receptor pharmacology. Specifically, a γ-turnsecondary structure involving the DPhe4 in the central position of the Tyr-c[â-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide may differentiate the mixed mMC3R antagonistand mMC4R agonist pharmacology.

IntroductionThe melanocortin pathway has been implicated in

mediating a variety of physiological responses includingpigmentation,1 obesity, energy homeostasis,2-4 andsexual function.5 The melanocortin system consists ofendogenous agonists derived from posttranslationalmodification of the proopiomelanocortin (POMC) genetranscript,6,7 five G-protein coupled receptors (MC1-5R),8-14 and the endogenous antagonists agouti15 andagouti-related protein (AGRP).16 The endogenous ago-nists R-, â-, γ-melanocyte-stimulating hormones (MSH)and adrenocorticotropin (ACTH) all contain a core “His-Phe-Arg-Trp” tetrapeptide sequence that has beenhypothesized to be important for melanocortin recep-tor molecular recognition and stimulation.17,18 Interest-ingly, the endogenous melanocortin receptor antagonistsagouti and AGRP have a core “Arg-Phe-Phe” tripeptidesequence that has been demonstrated to be importantfor agouti and AGRP to bind and antagonize themelanocortin receptors.19,20 It has been postulated thatthe antagonist hAGRP Arg-Phe-Phe amino acids maybe mimicking the agonist Phe-Arg-Trp residues. Chi-meric peptide ligands that utilize the melanocortinagonist NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-

Arg-Trp-Gly-Lys-Pro-Val-NH2)21 and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2)22,23 or the antago-nist hAGRP(109-118) Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr20,24 peptide templates have been examinedwhere the agonist Phe-Arg-Trp or antagonist Arg-Phe-Phe were substituted for the corresponding residues inthe normal peptide template.25,26 These latter studiesresulted in the identification of the novel melanocortinreceptor peptide template Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 that is equipotent toR-MSH at the mouse MC1, MC3, and MC5 receptors,but is more potent than R-MSH at the mMC4R.25 Uponthe basis of the discovery of this novel melanocortinreceptor template25 and previous structure-activitystudies on the agonist Ac-His-DPhe-Arg-Trp-NH2 tetra-peptide template,27-30 where incorporation of unusualamino acids at various positions of the tetrapeptidetemplate resulted in unique pharmacology, we designedthe study reported herein to determine if this is a“novel” peptide template or if instead it resembles the“classical” melanocortin peptide agonist ligand phar-macology and structure-activity relationships (SAR).This study utilizes the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 melanocortin agonist tem-plate that incorporates substitution of the His, DPhe,Arg, and Trp amino acids with natural and unnaturalamino acids that have been “classically” demonstratedto result in significant and consistent changes in mel-anocortin receptor pharmacology.

* Reprint requests should be addressed to Carrie Haskell-Luevano,University of Florida, Department of Medicinal Chemistry, P.O. Box100485, Gainesville, FL 32610-0485. Phone (352) 846-2722, Fax (352)392-8182, e-mail: [email protected].

† Department of Medicinal Chemistry.‡ Department of Biochemistry.

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

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

Results

Chemical Design, Synthesis, and Characteriza-tion. The chimeric peptide template examined in thisstudy is derived from the melanocortin receptor antago-nist hAGRP(109-118) sequence with the antagonistArg-Phe-Phe amino acids replaced with the melano-cortin agonist His-DPhe-Arg-Trp residues and has thedisulfide bridge of hAGRP(109-118) replaced by alactam bridge between Asp and Dpr resides. Thistemplate has been previously discovered by our labora-tory to result in full agonist pharmacology at themelanocortin receptors examined herein, and is aspotent as the endogenous melanocortin agonist R-MSH.25

Contrary to the latter studies where the templateutilized a lactam bridge cyclization between the sidechains of the Asp and diaminopropionic (Dpr) aminoacids, for this study, the lactam bridge is formed usingthe CR carboxylic acid of Asp instead of the typical sidechain carboxylic acid moiety (Figure 1). This differencein lactam cyclization has been previously identified asresulted in unique structural changes and pharmacologyfor AGRP decapeptides.31 Substitution and placementof the natural and unnatural amino acids used herein(Table 1, Figure 2) was based upon tetrapeptide studiesincorporating these amino acids and resulting in uniqueand unexpected melanocortin receptor pharmacology.27-30

These chimeric hAGRP-melanocortin peptides weresynthesized using standard tert-butyloxycarbonyl (Boc)methodology.32,33 The peptides were purified to homo-geneity using semipreparative reversed-phase high-pressure liquid chromatography (RP-HPLC). The pep-tides possessed the correct molecular weights asdetermined by mass spectrometry. The purity of thesepeptides (>95%) were assessed by analytical RP-HPLCin two diverse solvent systems.

Biological Evaluation. Table 2 summarizes thepharmacology at the mouse melanocortin receptors,mMC1R, mMC3R, mMC4R, and mMC5R, of the 21peptides synthesized in this study. Additionally, thecontrol melanocortin agonists and antagonist listed inTable 2 were assayed in parallel with the compoundsreported herein, unless otherwise noted. The melano-cortin receptor pharmacology of lead template, peptide1, is consistent with our previous results,25 with theligand possessing full sub nM agonist potencies at themMC1 and mMC3-5 receptors and is equipotent withR-MSH at these receptors (within the inherent experi-mental error).

Alanine Scanning of the His-DPhe-Arg-TrpAmino Acids. Since this is the first structure-activitystudy being performed using this potentially “novel”melanocortin receptor chimeric template, we performedan Ala scan of the His-DPhe-Arg-Trp sequence. Peptide2, with the His replaced with Ala, resulted in full

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

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

Ala Ala Ala AlaPro Pro Pro ProPhe (pI)DPhe Lys Nal(2′)Atc DNal(2′) DNal(2′)(rac) DNal(1′) Bip

DBip Tica 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 formedusing 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 hAGRP-melanocortin peptide template utilized in this study. The boldbonds and circle focuses upon the Asp amino acid where thelactam bridge involves the Asp backbone domain and the sidechain is incorporated into the peptide backbone. This config-uration is different from the typical incorporation of the Aspresidue that generally results in the residue side chainparticipating in the formation of the lactam bridge cycle,versus the peptide backbone, in melanocortin ligands.

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

AGRP-Melanocortin Chimeric Peptide Template Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3061

Tab

le2.

Fu

nct

ion

alA

ctiv

ity

ofth

eC

him

eric

An

tago

nis

th

AG

RP

-M

elan

ocor

tin

Ago

nis

tP

epti

des

atth

eM

ouse

Mel

anoc

orti

nR

ecep

tors

a

mM

C1R

mM

C3R

mM

C4R

mM

C5R

pept

ide

stru

ctu

reE

C50

(nM

)fo

lddi

ffer

ence

EC

50(n

M)

fold

diff

eren

ceE

C50

(nM

)fo

lddi

ffer

ence

EC

50(n

M)

fold

diff

eren

ce

R-M

SH

Ac-

Ser

-Tyr

-Ser

-Met

-Glu

-His

-Ph

e-A

rg-T

rp-G

ly-L

ys-P

ro-V

al-N

H2

0.50

(0.

110.

57(

0.08

1.93

(0.

390.

32(

0.09

ND

P-M

SH

Ac-

Ser

-Tyr

-Ser

-Nle

-Glu

-His

-DP

he-

Arg

-Trp

-Gly

-Lys

-Pro

-Val

-NH

20.

018

(0.

005

0.08

9(

0.01

30.

16(

0.03

0.09

5(

0.02

MT

IIA

c-N

le-c

[Asp

-His

-DP

he-

Arg

-Trp

-Lys

]-N

H2

0.02

2(

0.00

60.

13(

0.02

70.

053

(0.

011

0.06

8(

0.02

1S

HU

9119

*A

c-N

le-c

[Asp

-His

-DN

al(2

′)-A

rg-T

rp-L

ys]-

NH

20.

64(

0.26

part

iala

gon

ist

pA2

)9.

5pA

2)

10.4

2.31

(0.

45

hA

GR

P*

(109

-11

8)T

yr-c

[Cys

-Arg

-Ph

e-P

he-

Asn

-Ala

-Ph

e-C

ys]-

Tyr

-NH

251

20(

3040

>10

0000

pA2

)6.

8(

0.24

>10

0000

1T

yr-c

[â-A

sp-H

is-D

Ph

e-A

rg-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

0.22

(0.

141

0.97

(0.

491

0.13

(0.

041

0.37

(0.

261

2T

yr-c

[â-A

sp-A

la-D

Ph

e-A

rg-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

7.20

(2.

8933

29.0

(7.

530

0.36

(0.

073

0.46

(0.

181

3T

yr-c

[â-A

sp-H

is-A

la-A

rg-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

1400

0(

1700

6400

0S

A60

%@

100

µM14

700

(10

0011

3000

690

(98

1800

4T

yr-c

[â-A

sp-H

is-D

Ph

e-A

la-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

66.5

(26

.830

023

800

(18

900

2400

032

.5(

16.7

1000

2.06

(0.

676

5T

yr-c

[â-A

sp-H

is-D

Ph

e-A

rg-A

la-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

350

(16

015

9013

500

(37

0014

000

710

(17

054

0050

.1(

9.6

135

6T

yr-c

[â-A

sp-P

ro-D

Ph

e-A

rg-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

22.1

(18

100

part

iala

gon

ist

pA2

)7.

2(

0.2

0.63

(0.

205

7.16

(0.

2219

7T

yr-c

[â-A

sp-P

he-

DP

he-

Arg

-Trp

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

26.

04(

1.36

27pa

rtia

lago

nis

tpA

2)

7.2

(0.

60.

64(

0.17

53.

15(

1.38

9

8T

yr-c

[â-A

sp-(

rac)

Atc

-DP

he-

Arg

-Trp

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

269

0(

150

3100

SA

pA2

)6.

4(

0.2

40%

@10

0µM

115

(26

880

440

(23

012

00

9T

yr-c

[â-A

sp-H

is-P

ro-A

rg-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

1030

0(

3200

4680

013

200

(34

0014

000

1670

0(

1300

1280

0079

00(

4000

2100

010

Tyr

-c[â

-Asp

-His

-(p

I)D

Ph

e-A

rg-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

0.30

(0.

051

pA2

)8.

8(

0.1

part

iala

gon

ist

pA2

)9.

4(

0.3

2.33

(0.

966

11T

yr-c

[â-A

sp-H

is-D

Nal

(2′)-

Arg

-Trp

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

25.

56(

3.40

25pA

2)

8.2

(0.

2pa

rtia

lago

nis

tpA

2)

9.1

(0.

122

.3(

10.6

60

12T

yr-c

[â-A

sp-H

is-D

Nal

(1′)-

Arg

-Trp

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

29.

20(

0.97

4232

0(

250

330

0.57

(0.

084

3.13

(1.

898

13T

yr-c

[â-A

sp-H

is-D

Bip

-Arg

-Trp

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

23.

87(

0.85

1833

.0(

21.3

340.

57(

0.07

45.

27(

2.12

1414

Tyr

-c[â

-Asp

-His

-DP

he-

Pro

-Trp

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

299

0(

290

4500

1730

0(

290

1800

043

00(

1900

3300

032

00(

2400

8600

15T

yr-c

[â-A

sp-H

is-D

Ph

e-L

ys-T

rp-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

680

(82

3100

3080

0(

4200

3200

086

00(

2200

6600

080

0(

120

2100

16T

yr-c

[â-A

sp-H

is-D

Ph

e-A

rg-P

ro-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

1000

(17

045

0039

200

(13

200

4000

072

00(

1800

5500

070

0(

110

1900

17T

yr-c

[â-A

sp-H

is-D

Ph

e-A

rg-N

al(2

′)-A

sn-A

la-P

he-

Dpr

]-T

yr-N

H2

0.53

(0.

132

5.56

(2.

726

0.27

(0.

122

1.93

(1.

105

18T

yr-c

[â-A

sp-H

is-D

Ph

e-A

rg-D

Nal

(2′)-

Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

20.

20(

0.05

16.

50(

1.87

70.

67(

0.25

50.

95(

0.40

319

Tyr

-c[â

-Asp

-His

-DP

he-

Arg

-Bip

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

211

.6(

3.5

5345

0(

130

460

42.6

(9.

733

03.

43(

0.90

920

Tyr

-c[â

-Asp

-His

-DP

he-

Arg

-Tic

-Asn

-Ala

-Ph

e-D

pr]-

Tyr

-NH

221

.6(

10.3

9834

00(

1200

3500

260

(99

2000

42.7

(10

.011

521

Tyr

-c[â

-Asp

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-DP

he-

Arg

-Trp

-Asn

-Ala

-DP

he-

Dpr

]-T

yr-N

H2

2.61

(0.

1412

14.0

(1.

814

0.66

(0.

205

4.87

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3062 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 Wilczynski et al.

agonist activity at the melanocortin receptors, withpotency ranging from 29 to 0.4 nM, and was equipotentor possessed up to 33-fold decreased potency, as com-pared to 1. Peptide 3, with the DPhe substituted withAla 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 concen-trations). Peptide 4, with Ala substituting for the Argresidue, resulted in full nM agonist potency at themMC1 and mMC4-5 receptors, however at the mMC3R24000-fold decreased potency resulted, as compared to1. Replacement of Trp by Ala (peptide 5), resulted in130- to 14000-fold decreased potency at the melano-cortin receptors, as compared with the lead peptide 1.

Substitutions at the His Position. Modification atthe His position with the Pro (6), Phe (7), and racemic2-aminotetraline-2-carboxylic acid (Atc, 8) resulted infull mMC1, mMC4, and mMC5 receptor agonists, butwas a mMC3R antagonist, with some stimulatory ago-nist activity. Figure 3 shows the mMC3R antagonistpharmacology 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 and7, presumably due to the compound being tested as adiastereomeric mixture.

Substitutions at the Phe Position. Substitutionsof the Phe position by Pro (9), p-iodo-D-phenylalanine[(pI)DPhe, 10], D-2-naphthylalanine [DNal(2′), 11], D-1-naphthylalanine [DNal(1′), 12], and 4-phenyl-D-phenyl-alanine (DBip, 13) resulted in mixed melanocortinreceptor pharmacology. Peptides 10 and 11 resulted inmMC3R and mMC4R antagonists (Figure 4) with somestimulatory activity. The Pro containing peptide 9possessed µM melanocortin agonist potency that was>14000-fold less potent than 1. Peptide 12 containingthe DNal(1′) substitution at the DPhe position resultedin low nM agonist potency at the mMC1R, mMC4R, andmMC5R but possessed a mMC3R agonist EC50 value of320 nM. The DBip-containing peptide 13 resulted inpotent nM agonist activity at the melanocortin receptorsexamined in this study.

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

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


Substitutions at the Arg Position. Substitution ofthe Arg amino acid of peptide 1 with Pro (14) or Lys(15) resulted in significantly reduced agonist melano-cortin receptor potency at the mMC1 and mMC3-5receptors.

Substitutions at the Trp Position. Modification ofthe Trp indole side chain by the insertion of differentamino acids [Pro, Nal(2′), DNal(2′), Bip, or 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic)] resultedin a diverse range of melanocortin receptor agonistpharmacological profiles. The Pro (16) and Tic (20)peptides resulted in the least potent melanocortinreceptor ligands ranging from 98- to 55000-fold de-

creased potency compared with the lead peptide 1. TheNal(2′) containing peptide 17 and DNal(2′)-containingpeptide 18, possessed nearly identical pharmacologicalprofiles and possessed less than 10-fold decreasedpotency, as compared with 1. Peptide 19, containing theBip substitution for the Trp amino acid, resulted in arange of agonist EC50 values from 3 to 450 nM at themouse melanocortin receptors examined in this study.

Finally, peptide 21 which has a stereochemical inver-sion of the Phe at the 9 position to DPhe was designedto test a previous hypothesis that the differencesbetween the hAGRP decapeptides that were MC1Ragonists versus an MC1R antagonist is a reverse turn

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


putatively in the Ala-Phe-Dpr sequence.31 Precedentregarding the use of stereochemical conversion of L-Pheto D-Phe to stabilize a putative reverse turn in themelanocortin peptides has been previously documentedwith great success.21-23 Herein using this peptidetemplate and DPhe at the 9 position, full nM melano-cortin receptor agonists resulted that possessed 5- to 14-fold decreased potency, as compared to 1.

Structural Analysis by 1H NMR and Computer-Assisted Molecular Modeling. Figure 5 summarizesthe NOE intensities observed for peptides 1, 2, 6, 7, 11,and 12. Table 3 provides the NMR-derived protonassignments for peptides 1, 2, 6, 7, 11, and 12. Theâ-turn secondary structural motif consists of four con-secutive residues defined by positions i, i + 1, i + 2, i +3 that are not within an a-helix and the distancebetween CR (i) and CR (i + 3) is less than 7 Å.34 Basedupon this definition, a reverse turn structure wasidentified in almost all of the cyclic peptides studied.Another common feature that was observed in thesepeptides examined by NMR was a significant upfieldshift of â and γ methylene protons of the Arg5 residueside chain (especially distinct in peptides containingDNal residues peptides 11 and 12), suggesting thatthese protons may be shielded by aromatic rings. Thesedata are consistent with previous reports of NMR-basedstructural studies of the cyclic synthetic melanocortinpeptides MTII and SHU9119 in addition to linearR-MSH analogues that observed similar findings.35,36

Peptide 1 Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2. The representative structureof the most highly populated conformation family ofpeptide 1 (62% of total number of conformers) hasâ-hairpin-like structure (Figure 6), which may be sta-bilized by hydrophobic interactions between side chainsof the His3, DPhe4 and Phe9 aromatic residues. The His3

side chain has a perpendicular alignment with thephenyl ring of Phe9, whereas hydrophilic Arg5 and Asn7

residues are pointed to the other direction, suggestingthat this molecule possesses amphiphilic character. Areverse â-turn structure involving Trp6 and Asn7 resi-dues has been observed for this peptide. Examinationof the backbone phi and psi angles at these residuesshowed that this turn can be defined as type VIII, whichis the most common nonclassical â-turn occurring inpeptides and proteins.37 The i+2 position of this typeVIII â-turn is occupied by Asn7, which has been identi-fied as a “preferred” amino acid at this position. Thekey ligand residues DPhe5, Arg6 and Trp7 are positionedon one of the strands forming the â-hairpin structureand their side chains are located at opposite sides ofplane formed by â-hairpin. The core melanocortin-basedsequence, composed of His-DPhe-Arg-Trp amino acids,examined in this peptide has similar orientations aspreviously observed in previous NMR-based structuralstudies of MTII and other linear analogues of R-MSH.38,39

Peptide 2, Tyr-c[â-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2. The overall NMR structure ofthe peptide 2, which is a full agonist at melanocortinreceptors 1, 3, 4 and 5, is similar to that of peptide 1described above (Figure 6). Peptide 2 also possesses atype VIII â-turn with Trp6 and Asn7 residues at i+1 andi+2 positions, respectively. The His3, DPhe4 and Phe9

aromatic residues do not form a distinct hydrophobic

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

amino acid HN HR Hâ other

Peptide 1,Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2Tyr1 - 4.16 3.06 7.07 6.38Asp2 8.67 4.55 2.59 2.72His3 8.45 4.64 3.03 3.11 6.99DPhe4 8.64 4.57 2.89 7.34 7.22Arg5 8.40 4.02 1.5 1.32 1.04 0.92Trp6 8.43 4.50 3.29 7.24Asn7 7.76 4.30 2.1 2.53 7.46 6.78Ala8 7.83 3.99 1.16Phe9 8.18 4.38 2.90 7.09Dpr10 R-7.93 â-7.97 4.42 3.52 3.14Tyr11 8.17 4.44 2.87 3.08 7.16 7.64

Peptide 2,Tyr-c[â-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2Tyr1 - 4.15 3.06 7.08Asp2 8.68 4.57 2.61Ala3 8.32 4.29 1.25DPhe4 8.58 4.59 3.02 7.23 7.34Arg5 8.38 4.00 1.5 1.3 1.05 0.92Trp6 8.40 4.54 3.33 7.58 7.27Asn7 7.62 4.30 1.98 2.52 7.48Ala8 7.84 3.98 1.16Phe9 8.18 4.42 2.93 7.1Dpr10 R-7.88 â-8.13 4.43 3.54 3.11Tyr11 8.26 4.43 2.88 3.1 7.18

Peptide 6,Tyr-c[â-Asp-Pro-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2Tyr1 - 4.47 2.92 3.13Asp2 8.73 4.43 2.78Pro3 - 4.30 2.17 1.63 3.52DPhe4 8.52 4.66 3.00Arg5 8.41 4.07 1.57 1.39 1.17 1.04Trp6 8.56 4.48 3.29 7.55 7.25Asn7 7.60 4.33 2.05 2.50 7.5Ala8 7.87 3.93 1.19Phe9 8.22 4.38 2.93Dpr10 R-7.79 â-7.95 4.39 3.51 3.10Tyr11 8.78 4.48 3.09 2.84

Peptide 7,Tyr-c[â-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2Tyr1 - 3.99 3.05Asp2 8.58 4.60 2.61Phe3 8.43 4.60 3.00 2.93DPhe4 8.40 4.36 2.59 2.67 7.32 7.15Arg5 8.28 3.93 1.42 1.2 0.92 0.75Trp6 8.38 4.53 3.33 7.26Asn7 7.58 4.26 1.92 2.54 7.58Ala8 7.82 3.98 1.14Phe9 8.17 4.41 2.93Dpr10 R-7.87 â-8.17 4.42 3.54 3.09Tyr11 8.28 4.39 2.86 3.07

Peptide 11,Tyr-c[â-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

Tyr1 - 4.15 3.04Asp2 8.67 4.54 2.58 2.7His3 8.42 4.66 3.00 3.13 6.95DNal2′ 8.73 4.68 3.07Arg5 8.30 3.95 1.34 1.14 0.66 0.55Trp6 8.41 4.49 3.29 7.23Asn7 7.73 4.30 2.16 2.51 7.48Ala8 7.81 3.98 1.16Phe9 - -Dpr10 R-7.92 â-8.00 4.00 3.13 3.51Tyr11 - -

Peptide 12,Tyr-c[â-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2

Tyr1 - 4.16Asp2 8.68 4.54 2.6 2.7His3 8.47 4.68 3.02 3.14DNal1′ 8.73 4.73 3.36Arg5 8.03 3.78 1.27 1.00 0.57 0.35Trp6 8.38 4.45 3.27 7.21Asn7 7.65 4.26 1.95 2.48 7.43Ala8 7.75 3.95 1.13Phe9 - -Dpr10 R-7.90 â-8.00 4.40 3.1 3.5Tyr11 - -


cluster as observed for peptide 1. The putative disrup-tion of the â-hairpin structure observed in peptide 1,but absent in peptide 2, is postulated to be a result ofthe His substitution by Ala, that resulted in 30-folddecreased agonist potency at MC3R as compared to theanalogue 1.

Peptide 6, Tyr-c[â-Asp-Pro-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2. Cluster analysis of the mo-

lecular dynamics trajectory of peptide 6 identified twomajor conformational families. Both representative con-formational family structures are characterized by areverse â-turn with Arg6 and Trp7 residues at i+1 andi+2 positions, respectively. Analysis of the amino acidphi and psi backbone dihedral angles revealed that thisturn most likely resembles a type I â-turn (Figure 6).It was previously demonstrated that the cyclic melano-

Figure 6. Different conformers of chimeric AGRP-melanocortin peptides 1, 2, 6, 7, 11, and 12. (A) Sausage representations ofbackbone 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 areshown in blue, DPhe (DNal1′ and DNal2′) in yellow, and Trp in red. Residues involved in lactam bridge (Asp and Dpr) and bothN- and C-terminal Tyr amino acids are omitted.


cortin MTII analogue containing Pro6 instead of His6

(R-MSH numbering)40,41 lacks a clearly defined classicalturn structure, however this analogue displays a dif-ferent pharmacological profile at melanocortin receptorsas compared to peptide 6 (peptide 6 is a partial agonistat MC3R whereas MTII analogue is a full agonist).

Peptide 7, Tyr-c[â-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2. The largest conformationalfamily (60% of total) identified for peptide 7 possesseda well-defined structure with Phe, DPhe and Argresidues participating in a γ-turn secondary structuralmotif (Figure 6). Based on values of phi and psi anglesof DPhe residue, this type of turn can be classified as aclassic γ-turn.34 The very close proximity of Phe3 andTrp6 may suggest that the structure is additionallystabilized by aromatic interactions involving side chainsof these residues. The examination of dynamics trajec-tory revealed that the distance between centroids of thearomatic rings of Phe3 and Trp6 was conserved andranged between 4 and 7 Å which is in good agreementwith previous reported data.42 Additionally, the orienta-tion of the side chains indicates that there is a parallelarrangement of aromatic rings.43

Peptide 11, Tyr-c[â-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2. XCluster analysis of pep-tide 11 which possesses message sequence His-DNal(2′)-Arg-Trp resulted in two distinct families of structures(approximately 40% and 50% of total population ofstructures derived from dynamics trajectory). Within therepresentative structure of family 1 we can identify thecluster of aromatic residues His3, DNal(2′),4 Trp6 andPhe9 and a hydrophilic patch involving Arg5 and Asn7

amino acids (Figure 6). Both patches are located at theopposite sides of backbone plane. This arrangement tosome extent resembles that in peptide 1. Additionallya â-turn of type I spanning Trp6 and Asn7 residues wasidentified.

In the representative structure of family 2 the reverseturn is located at Arg5 and Trp6 position (Figure 6).Values of the phi and psi backbone angles allow us tocategorize this as a type II turn. Generally, the confor-mation of family 2 of the peptide 11 is similar to thepeptide 6 which contains Pro instead of His3.

Peptide 12, Tyr-c[â-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2. Clustering analysis ofpeptide 12 resulted in one main family which contains80% of the structures (Figure 6). Two consecutive turnswere identified within the representative structure ofthis family. The first turn belongs to the type II′ withDNal(1′)4 and Arg5 amino acids, whereas the secondturn involves residues Arg5 and Trp6 and belongs to thetype I.

Discussion

Comparative Ligand-Melanocortin ReceptorStructure-Activity Relationships. Melanocortin-1Receptor. The peripheral skin melanocortin receptor,MC1R, is involved in human skin pigmentation1,8,14 andanimal 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[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide templateresulted in equipotent agonists at the mMC1R, ascompared with the lead peptide 1. Substitution of the

His imidazole side chain with the Ala (2) or Phe (7) sidechains resulted in ca. 30-fold decreased mMC1R po-tency, compared with 1, consistent with previous mel-anocortin agonist pharmacology at the MC1R.27,45,46

Incorporation of Pro (6) and racemic Atc (8) at the Hisposition in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in 100- to3100-fold decreased mMC1R potency, as compared with1, respectively. Modification of the His residue in theTyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 template is consistent with previous MC1R dataupon incorporation of the racemic Atc amino acid at thisposition in the cyclic c[â-Asp-His-DPhe-Arg-Trp-Lys]-NH2 template,47 but resulted in a notable difference inmelanocortin receptor pharmacology, based upon incor-poration of these modifications into the linear tetra- andpentapeptide templates.27,46

Replacement of the DPhe residue in Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 with Ala, pep-tide 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-Pro-Val-NH2) there was a 509-fold decrease in bindingaffinity at the mouse B16 melanoma cells (putativeMC1R), and this analogue was 178-fold less potent thanthe native hormone in functional activity.45 Comparisonof these data suggest that the DPhe residue may servea more crucial functional role of agonism at the MC1Rin the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 cyclic template versus the linear R-MSH-basedtemplate. Substitution of the DPhe benzyl side chainwith (pI)DPhe (10), DNal(2′) (11), DNal(1′) (12) andDBip (13) resulted in up to a 40-fold decreased mMC1Rpotency in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template. Modification of theDPhe in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template (peptides 10-13)resulted in different MC1R potency as compared withthe linear melanocortin-based agonist templates previ-ously reported,28,48 but is consistent with studies usingthe cyclic MTII or SHU9119 templates.22,23,49,50

Replacement of the Arg side chain in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptidetemplate with Ala (4), Pro (14) or Lys (15) resulted in300-. 4500-, and 3100-fold decreased mMC1R agonistpotency, respectively, compared to 1. Interestingly, themethyl side chain of Ala (4) resulted in a compound thatwas 10-fold more potent than the basic amine moietyof Lys (15). This latter observation is in contrast tosimilar substitutions in the Ac-His-DPhe-Arg-Trp-NH2template where substitution of the Arg side chain withthe Lys moiety was slightly more potent than the Alasubstitution.30 At the MC1R, modification at the Argposition in the chimeric AGRP-melanocortin templatepresented herein results in different structure-activityrelationships (SARs) than those previously reported forthe 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 theTyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in equipotent or up toca. 100-fold decreased mMC1R potency, compared to 1(Table 2). When the Ala methyl side chain (5) replacedthe Trp indole side chain (1), a 1500-fold decreased


mMC1R potency resulted, significantly different com-pared to the functional activity of the homologoussubstitutions in the R-MSH and Ac-His-DPhe-Arg-Trp-NH2 linear templates at the MC1R.30,45 SAR compari-sons demonstrate that substitution at the Trp positionin the chimeric template with amino acid substitutionsthat have been previously reported to possess equipotentMC1R agonists results in an overall different pharma-cological MC1R profile.29,48

Melanocortin-3 Receptor. The MC3R is expressedboth peripherally and centrally and appears to beinvolved in metabolism and energy homeostasis.9,10,51,52

Alanine scanning of the His, DPhe, Arg, and Trpresidues in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template were not well toler-ated at this receptor. Analogue 2, where the His isreplaced by the Ala amino acid, resulted in 30-folddecreased mMC3R potency, compared to 1. Whereassubstitution of Ala at the DPhe (3), Arg (4), and Trp (5)positions resulted in µM agonist EC50 values, or in thecase of peptide 3, was only able to stimulate the mMC3Rto 60% at 100 µM concentrations (Table 2). The MC3Rpharmacological profile, even within the various mel-anocortin peptide-based templates, appears to varysignificantly at the His position.27-29,53-56 Remarkablyhowever, replacement of His by Pro (6), Phe (7), andracemic Atc (8) in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 template all resulted inmMC3R antagonists, with or without (8) partial agonistactivity (Figures 3 and 4). These results were unex-pected, as it has been well demonstrated in the litera-ture that modification of the melanocortin agonist Hisposition results in MC4R versus MC3R agonist ligandselectivity.27,41,56-59

Peptide 10. Tyr-c[â-Asp-His-(pI)DPhe-Arg-Trp-Asn-Ala-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-DPhe-Arg-Trp-NH2 template.28,50 Surprisingly, peptide 9, Tyr-c[â-Asp-His-Pro-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2, inwhich the Pro was substituted for the DPhe, retainedfull agonist activity at the MC3R. This is surprising, asit is well documented in the melanocortin-based peptidetemplates that the Phe seven position is critical forbiological activity and the substitution of this keyresidue by amino acids other than aromatic or “bulky”result in a loss of ligand activity. Modification of thePhe seven position with DBip and DNal(1′) resulted in13-and 29-fold decreased mMC3R potency in the Ac-His-DPhe-Arg-Trp-NH2 template,28 whereas in the chi-meric template Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 (1), these substitutions resulted in34- and 330-fold decreased mMC3R potency. Charac-teristically, the DNal(2′) substitution for DPhe in thetemplate used herein (11) resulted in MC3R antago-nism, typical for this substitution in melanocortin-basedpeptide templates.50,60 Generally, as observed above,modifications of the chimeric template used hereinresult in different MC3R pharmacological profiles thanpreviously observed for the melanocortin peptides, withthe specific exception of the DNal(2′) substitution thatwill be discussed further in the MC4R section below.

Substitution of Arg in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 template with Ala (4),

Pro (14), and Lys (15) resulted in 24000-, 18000-, and32000-fold decreased mMC3R potency, respectively.Significant differences in MC3R pharmacology result forthe same amino acid substitutions of the Arg residuein 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[â-Asp-His-DPhe-Arg-Trp-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 3400-fold decreased mMC3R potency, respectively. Bothdifferences and similarities exist between the chimerictemplate 1 versus the melanocortin peptide templatesat the MC3R, depending upon the specific amino acidmodification examined.29,41,53-55

Melanocortin-4 Receptor. The central MC4R hasbeen identified as physiologically participating in foodconsumption3 and obesity in mice2 with several poly-morphisms of the MC4R observed in obese humans.62-67

Substitution with Ala (2), Pro (6), or Phe (7) at the Hisposition, DNal(1′) (12) or DBip (13) at the DPhe position,and Nal(2′) (17) or DNal(2′) (18) at the Trp position inthe Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in equipotent ago-nist potency at the mMC4R, as compared with the leadpeptide 1 (within experimental error). These resultswere unexpected, as it has been well demonstrated inthe literature that modification of the melanocortinagonist His position results in MC4R versus MC3Rselectivity.27,41,56-59 Specifically, modification of theHis position by the Pro side chain in the MTIItemplate resulted in the identification of modificationsthat might lead to increased MC4R selectivity versusthe MC3R.41,58,59 Peptide 8, containing racemic Atc atthe His position in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template, resultedin 880-fold decreased mMC4R potency, compared with1, and was a ca. 400 nM potent MC3R antagonist,versus a MC4R selective agonist as demonstrated previ-ously in different melanocortin peptide templates.27,46,47,57

Consistent with previous reports of homologous substi-tutions in the cyclic MTII template,41,54 peptides 6 (Pro)and 7 (Phe) modifications at the His position in theTyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in nearly equipotent (5-fold) mMC4R potency as the control compound 1.However, the mMC4R pharmacology of peptides 6 (Pro)and 7 (Phe) was significantly different from the lineartetrapeptide template27 and cyclic (c[CO-CH2CH2CO-His-DPhe-Arg-Trp-Lys]-NH2)55 templates containingidentical substitutions at the His position.

Substitution of DPhe with Ala (3) in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptidetemplate resulted in 113000-fold decreased mMC4Ragonist potency. Replacement of Phe by Ala in theendogenous melanocortin agonist γ2-MSH (Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly) template re-sulted in a loss of full hMC4R agonist activity at up to10µM concentrations.53 Replacement of the DPhe resi-due of MTII (Ac-Nle-[Asp-His-DPhe-Arg-Trp-Lys]-NH2)with Ala resulted in a modification that possessed onlyslight agonist activity at the hMC4R.54 Thus, while allthe 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 tofully stimulate the mMC4R at high concentrations.Similarly, in peptide 9 the DPhe substitution with Proin the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in 16700-fold de-creased mMC4R agonist potency, but still maintain fullagonist stimulation at high concentrations. Surprisingly,substitution with DNal(2′), peptide 11, at the DPheposition in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template, resulted in partialagonist activity at the mMC4R (Figure 3), in additionto the expected potent competitive antagonist pharma-cology. In all the various melanocortin templates, bothlinear and cyclic, substitution of L/DPhe with DNal(2′)resulted in potent MC4R competitive antagonists devoidof 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 theTyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template, resulted in partial agonist activ-ity at the mMC4R (Figure 4) in addition to the expectedpotent competitive antagonist pharmacology. These dataare consistent with previous reports that (pI)DPhesubstitution in the linear tridecapeptide NDP-MSH andcyclic MTII templates result in mixed partial agonistand antagonist MC4R pharmacology.50,69 However, thetetrapeptide Ac-His-(pI)DPhe-Arg-Trp-NH2 is a potentmMC4R agonist.28 Thus, at the MC4R, peptide 10is consistent with previous melanocortin templatesNDP-MSH and MTII that incorporate the (pI)DPhe atthe seven position (R-MSH numbering). Peptides 12[DNal(1′)] and 13 (DBip), modified at the DPhe posi-tion in the chimeric template, resulted in equipotent(4-fold) mMC4R agonist potency as compared with thecontrol (1), similar to homologous substitutions inlinear tetra- and pentapeptide melanocortin-based tem-plates.28,48

Modification of the Arg position in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptidetemplate with Ala (4), Pro (14), and Lys (15) resultedin 1000- to 66000-fold decreased mMC4R potency (Table2). Modification at the Arg position in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptidetemplate results in MC4R pharmacology different thanpreviously reported homologous substitutions in othermelanocortin peptide templates.30,41,53,54,61

The functional role of the Trp indole side chain in theTyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template at the mMC4R was explored bysubstitution with the following amino acids: Ala (5,5400-fold decreased mMC4R potency), Pro (16, 55000-fold decreased mMC4R potency), Nal(2′) (17, equi-potent), DNal(2′) (18, equipotent), Bip (19, 330-folddecreased mMC4R potency), and Tic (20, 2000-folddecreased mMC4R potency). In the chimeric peptidetemplate examined herein (Table 1), Trp modificationswith Nal(2′), peptide 17, and DNal(2′), peptide 18resulted in consistent MC4R pharmacology as previ-ously reported.29,48,55 However, the Ala (5), Pro (16), Bip(19), and Tic (20) amino acid substitutions of the Trpamino acid in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted inMC4R pharmacology different from that previously

reported for homologous substitutions in other melano-cortin peptide templates.29,41,53,54

Melanocortin-5 Receptor. The peripheral MC5R isexpressed in a variety of tissues and has been implicatedas physiologically participating in the role of exocrinegland function.12,70,71 Substitution with Ala (2), at theHis position, (pI)DPhe (10) at the DPhe position, Nal(2′)(17) or DNal(2′) (18) at the Trp position, and Ala (4) atthe Arg position in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted inequipotent agonist potency at the mMC5R, as comparedwith the lead peptide 1 (within experimental error). Dueto the fact that substitution with identical amino acidsat homologous positions in different melanocortin recep-tor peptide templates result in drastically differentMC5R pharmacology,27,41,53-55 from equipotent to noactivity for the same modification in different templates,comparisons of His modifications in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide tem-plate with homologous substitutions in other melano-cortin templates becomes confounding.

Modification of the DPhe residue in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptidetemplate with Ala (3) resulted in a 690 nM full agonistwith 1800-fold decreased mMC5R potency. Thus, Alasubstituted for DPhe in the chimeric peptide 3 retainedhigh nM MC5R agonist potency, while other melano-cortin receptor templates with the identical modificationresulted in compounds that were unable to stimulatethe 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 previ-ously reported results.28 However, the Tyr-c[â-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide12, resulted in 60-fold decreased mMC5R potency whichis inconsistent with previously published DNal(2′) sub-stitutions at the DPhe position in the tetrapeptidetemplate.28 The Tyr-c[â-Asp-His-Pro-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide 9, resulted in µM MC5Ragonist potency but was 21000-fold less active than thecontrol peptide 1.

Modification of the Arg side chain with Ala (4), Pro(14), and Lys (15) in the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template resulted in6-, 8600-, and 2100-fold decreased MC5R potency. TheTyr-c[â-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide 4 possesses unique MC5R pharmacologyas compared with homologous modifications in othermelanocortin peptide templates.30,53,54 Peptide 14, con-taining the Pro amino acid modification for the Arg,resulted in 8600-fold decreased MC5R potency, consist-ent with the same substitution in the tetrapeptidetemplate,30 but different in the MTII template.41 Peptide15, containing the Lys residue substitution for the Arg,resulted in 2100-fold decreased MC5R potency, whichis different than the homologous modifications in thetetrapeptide and MTII templates.30,61

In the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template, the Trp amino acid wassubstituted with Ala (5), Pro (16), Nal(2′) (17), DNal(2′)(18), Bip (19), and Tic (20) residues. The Ala-containingpeptide 5 resulted in 135-fold decreased mMC5R po-tency. Peptide 16, Tyr-c[â-Asp-His-DPhe-Arg-Pro-Asn-


Ala-Phe-Dpr]-Tyr-NH2, possessed 700 nM mMC5R ag-onist potency, but was 1900-fold less potent than 1.Comparison of the chimeric versus the MTII templates41

with Pro substituted for Trp resulted in dramaticallydifferent MC5R pharmacology. The Trp amino acidsubstitution by Nal(2′) (17), DNal(2′) (18), and Bip (19)resulted in nearly equipotent MC5R pharmacology ascontrol (1) and is consistent with identical substitutionsin the tetrapeptide template,29 but the Nal(2′) substitu-tion in the (c[CO-CH2CH2CO-His-DPhe-Arg-Nal(2′)-Lys]-NH2) peptide resulted in significantly differentMC5R pharmacology.55

In summary, these structure-function substitutioncomparisons of the chimeric template examined herein,versus other various linear and cyclic melanocortin-based peptide agonist templates, demonstrate that thechimeric Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide is a novel melanocortin agonisttemplate possessing distinct receptor pharmacologicalprofiles.

Melanocortin Receptor Selectivity. The melano-cortin pathway consists of five known receptor isoforms,located in a variety of tissues, and is involved in avariety of physiological functions.72 The melanocortinreceptor knock out mice have provided valuable infor-mation attributing a melanocortin receptor to a par-ticular phenotype, but not all the physiological functionsattributed to the melanocortin pathway have beenlinked to a specific melanocortin receptor(s). There-fore, the search for novel melanocortin receptor selec-tive ligands with unique pharmacology is being pur-sued by both academic and industrial researchteams.27,28,41,55-57,73-75 Current emphasis regarding mel-anocortin receptor selectivity focuses upon distinctionbetween the MC3 and MC4 receptors that are bothexpressed in the hypothalamus of the brain and areinvolved in the regulation of energy homeostasis. Herein,peptide 4, Tyr-c[â-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2, resulted in a ca. 24 µM MC3R agonistbut possessed 2 to 70 nM agonist potencies at themMC1R, and mMC4-5Rs, and is 730-fold mMC4Rversus mMC3R selective. Table 4 summarizes thepeptides examined in this study that possess greaterthan 10-fold selectivity for the MC4R versus the MC3R.Modifications at each of the His-DPhe-Arg-Trp positionsin the Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 chimeric peptide template resulted in someMC4R versus MC3R selectivity. Interestingly, withexception of the Pro and Ala substitutions, modificationat the Trp position resulted in MC4 versus MC3 receptorselectivity not previously identified by identical amino

acid substitutions in other melanocortin receptor tem-plates.29,41,48,53-55 Additionally, previous reports identi-fying that modification at the His position with Pro andother residues in cyclic melanocortin peptides resultsin MC4R versus MC3R selective agonists.27,41,56-59

However, in the chimeric peptide template, substitutionof His with Pro (6), Phe (7), and racemic Atc (8) allresulted in ligands that were mMC3R antagonists butmMC4R agonists, clearly possessing different pharma-cological profiles compared with other published mel-anocortin receptor templates.

1H NMR and Computer-Assisted Molecular Mod-eling-Based Structural Studies. In attempts to cor-relate the melanocortin receptor functional studies withpeptide structure, we performed 2D 1H NMR andCAMM experiments. Six peptides were selected fromthe 21 peptides pharmacologically evaluated in Table2, based upon controls (peptides 1 and 2), unique andunanticipated MC3R versus MC4R differences in func-tion (peptides 6, 7, and 11), and comparisons withsimilar substitutions in other NMR-based structuralstudies of melanocortin ligands MTII and SHU911936,38,76

(peptides 11 and 12). For all the peptides examined byNMR in this study, a reverse turn conformation withinthe putative His-DPhe-Arg-Trp message sequence wasfound. Our results are consistent with previous studiesthat identified the presence of a reverse turn in this His-DPhe-Arg-Trp domain of various melanocortin agonistligands.36,38,77-81

NMR-Based Structural Features of ChimericAgonist Peptides. Peptides 1 and 2 possessing agonistactivity at all the mouse melanocortin receptor exam-ined in this study and are generally characterized topossess a â-turn involving the Trp6 and Asn7 residues.The 30-fold decreased potency of peptide 2 at themMC1R and mMC3 may suggest that stacking interac-tion between side chains of His3 and DPhe4 observed inpeptide 1 (Figure 6) may be important for the agonistpotency at these receptors. NMR studies of melanocortinligands has shown that this type of â-turn secondarystructure may also exist in MTII, which contains thesame message sequence His-DPhe-Arg-Trp, as peptide1.38 Another interesting structural feature of peptide 1is the positioning of the side chain of the Arg5 residuewhich is directed in an opposite orientation from theDPhe4 and Trp6 residues (Figure 6). This side chainorientation may explain how peptide 1 possesses theamphiphilic characteristic similar to structures observedin other melanocortin ligands.38,78,82 Peptide 12, con-taining a bulkier DNal(1′) residue instead of the DPheamino acid, is a full agonist at mMC1R and mMC3-

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

peptide structuremMC3R

EC50 (nM)mMC4R

EC50 (nM)

selectivity ratio:mMC3R EC50/mMC4R EC50

4 Tyr-c[â-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 23800 32.5 73012 Tyr-c[â-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 320 0.57 5602 Tyr-c[â-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 29.0 0.36 8013 Tyr-c[â-Asp-His-DBip-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 33.0 0.57 5817 Tyr-c[â-Asp-His-DPhe-Arg-Nal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.56 0.27 2121 Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-DPhe-Dpr]-Tyr-NH2 14.0 0.66 215 Tyr-c[â-Asp-His-DPhe-Arg-Ala-Asn-Ala-Phe-Dpr]-Tyr-NH2 13500 710 1920 Tyr-c[â-Asp-His-DPhe-Arg-Tic-Asn-Ala-Phe-Dpr]-Tyr-NH2 3400 260 1319 Tyr-c[â-Asp-His-DPhe-Arg-Bip-Asn-Ala-Phe-Dpr]-Tyr-NH2 450 42.6 1118 Tyr-c[â-Asp-His-DPhe-Arg-DNal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 6.50 0.67 10


5Rs, albeit with 40-fold decreased potency at the mMC1and 330-fold decreased agonist potency at the mMC3,as compared to peptide 1. Interestingly, this analoguecontains two reverse turns involving His-DNal(1′)-Arg-Trp-Asn residues, which may be defined as consecutiveturns of a 310-helix.37 As a result of this putative 310-helical secondary structure for peptide 12, the sidechains of the message His-DNal(1′)-Arg-Trp sequenceare oriented differently than observed in peptide 1(Figure 6). Both the differences in the peptide backboneand side chain orientations may account for the differ-ences observed in melanocortin receptor potency.

NMR-Based Structural Features of ChimericmMC3 and mMC4 Receptor Partial Agonist andAntagonist Peptides. The common feature of thisgroup of peptides possessing partial agonist or antago-nist melanocortin receptor pharmacology is a reverseturn including the Arg5 and Trp6 residues (Figure 6).Additionally, these peptides are generally more flexible,as compared with the full agonist peptides discussedabove. Peptides 6 and 7, with Pro and Phe substitutionsat the His position respectively, possess strikinglyidentical melanocortin receptor pharmacological profilesin which both peptides are partial agonist/antagonistat the mMC3R and full agonists at the mMC1R,mMC4R, and mMC5R (Table 2). These peptides possessdifferent overall tertiary structures with peptide 7possessing a more ordered conformation as comparedwith peptide 6. However, comparison of the peptidefragments containing the melanocortin “key” DPhe-Arg-Trp residues (Figure 7) identified that both peptides 6and 7 possess similar side chain orientations in thislocal domain, supporting the hypothesis that both thesepeptides may be interacting with the melanocortinreceptors in a similar molecular recognition motif.Peptide 11 is a potent competitive antagonist at mMC3R,is a partial agonist and antagonist at mMC4R (Figure4) and possesses full nM agonist potency at the mMC1and mMC5 receptors (data not shown). This “mixed-agonist/antagonist ligand pharmacology” of peptide 11may be attributed to the conformational equilibriumbetween two identified conformational families (Figure6). Family 1 conformer contains a reverse turn involvingthe Trp6-Asn7 residues, which is similar to the analo-gous turn existing in the full agonist peptide 1. Ad-ditionally however, a highly populated family 2 con-former possesses a reverse turn involving the Arg5-Trp6

amino acids that resembles that major structural familyobserved for peptide 6, which also possesses partialagonist activity at MC3R (Figure 3). Peptide 7, whichis a partial agonist and antagonist at the MC3R (Figure3) but is a full nM mMC4R agonist, possesses a γ-turn

with the DPhe4 in the central position, possibly explain-ing the difference between mMC3R antagonist andmMC4R agonist pharmacology.

ConclusionsThis study reports incorporation of selected amino

acid substitutions into a chimeric antagonist AGRP-melanocortin agonist peptide template Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 to determineif the pharmacological profiles of these chimeric mol-ecules resemble the published “classical” melanocortinreceptor antagonists or antagonists. Detailed melano-cortin receptor comparison of amino acid substitutionsincorporated into homologous positions in the chimerictemplate versus other published melanocortin peptidetemplate structure-activity studies has resulted in theconclusion that the melanocortin chimeric Tyr-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 templateresults in novel receptor pharmacology profile and is anew lead template for further structure-activity stud-ies, both from the in vitro ligand-receptor standpointas well as providing novel tools to study the in vivomelanocortin pathways. The conformational studiespresented herein suggest that single amino acid sub-stitutions can induce a broad range of structural con-formations and as a consequence may cause differentpharmacological function at the melanocortin receptors,and that perhaps specifically a γ-turn secondary struc-ture involving the DPhe4 in the central position candifferentiate mMC3R antagonist and mMC4R agonistfunctions.

Experimental SectionPeptide 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, Boc-Trp(For), Boc-Asn, Boc-Ala, Boc-Phe, Boc-Pro, Boc-(2-Naph-thyl)-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 Boc-Arg(Tos) and Boc-diaminopropionic (Dpr) amino acids werepurchased from Peptides International (Louisville, KY). Theamino acids Boc-p-iodo-D-phenylalanine [(pI)DPhe], Boc-4-phenyl-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). Theracemic amino acid 2-N-Boc-amino-tetrahydro-2-naphthyl car-boxylic acid (Atc) was purchased from Pharmacore (High Point,NC). p-Methylbenzhydrylamine Resin (p-MBHA Resin, 0.28mequiv/g substitution) was purchased from Peptides Interna-tional. The coupling reagents: benzotriazol-1-yl-N-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP), and1-hydroxybenzotriazole (HOBt) were obtained from PeptidesInternational. Glacial acetic acid (HOAc), dichloromethane(DCM), methanol (MeOH), acetonitrile (ACN), and anhydrousethyl ether were purchased from Fisher (Fair Lawn, NJ). N,N-Dimethylformamide (DMF) was purchased from Burdick andJackson (McGaw Park, IL). Trifluoroacetic acid (TFA), 1,3-diisopropylcarbodiimide (DIC), and piperidine were purchasedfrom Sigma (St. Louis, MO). N,N-Diisopropylethylamine (DIEA)was purchased from Aldrich (Milwaukee, WI). All reagents andchemicals were ACS grade or better and were used withoutfurther purification.

The syntheses were performed using a 40-well Teflonreaction block with a coarse Teflon frit. Approximately 200mg 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 anddeprotected using 4 mL of 50% TFA and 2% anisole in DCM

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


for 3 min followed by a 20 min incubation at 500 rpm andwashed with DCM (4.5 mL, 2 min, 500 rpm three times). Thepeptide-resin salt was neutralized by the addition of 4 mL of10% DIEA in DCM (3 min, 500 rpms, two times) followed bya DCM wash (4.5 mL, 2 min, 500 rpm four times). A positiveKaiser83 test resulted indicated free amine groups on the resin.The growing peptide chain was added to the amide-resin usingthe general amino acid cycle as follows: 500 µL of DMF wasadded to each reaction well to “wet the frit,” 3-fold excessamino acid starting from the C-terminus is added [400 µM of0.5 M solution in 0.5 M HOBt and BOP in DMF] followed bythe addition of 400 µL of 0.5 M DIC in DMF and the reactionwell volume is brought up to 3 mL using DMF. The couplingreaction was mixed for 1 h at 500 rpm, followed by emptyingof the reaction block by positive nitrogen gas pressure. Asecond coupling reaction was performed by the addition of 500µL of DMF to each reaction vessel, followed by the addition of400 µL of the respective amino acid (3-fold excess), 400 µL of0.5 M HOBt and BOP, 300 µL of 1 M DIEA, the reaction wellvolume was brought up to 3 mL with DMF, and mixed at 500rpm for 1 h. After the second coupling cycle, the reaction blockwas emptied and the resin-NR-protected peptide was washedwith DCM (4.5 mL 4 times). NR-Boc deprotection was per-formed by the addition of 4 mL of 50% TFA, 2% anisole inDCM and mixed for 5 min at 500 rpm followed by a 20 mindeprotection at 500 rpm. The reaction well was washed with4.5 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 asdescribed above. The Fmoc and OFm protecting groups wereremoved from Dpr and Asp, respectively by treatment with4.5 mL of 25% piperidine in DMF (20 min at 500 rpm) with apositive Kaiser test resulting. The lactam bridge between theAsp and Dpr amino acids was formed using 5-fold excess BOPand 6-fold excess DIEA as coupling agents and mixing at 500rpm and monitored for cyclization completion by a negativeKaiser test. Deprotection of the remaining amino acid sidechains and cleavage of the amide-peptide from the resin wasperformed by incubation the peptide-resin with anhydroushydrogen fluoride (HF, 5 mL, 0°C, 1 h) and 5% m-cresol, 5%thioanisole as scavengers. After the reaction was complete andthe HF was distilled off, the peptide was ether precipitated

(50 mL × 1) and washed with 50 mL of cold (4°) anhydrousethyl ether. The peptide was filtered off using a coarse fritglass filter, dissolved in glacial acetic acid, frozen and lyo-philized. The crude peptide yields ranged from 60% to 90% ofthe theoretical yields. A 40 mg sample of crude peptide waspurified by RP-HPLC using a Shimadzu chromatographysystem with a photodiode array detector and a semipreparativereversed phase high performance liquid chromatography (RP-HPLC) C18 bonded silica column (Vydac 218TP1010, 1.0 × 25cm) and lyophilized. The purified peptide was >95% pure asdetermined by analytical RP-HPLC and had the correctmolecular mass (University of Florida protein core facility)Table 5.

Cell Culture and Transfection. HEK-293 cells weremaintained in Dulbecco’s modified Eagle’s medium (DMEM)with 10% fetal calf serum and seeded 1 day prior to transfec-tion at 1 to 2 × 106 cell/100-mm dish. Melanocortin receptorDNA in the pCDNA3 expression vector (20 µg) were transfectedusing the calcium phosphate method. Stable receptor popula-tions were generated using G418 selection (1 mg/mL) forsubsequent bioassay analysis.

Functional Bioassay. HEK-293 cells stably expressing themelanocortin receptors were transfected with 4µg CRE/â-galactosidase reporter gene as previously described.18,69,84

Briefly, 5000 to 15000 posttransfection cells were plated into96-well Primera plates (Falcon) and incubated overnight.Forty-eight hours posttransfection the cells were stimulatedwith 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 BSAand 0.1 mM isobutylmethylxanthine) for 6 h. The assay mediawas aspirated and 50 µL of lysis buffer (250 mM Tris-HCl pH) 8.0 and 0.1% Triton X-100) was added. The plates werestored at -80 °C overnight. The plates containing the celllysates were thawed the following day. Aliquots of 10 µL weretaken from each well and transferred to another 96-well platefor relative protein determination. To the cell lysate plates,40 µL of phosphate-buffered saline with 0.5% BSA was addedto each well. Subsequently, 150 µL of substrate buffer (60 mMsodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM â-mer-captoethanol, 200 mg/100 mL ONPG) was added to each welland the plates were incubated at 37°. The sample absorbance,OD405, was measured using a 96-well plate reader (Molecular

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

peptide structureHPLC k′

(system 1)HPLC k′

(system 2)M +1(calcd)

mass spectralanalysis(M + 1)

purity,%

1 Y-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 4.8 8.1 1486.61 1486.06 >962 Y-c[â-Asp-Ala-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.2 8.7 1420.54 1419.60 >963 Y-c[â-Asp-His-Ala-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 4.9 8.9 1410.51 1408.53 >964 Y-c[â-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.5 9.2 1401.50 1399.94 >985 Y-c[â-Asp-His-DPhe-Arg-Ala-Asn-Ala-Phe-Dpr]-Tyr-NH2 3.9 7.0 1371.47 1370.59 >966 Y-c[â-Asp-Pro-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peak 1: 5.5

peak 2: 5.8peak 1: 8.7peak 2: 9.2

1446.58 1445.83 >96

7 Y-c[â-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 6.8 9.5 1496.64 1495.62 >958 Y-c[â-Asp-(rac)Atc-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peak 1: 6.4

peak 2: 6.6peak 1: 11.3peak 2: 12.7

1521.68 1523.31 >95

9 Y-c[â-Asp-His-Pro-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.0 8.8 1436.55 1435.62 >9510 Y-c[â-Asp-His-(pI) DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.0 9.2 1612.50 1610.89 >9511 Y-c[â-Asp-His-DNal(2′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.0 9.2 1536.66 1535.24 >9512 Y-c[â-Asp-His-DNal(1′)-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 4.9 8.6 1536.66 1535.33 >9813 Y-c[â-Asp-His-DBip-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.3 9.1 1562.70 1561.34 >9614 Y-c[â-Asp-His-DPhe-Pro-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 3.2 11.2 1427.54 1448.91 >9515 Y-c[â-Asp-His-DPhe-Lys-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 4.0 7.3 1458.59 1457.83 >9616 Y-c[â-Asp-His-DPhe-Arg-Pro-Asn-Ala-Phe-Dpr]-Tyr-NH2 3.9 7.4 1397.51 1397.46 >9817 Y-c[â-Asp-His-DPhe-Arg-Nal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.3 8.9 1497.63 1497.82 >9618 Y-c[â-Asp-His-DPhe-Arg-DNal(2′)-Asn-Ala-Phe-Dpr]-Tyr-NH2 4.9 8.7 1497.63 1498.43 >9819 Y-c[â-Asp-His-DPhe-Arg-Bip-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.5 9.4 1523.67 1524.64 >9620 Y-c[â-Asp-His-DPhe-Arg-Tic-Asn-Ala-Phe-Dpr]-Tyr-NH2 5.6 9.6 1459.58 1461.00 >9521 Y-c[â-Asp-His-DPhe-Arg-Trp-Asn-Ala-DPhe-Dpr]-Tyr-NH2 4.7 8.1 1486.61 1485.86 >96a 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% trifluoroaceticacid/water and a gradient to 90% methanol over 35 min). An analytical Vydac C18 column (Vydac 218TP104) was used with a flow rateof 1.5 mL/min. The peptide purity was determined by HPLC at a wavelength of 214 nm. Multiple peaks indicate a racemic mixture (bothpeaks having the same mass spectral M + 1).


Devices). The relative protein was determined by adding 200µL of 1:5 dilution Bio Rad G250 protein dye:water to the 10µL cell lysate sample taken previously, and the OD595 wasmeasured on a 96-well plate reader (Molecular Devices). Datapoints were normalized both to the relative protein contentand nonreceptor dependent forskolin stimulation. The antago-nistic properties of these compounds were evaluated by theability of these ligands to competitively displace the MTIIagonist (Bachem) in a dose-dependent manner, at up to 10 µMconcentrations.69 The pA2 values were generated using theSchild analysis method.85

Data Analysis. EC50 values represent the mean of duplicateexperiments performed in quadruplet or more independentexperiments. EC50 value estimates, and their associatedstandard errors, were determined by fitting the data to anonlinear least-squares analysis using the PRISM program(v3.0, GraphPad Inc.). The results are not corrected for peptidecontent.

NMR Spectroscopy. Peptide NMR samples were preparedby 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 internalstandard (0.0 ppm). NMR data were collected using BrukerAvance spectrometer operating at 600 MHz at the AdvancedMagnetic Resonance and Imaging and Spectroscopy (AMRIS)Facility in the McKnight Brain Institute at the University ofFlorida. All NMR data were collected at 13 °C. Standard protonTOCSY and NOESY 2D NMR datasets were collected, pro-cessed, and analyzed as described previously.31 NOESY datawere collected at both 250 and 400 ms mixing times, andproton-proton distances were obtained from the 400 msdataset.

Computer-Assisted Molecular Modeling (CAMM).Proton-proton distances were calibrated using the well-resolved Arg or Dpr amino acid methylene protons based onthe relationship r ) rref *(ηref/η)1/6, where r is the distancebetween atoms, η is the NOESY cross-peak volume, rref is theknown distance, and ηref is the corresponding volume of theNOESY calibration cross-peak. The NOE volumes were cat-egorized 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 SYBYLv6.9 software from Tripos Inc. (St. Louis, MO) on a SiliconGraphics workstation. Restrained molecular dynamics (RMD)simulations were run in vacuo with a dielectric constant of4.0 and a temperature of 500 K using the Tripos force fieldand the Gasteiger-Huckel method of calculation of partialatomic charges.

The peptides were built in fully extended linear conforma-tions. In the first step of modeling, only sequential constraintswere used and the RMD simulations were run for 1 ns. Theresulting dynamics trajectories were evenly sampled andenergy minimized, and structures consistent with mediumrange NOESY restraints were used for the next simulation.The next step was a 1 ns RMD simulation using medium rangeNOE restraints, structures were identified in which the sidechains of aspartic acid and diaminopropionic acid were closein space, and lactam bridges were then formed. These stepsensured that the cyclization steps did not trap the moleculein local energy minima. The resulting cyclic peptides were thenenergy minimized without restraints. Finally, all the NOErestraints were included, and 20 ns RMD trajectories werecollected. Following the RMD simulations, structures from 100equally spaced points along the dynamics trajectory wereenergy minimized, analyzed, and grouped into conformationalfamilies using the XCluster program.86 The clustering wasdone by comparison of backbone phi and psi dihedral anglesin the loop region corresponding to residues 3-9.

Acknowledgment. This work has been supportedby NIH Grants DK57080 and DK64250. AndrzejWilczynski is a recipient of an American Heart Associa-tion Postdoctoral Fellowship.

Appendix

Abbreviations. Tyr-c[â-Asp-Xaa-Yaa-Zaa-Baa-Asn-Ala-Phe-Dpr]-Tyr-NH2 (cyclo Asp RCOOH-Dpr âNH):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.

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