Structure-activity relationships of peptides incorporating a bioactive reverse-turn heterocycle at the melanocortin receptors: Identification of a 5800-fold mouse melanocortin-3 receptor (mMC3R) selective antagonist/partial agonist versus the mouse melanocortin-4 receptor (mMC4R)

Article

pubs.acs.org/jmc

Structure−Activity Relationships of Peptides Incorporating a

Bioactive Reverse-Turn Heterocycle at the Melanocortin Receptors:

Identiﬁcation of a 5800-fold Mouse Melanocortin‑3 Receptor

(

mMC3R) Selective Antagonist/Partial Agonist versus the Mouse

Melanocortin‑4 Receptor (mMC4R)

Anamika Singh,^†^,^‡Marvin Dirain, Rachel Witek, James R. Rocca, Arthur S. Edison,

and Carrie Haskell-Luevano*

†

§

#

,†,‡

†

Departments of Medicinal Chemistry and Pharmacodynamics, University of Florida, Gainesville, Florida 32610, United States

Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States

McKnight Brain Institute, University of Florida, Gainesville, Florida 32611, United States

‡

§

#

Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville,

Florida 32610, United States

*

S Supporting Information

ABSTRACT: The melanocortin-3 (MC3) and melanocortin-4

MC4) receptors regulate energy homeostasis, food intake, and

associated physiological conditions. The melanocortin-4 receptor

MC4R) has been studied extensively. Less is known about speciﬁc

(

physiological roles of the melanocortin-3 receptor (MC3R). A major

obstacle to this lack of knowledge is attributed to a limited number of

identiﬁed MC3R selective ligands. We previously reported a spatial

scanning approach of a 10-membered thioether-heterocycle ring

incorporated into a chimeric peptide template that identiﬁed a lead

nM MC4R ligand. Upon the basis of those results, 17 compounds

were designed and synthesized that focused upon modiﬁcation in the

pharmacophore domain. Notable results include the identiﬁcation of

a 0.13 nM potent 5800-fold mMC3R selective antagonist/slight

partial agonist versus a 760 nM mMC4R full agonist (ligand 11).

1

Biophysical experiments (two-dimensional H NMR and computer-assisted molecular modeling) of this ligand resulted in the

identiﬁcation of an inverse γ-turn secondary structure in the ligand pharmacophore domain.

INTRODUCTION

GPCRs stimulate the cAMP signal transduction pathway in

response to agonist ligand stimulation.

■

Peptide ligands for G-protein coupled receptor (GPCR)

molecular probes have been well documented as valuable

tools for studying ligand−receptor interactions and the

diﬀerentiation of ligand-based agonist versus antagonist design

strategies. The melanocortin GPCRs (consisting of ﬁve

Extensive structure−activity relationship (SAR) studies since

21,22

the 1960s

have characterized the presence of a reverse turn

in the purported His-Phe-Arg-Trp pharmacophore domain of

melanocortin receptor agonists. As the endogenous agonist

ligands are linear in structure, they possess a plethora of

possible “bioactive” conformations depending upon their local

changing environment (aqueous or receptor bound). Successful

strategies designed to decrease ligand ﬂexible conformations

and directed toward identiﬁcation of the “bioactive” con-

1

−7

isoforms ) are involved in the regulation of a variety of

8

physiological functions including pigmentation, cardiovascular

9

,10

11,12

13,14

eﬀects,

food intake,

and erectile function.

These

receptors are stimulated by the α-, β-, γ-melanocyte stimulating

hormones (MSH) and adrenocorticotropin (ACTH) endoge-

nous agonists that are derived from the proopiomelanocortin

2

3−27

formation include introduction of cyclic constraints,

2

8

29

30

peptoids, aza peptides, D-amino acids, constrained

1

5,16

27,31,32

33,34

(

POMC) gene.

Additionally, the melanocortin-1 receptor

amino acids,

as well as N-methylation

synthetic

(

MC1R) is endogenously antagonized by the Agouti

modiﬁcations. These strategies share a common underlying

1

7,18

peptide.

The Agouti-related protein (AGRP) functions as

both a competitive antagonist and an inverse agonist at the

Received: August 30, 2012

1

9,20

melanocortin-4 receptor (MC4R).

These melanocortin

©

XXXX American Chemical Society

A

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theme of stabilizing secondary structure(s) and are commonly

used to restrict peptide conformations, aid in conformational

analysis, and often result in selective and potent ligands. As a

complementary strategy, our laboratory examined the pharma-

cological and structural consequences of incorporating a

derivative at the Trp position due to steric constraints during

the synthesis. The second design strategy incorporated into this

study includes the incorporation of amino acid side chain

modiﬁcations (Figure 2) that have been previously identiﬁed as

35

bioactive heterocyclic moiety into a cyclic peptide tem-

3

6−38

plate

(Figure 1) in attempts to increase structural

Figure 2. Structure of amino acid building blocks used in this study.

important for melanocortin receptor selectivity, and antagonist

versus agonist melanocortin-3 receptor (MC3R) and melano-

cortin-4 receptor (MC4R) pharmacological proﬁles. These

amino acid modiﬁcations resulted in several compounds with

low nM potencies and mixed MC3R/MC4R pharmacological

proﬁles at the mouse melanocortin receptor subtypes. The

compounds with DPhe substitutions were selected for

Figure 1. Illustration of the control ligand 2 incorporating the

bioactive heterocycle (pink) into the peptide template. The amino acid

substitutions at each of the R positions [R = His (green), R = DPhe

1

biophysical conformational experiments (2D H NMR and

CAMM) with the goal to correlate structure with mixed

MC3R/MC4R pharmacological proﬁles and attempt to diﬀer-

entiate the structure of the MC3R antagonist/agonist versus

MC4R full agonist ligands.

1

2

(

orange), R =Arg (blue), R =Trp (red) of the melanocortin ligand

3

4

pharmacophore domain are indicated. The library design approach was

based upon the positional scanning method where one R position is

modiﬁed while holding the other positions constant.

RESULTS

■

backbone rigidity and probe the His-Phe-Arg side chain

Chemistry. The ligands examined herein were either

synthesized manually using standard Nα-9-ﬂuorenylmethox-

3

9

orientations. Unexpectedly, the conclusions from that study

39

were that more conformationally ﬂexible ligands resulted [as

ycarbonyl (Fmoc) synthetic methodology as reported or

using a microwave synthesizer (Discover SPS, CEM Corp.),

Scheme 1. Microwave synthetic experimental conditions

facilitated amino acid coupling and Fmoc deprotection

reactions in a more time eﬃcient manner (Fmoc removal: 30

min under conventional conditions to 4 min using the

microwave; amino acid coupling: 60−120 to 5 min using the

microwave). Use of the microwave versus the manual synthetic

approach reduced the overall reaction time signiﬁcantly from 50

to 15 h without aﬀecting the yield of the crude molecules.

Cleavage of the compounds from the resin and removal of the

side chain protecting groups was achieved by a 3 h treatment

with the cleavage cocktail (TFA/triisopropylsilane/EDT/water

91:3:3:3) at room temperature. The disulﬁde bond was formed

after cleavage by dissolving the crude product in 20% DMSO/

water and mixing at room temperature. Progress of the disulﬁde

cyclization was monitored by analytical reversed-phase high-

1

determined by two-dimensional (2D) H NMR and computer-

assisted molecular modeling (CAMM)] compared to the

control cyclic peptide devoid of the heterocyclic backbone

moiety. That study did provide some interesting SAR insights

but also resulted in the identiﬁcation of a nM full agonist

template that could be used for additional SAR experiments to

probe the importance of the His-Phe-Arg-Trp side chain

orientation. Herein, we have further utilized the lead template

(

Figure 1, control ligand 2) and performed additional SAR

studies by maintaining the heterocyclic template constant and

modifying the side chains at the R (His), R (Phe), R (Arg),

1

2

3

and R (Trp) positions. The initial design strategy was to

4

compare an Ala and Pro amino acid scan at each of the four

positions to identify the pharmacological consequences of these

modiﬁcations at the melanocortin receptors examined.

Unfortunately, we were unable to obtain the Pro containing

B

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a

Scheme 1

a

Reagents and conditions: (a) 20% piperidine/DMF; (b) 3 equiv of Fmoc-Xaa-OH, 3 equiv of BOP or HBTU, 6 equiv of DIEA, DMF; (c) Fmoc-

Yaa-aldehyde, NaBH CN, AcOH, DMF; (d) Fmoc-Cys(SBu )-OH, BOP or HBTU, DIEA, DMF; (e) (ClCH CO) O, NEM, DCM; (f) Bu P/H O/

3

t

2

3

2

THF; (g) NEM, DMF, heat 55−60 °C; (h) TFA/TIS/EDT/H O 91:3:3:3.

2

Figure 3. Melanocortin receptor pharmacology of the control NDP-MSH peptide and analogues 6−11 that were modiﬁed at the DPhe position.

Agonist proﬁles at the mMC1R and mMC3−5Rs are shown in the top four panels. The mMC3R antagonist proﬁles for molecules 9 and 11 are

shown in the bottom two panels. Both ligands 9 and 11 also possess partial mMC3R agonist activity at the higher concentrations examined.

pressure liquid chromatography (RP-HPLC) at a ﬂow rate of

assessed by analytical RP-HPLC in two diverse solvent systems

(Supporting Information).

Melanocortin Receptor Pharmacology. All the com-

pounds were tested for agonist or antagonist functional activity

at the mouse MC1, MC3, MC4, and MC5 melanocortin

receptors using a cAMP based β-galactosidase reporter gene

1

3

.5 mL/min (λ = 214 nm) and was generally completed in 24−

6 h. The peptides were puriﬁed to homogeneity using

semipreparative RP-HPLC and a ﬂow rate of 5 mL/min. The

peptides possessed the correct molecular weights as determined

by mass spectrometry. The purity of these peptides (>95%) was

C

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a

Table 1. Agonist EC (nM) and Antagonist pA Pharmacology of the Modiﬁed Peptides at the Mouse Melanocortin Receptors

5

0

2

a

The indicated errors represent the standard error of the mean determined from at least three independent experiments. The potency of the

antagonist was determined by using the equation pA2 = −log K where K is estimated as the ligand potency in nM. A parentheses ( ) indicates that

i

an increase in potency has resulted.Changes less than 3-fold are considered to be within the inherent experimental assay error.

4

0

bioassay. The MC2R is only stimulated by the endogenous

ACTH agonist and was excluded from the present study. The

NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-

One of the ﬁrst experiments we performed was to determine

the eﬀect of an Ala side chain replacement scan at each of the

His-DPhe-Arg-Trp positions in the molecular control template

30

39

Lys-Pro-Val-NH ) peptide is one of the standard melano-

2. Substitution of the His with Ala resulted in no change in

2

cortin agonists used in the study of these receptors and is

included herein as a reference control and as an internal control

to for maximal ligand eﬃcacy (100%). For the compounds

examined in this study, with the exceptions of 9 and 11, which

are partial agonists and antagonists at the mMC3R (Figure 3),

fully eﬃcacious agonists resulted (Table 1). The superpotent

MTII peptide [Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-

potency at the mMC4R with 12−22-fold reduced agonist

potency at the mMC1R, mMC3R, and mMC5R (Table 1).

Replacement of the DPhe side chain with the corresponding

methyl of the Ala amino acid in 7 however resulted in 24- to

>5000-fold decreased ligand potency at the melanocortin

receptors with the most notable decreased potency at the

mMC1R. The reduced potency of this ligand may be associated

with the side chain modiﬁcation, but also the L-stereochemistry

versus the D- in the control molecule at this position.

4

1,42

4

NH₂]

is used as the agonist in the Schild antagonist

3

bioassays to assess the potency of the mMC3R antagonists

instead of α-MSH. The reason is that MTII is ca. 10−100-fold

more potent than α-MSH, and when performing antagonist

assays on weakly potent compounds (i.e., compound 9), the

use of the α-MSH agonist may not be able to generate the

required three agonist shifted dose−response curve EC values

Incorporation of the Ala side chain at the Arg R position in

3

analogue 12 resulted in 6−25-fold decreased melanocortin

receptor potency. Removal of the Trp indole side chain and

replacement by the Ala methyl side chain in analogue 16

resulted in no notable decreased potency at the mMC4 and

mMC5 receptors and approximately a 7−33-fold reduced

potency at the mMC3R and mMC1R, respectively.

50

in the presence of increasing concentrations of antagonist

peptide. A minimum of three dose−response shifted EC value

50

data points are needed to generate a straight line in order to

The His residue has been previously studied in pentapep-

4

3

44−49

50

21,51

determine the pA value according to the Schild method. As

tides,

tetrapeptides, and other peptide templates.

2

38

additional controls, the peptide template 1, which does not

Herein, the His side chain was substituted with Ala, discussed

above, as well as with the Phe (4) and Pro (5) side chains. The

Phe substitution was a well-tolerated modiﬁcation that resulted

in equipotent agonist pharmacology at the mMC1R and

mMC4R, as compared to the control template 2. At the

mMC3R, substitution of the benzyl moiety in 4 resulted in 14-

contain the heterocyclic moiety, as well as the previously

39

reported lead template 2 have been included. All the control

values are consistent with previously reported values and are

within the inherent 3-fold experimental error associated with

this assay in our laboratory over the past decade.

D

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fold decreased potency but only 9-fold decreased mMC5R

potency. The Pro substitution at the His position (5) resulted

in equipotent mMC4R agonist potency, ca. 6-fold decreased

agonist potency at both the mMC3R and mMC5R, and 13-fold

decreased mMC1R potency as compared to control 2.

MeOH/DMSO, (vi) H O/D O]. All two−dimensional (2D)

2

total correlation spectroscopy (TOCSY), nuclear Overhauser

eﬀect (NOESY) and rotating-frame Overhauser eﬀect

(ROESY) spectra were recorded at 34 °C using a 600 MHz

Bruker NMR instrument. The watergate 3−9−19 sequence was

applied for water suppression. Data were processed using

NMRPipe and NMRDraw software on a Silicon Graphics

workstation. Sequential resonance assignment was performed

by the combined use of 2D-TOCSY and NOESY spectra

(ROESY was used as a complementary approach to NOESY).

Only molecule 7, containing the Ala substitution of DPhe, did

not result in experimental spectra possessing resolved peaks

necessary for proton assignment and structural information

under the conditions described above for the other molecules

and therefore was excluded from further biophysical NMR

experiments.

The Phe side chain in the melanocortin peptide ligands has

been a key feature of SAR experiments and has resulted in the

conversion of diﬀerent agonist scaﬀolds into compounds

possessing antagonist and partial agonist activities at the

MC3 and MC4 receptors. Such key modiﬁcations include β-(2-

26

naphthyl)-D-alanine [DNal(2′)] in SHU9119 as well as

3

7,52,53

(

pI)DPhe.

Replacement of the Phe side chain with the

constrained Pro side chain in 6 resulted in 24−32-fold

decreased potency at the mMC3−5 receptors with a signiﬁcant

drop in agonist potency at the mMC1R of ca. 2000-fold, as

compared to control 2. The Ala substitution containing ligand

7

, as discussed above, resulted in reduced potency as well at all

The NOE intensities observed for NMR-derived proton

assignments for peptide analogues 6, 8−11, control 2 are

summarized in the Supporting Information. Despite the fact

the melanocortin receptors examined and could be a

combination of stereochemical inversion (L- instead of D-) as

well as the methyl side chain substitution. Interestingly, the

DNal(1′) substitution of compound 8 resulted in equipotent

pharmacology at the melanocortin receptors examined as

compared to the control 2. Substitution of DPhe with DNal(2′)

in 9 resulted in a potent (25nM) mMC3R antagonist that

possessed equipotent agonist pharmacology as control 2 at the

mMC1, mMC4, and mMC5 receptors. The (pI)DPhe

containing molecule 10 resulted in a full agonist ligand at the

melanocortin receptors with ca. 4−9-fold increased potency at

the mMC1R, mMC3R, mMC4R, and mMC5R, as compared to

control 2. Molecule 11 with the DPhe side chain replaced by

the DBip (Figure 2) residue, resulted in equipotent agonist

pharmacology at the mMC1R, mMC4R, and mMC5R while

unexpectantly resulting in a 0.13 nM mMC3R antagonist/slight

partial agonist that is 2400-fold more potent than control 2.

Additionally, this ligand is a >5800-fold mMC3R selective

antagonist/slight partial agonist (Figure 3) versus the mMC4R.

Modiﬁcation of the Arg side chain residue by the Ala (12),

Lys (13), homoArg (14), and Pro (15) side chains resulted in a

range of agonist activities from no changes in ligand potency to

ca. 25-fold decreased agonist potency depending upon the

ligand and melanocortin receptor (Table 1).

4

that only one amino acid at DPhe position is changing, the

NOE pattern of connectivity is very diﬀerent in all ﬁve

experimental compounds. It was observed that d_N_N(i, i+8) and

d_N_N(i, i+9) NOEs in analogues 6 and 10 are consistent with

37

our earlier study. Compounds 9 and 11 have less NOE

connectivity than observed for the other compounds examined.

5

4

Diﬀerences between random coil (Δ-RC) shifts and

experimental chemical shifts values of compounds 6, 8−11,

control 2 are presented in the Supporting Information. The Δ-

RC shift values in compounds 9 and 11 suggest well-ordered

structures in both compounds. Both analogues 9 and 11 have

mixed pharmacology at the mMC3R/mMC4R subtypes

showing partial agonist/antagonist properties at the mMC3R

and full agonist at the mMC4R (Figure 3). The deviations of

chemical shifts value for the amino acid residues (His, DBip,

Arg for analogue 11 and His, DNal(2′), Arg for analogue 9)

from the melanocortin His-Phe-Arg-Trp message sequence are

very similar in both compounds. Compounds with full agonist

activity at all four melanocortin receptors 6, 8, 10, and control 2

have similar trends at the His, DPhe/Pro/DNal(1′)/(pI)DPhe

and Ala residues. These data indicate that the compounds in

this study are tending to form more ordered structures rather

than random coils and possess subtle diﬀerences in structures

for antagonist and agonist activity.

Substitution of the Trp residue with Ala (16), Nal(2′) (17),

DNal(2′) (18), and Bip (19) amino acids resulted in up to 33-

fold decreased agonist potency at the mMC1R as compared to

Figure 4 depicts the stereoview of sausage representations of

the ﬁve experimental compounds 6, 8−11, control 2 analyzed

by NMR and computer molecular modeling in the present

study. The structures are represented as the superposition of

the conformers of the major family of compounds aligned on

backbone heavy atoms of residues 3−7. Cluster analysis of the

molecular dynamics trajectory of compound 6 resulted in one

major family with 49% of all the conformers. Analysis of the φ

− ψ backbone dihedral angles did not result in the

identiﬁcation of any classical turn structure. Compound 8,

which possesses DNal(1′) in place of DPhe, resulted in one

major family with a population of 29% of the conformers. Using

a representative structure of the major family 8, we identiﬁed a

β-turn of type I′ spanning His and DNal(1′) residues.

Additionally, we observed a cluster of hydrophobic residues

2. At the mMC3 and mMC4 receptors, these Trp modiﬁcations

did not signiﬁcantly aﬀect potency as compared to the control

ligand 2. At the mMC5R, 19 resulted in 18-fold decreased

potency, but the other side chain modiﬁcations possessed

equipotent agonist activity as compared to control.

1

Biophysical Studies: 2D H NMR and Computer-

Assisted Molecular Modeling (CAMM) structures. Li-

gands studied in these experiments (2, 6, 8−11) were dissolved

in 90% (v/v) acetonitrile-d and 10% H O (pH = ∼3.0) for

proton NMR experiments. This solvent was selected based

upon the criteria that (1) it must completely dissolve all the

peptides examined that possessed diﬀerent biophysical proper-

ties, (2) similar solvents have been used previously so that

structural comparisons could be performed and discussed, and

3

2

4

7

10

(

3) the amide protons were well resolved in this system versus

DNal(1′) , Trp and Phe at one side and hydrophilic residues

6 8

the other solvents attempted. Other solvents that were

attempted for this study but did not result in all the peptides

examined being fully dissolved include (i) DMSO-d , (ii)

Arg and Asn on the other side of backbone plane in ligand 8.

Molecular modeling studies of α-MSH and other ligands

7

previously suggested the stacking of the aromatic rings of Phe

and Trp and orientation of the Arg side chains away from the

6

9

8

CDCl /DMSO, (iii) CD CN/H O, (iv) MeOH-d , (v)

3

2

3

E

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Figure 4. Sausage stereoview representations of the representative major families members of compounds (A) control 2 (RMSD = 0.97 ± 1.01 Å)

(

B) 6 (RMSD = 0.78 ± 0.26 Å), (C) 8 (RMSD = 1.66 ± 0.56 Å), (D) 9 (RMSD = 1.67 ± 0.53 Å), (E) 10 (RMSD = 1.08 ± 0.29 Å), and (F) 11

RMSD = 2.84 ± 0.53 Å), aligned on the backbone heavy atoms of residues 3−7. The His side chain is indicated in green, the DPhe side chain in

orange, the Arg side chain in blue, and the Trp side chain in red. A wide gray backbone indicates greater ﬂexibility in that domain. Following

restrained molecular dynamics (RMD) simulations for 10 ns, 200 equally spaced structures were energy minimized with the NMR based restraints.

The energy minimized structures were grouped into conformational families by comparison of the backbone dihedral angels within the His-Phe-Arg-

Trp domain. For comparative purposes, the energy ranges for each of the representative families illustrated is as follows. (A) control 2 33−110 kCal/

mol, (B) 6 157−513 kCal/mol, (C) 8 208−233 kCal/mol, (D) 9 166−190 kCal/mol, (E) 10 173−197 kCal/mol, and (F) 11 196−252 kCal/mol.

6

7

8

55,56

His , Phe and Trp (α-MSH numbering) side chains.

main family possessing a 32% population of the conformers

Cluster analysis of compound 9 resulted in one major family

that contains 27% of total conformers and ﬁve more families

with populations less than 5% that did not possess any classical

turn structure. The largest conformational family (25% of total)

identiﬁed for compound 10 possessed a well-deﬁned structure

after cluster analysis. Further analysis indicated an inverse γ-

8

9

10

turn encompassing Asn , Ala , and Phe residues and isolated

2

5

beta-bridges were found at the Cys and Cys amino acids. The

control compound 2 resulted in four families in this study. The

major family has 21% of the conformer population and other

three families have 13%, 6%, and 5%, respectively. The dihedral

angle analysis of the major family indicated a hydrogen bonded

turn structure spanning the Arg and Trp residues. Isolated β-

3

4

5

with residues His , (pI)DPhe and Cys participating in a classic

57

γ-turn secondary structure motif (Figure 4). Compound 11,

which has a DBip residue in the place of DPhe, resulted in one

F

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Figure 5. CAMM based φ−ψ angle distribution for ligands (A) control 2, (B) 6, (C) 8, (D) 9, (E) 10, and (F) 11 conformational families. The His

residue is indicated in green, the DPhe residue in orange, the Arg residue in blue, and the Trp residue in red. All other residues are indicated in

purple.

6

2−64

bridges were also observed at the His and Phe amino acids. The

root-mean-square superposition deviations of the backbone

heavy atoms for the conformers from the major family to the

lowest energy member are 0.78, 1.66, 1.67, 1.08, 2.84, and 0.97

Å for compounds 6, 8−11, as compared to control 2,

respectively. The φ−ψ distribution of the putative pharmaco-

phore residues His-DPhe-Arg-Trp is represented in Figure 5.

All the ligand residues were included, except the terminal Tyr

residues. The φ−ψ distribution patterns are very diﬀerent in the

pharmacophore region spanning the His, DPhe/Pro/

DNal(1′)/DNal(2′)/(pI)DPhe/DBip, Arg and Trp residues.

In particular, the DPhe substitutions and the Trp residues in all

compounds indicate the most dramatic diﬀerences. Apart from

(heterodimerization),

there is a continuing need for the

development and identiﬁcation of selective MC3 versus MC4

receptor agonists, antagonists, and/or mixed functional ligands.

It has been established that it is diﬃcult to design MC3R versus

MC4R selective ligands based on conventional modiﬁcations

using the MTII and SHU9119 molecular templates.

Even when it is possible to get somewhat selective and potent

4

9,65−70

ligands through other novel templates reported in the literature

6

9,70

by other laboratories,

precise structural and conformational

features that diﬀerentiate SAR between the MC3 and MC4

receptor subtypes is diﬃcult to predict a priori. We continue

these eﬀorts in the present study to probe ligand structural

features that can diﬀerentiate the ligand−receptor pharmaco-

logical proﬁles of the melanocortin receptors (MC1, MC3−

8

9

10

pharmacophore region, Asn , Ala , and Phe have prominent

diﬀerences in φ−ψ angle distribution patterns.

5

R). In a previous study we reported a novel hybrid peptide-

heterocyclic moiety template that resulted in a potent nM

39

MC4R analogue, control 2 herein. This compound, however,

was not selective toward the melanocortin MC3 and MC4

receptor subtypes. On the basis of these earlier results, and

reports from other laboratories, we designed the present library

by replacing the putative His-Phe-Arg-Trp side chain

pharmacophores with the natural and unnatural amino acid

side chains shown in Figure 2. These selected amino acid side

chains have been previously identiﬁed to increase melanocortin

receptor selectivity, potency, and agonist or antagonist

DISCUSSION

■

The MC4R is expressed centrally in the hypothalamus region of

brain and has been shown to regulate food intake upon i.c.v.

administration of agonists (decrease food intake) and

1

1,12,58

antagonists (increase food intake).

The MC3R, which

is expressed both centrally (including the hypothalamus) and

3

,5

peripherally, has been postulated to play fundamental roles in

59,60

metabolism and energy homeostasis,

as well as food

1

2

26,37,41,52,71,72

intake. However, the physiological mechanism(s) of action

for the MC3 receptor in the diﬀerent tissues and the periphery

are still unclear. In recent years, the role of the MC3R in other

pharmacology.

Melanocortin-1 Receptor. The MC1R is primarily

expressed in melanocytes and leukocytes and is involved in

skin and hair pigmentation. Mounting experimental evidence

suggests that the MC1R may also be involved in pain

modulation, inﬂammation, and control of the immune

61

functions like inﬂammation are becoming more evident.

Upon the basis of the unclear physiological roles of the

melanocortin receptors alone or in synergistic combinations

G

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Figure 6. Histogram comparing the relative fold change in EC₅₀values of the indicated amino acid relative to the appropriate control ligand at the

mMC1R. Color coding - the black bar represents the tetrapeptide template Ac-His-DPhe-Arg-Trp-NH . The blue bar represents the KAR template

2

‡

Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH . The red bar represents the control ligand 2 in this study. Signiﬁes that this amino acid

2

@

substitution was not reported in one of the templates used for the comparative analysis in this study. Indicates that the modiﬁcation results in an

increase in potency and is shown as bars pointing down.

1

,2,8,73−75

system.

2

Substitution of His with Ala (3) resulted in

DPhe phenyl moiety resulted in a modest, ca. 4-fold more

potent, compound at the mMC1R. These results indicate that

an aromatic group at this position is important for activity at

the mMC1R, but bulky aromatic residues do not show any

additional beneﬁts.

2-fold decreased potency at the mMC1R, as compared with

the lead control compound 2. Substitution of His with Phe (4)

in the cyclic peptide-heterocyclic template gave equipotent

agonist activity at this receptor subtype which is in contrast to

the AGRP-melanocortin chimeric peptide template KAR Tyr-

Substitution of the Arg and Trp side chains did not give any

dramatic changes with the exception of the Trp to Ala

modiﬁcation (compound 16) that resulted in 33-fold decreased

mMC1R activity. Other side chain substitutions resulted in

nearly equipotent mMC1R analogues as compared to the

control reference molecule. Interestingly, replacement of the

Arg side chain did not have any eﬀect on mMC1R activity in

this template, which is in contrast to some previous reports

3

7

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

which resulted in 27-fold decreased activity when the His

residue was replaced with a Phe (Figure 6). The Phe

substitution of His in tetrapeptide Ac-His-DPhe-Arg-Trp-NH₂

template also resulted in 25-fold decreased mMC1R activity,

37

consistent with the KAR template. Incorporation of a Pro

residue in place of the His (5) resulted in 13-fold decreased

potency as compared to the control template 2, unlike the 100-

fold decreased potency observed in the chimeric KAR

7

1,76

(Figure 6).

Results for Trp substitutions in the currently

used template are consistent with the chimeric peptide template

KAR that does not contain the heterocyclic moiety.

3

7

peptide. This SAR at the His position demonstrates that

His side chain at position 3 is not critical in the present

template for MC1R activity.

Melanocortin-3 Receptor. Alanine scanning of the His,

DPhe, Arg, and Trp residues in the peptide-heterocycle

template 2 resulted in 7−25-fold decreased potency at the

MC3R. All of these compounds, 3, 7, 12, and 16 resulted in full

agonists with μM EC₅₀values (Table 1). Substitution of His

with Pro (5) and Phe (4) did increase MC4R versus MC3R

Replacement of the DPhe side chain at position 4 with Ala

peptide 7) resulted in 5000-fold decreased activity at the

MC1R. The Pro substitution (6) resulted in ca. 2000-fold

decreased potency. These values are consistent with earlier

(

37−39,52

44,50

reports for tetrapeptides (Figure 6).

Since the L-

agonist ligand selectivity, consistent with previous reports,

conﬁguration in 6 and 7 was used, as opposed to the D- in

the parent compound, this may also be a factor for the resulting

reduced potency by this modiﬁcation. Substitution with the

but were diﬀerent than previous results using the chimeric

peptide template KAR, which resulted in mMC3R antagonists

37

(Figure 7). Compound 6, with Pro substitution of the DPhe,

resulted in a full MC3R agonist with 32-fold decreased activity,

with a similar agonist potency of 13200 nM as observed in the

“

bulkier” side chain DNal(1′) compound 8, DNal(2′)

compound 9, and DBip compound 11 resulted in equipotent

MC1R agonist activity compared to the control 2. Additionally,

compound 10 with the (pI)DPhe side chain instead of just the

37

KAR template. Both compounds 6 and 7 retained full agonist

activity at the MC3R, which is in contrast with the studies on

H

dx.doi.org/10.1021/jm301253y | J. Med. Chem. XXXX, XXX, XXX−XXX

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Figure 7. Histogram comparing the relative fold change in EC₅₀values of the indicated amino acid relative to the appropriate control ligand at the

‡

mMC3R. For the color coding scheme, refer back to the legend in Figure 6. Signiﬁes that this amino acid substitution was not reported in one of the

@

templates used for the comparative analysis in this study. *Indicates that the amino acid change in the template resulted in an antagonist. Indicates

that the modiﬁcation results in an increase in potency and is shown as bars pointing down.

melanocortin-based peptide templates where it was concluded

that replacement of Phe at the seventh position with anything

other than aromatic or “bulky” residue would result in loss of

ligand activity (Figure 7). Modiﬁcation of the DPhe phenyl ring

with the with DNal(1′) modiﬁcation in the chimeric template

the present template resulted in equipotent agonist proﬁles at

the mMC4R, as compare with the control 2. It has been

reported previously in the literature that modiﬁcation at the His

side chain position could result in MC4R versus MC3R

4

4,50,78,79

selectivity.

In the peptide-heterocyclic template exam-

37

KAR resulted in 330-fold decreased mMC3R potency,

ined herein, replacement of imidazole side chain of His with the

amino acids Ala, Phe, and Pro resulted in a modest reduction in

MC3R agonist activity, equipotent MC4R, and minor (up to

whereas compound 8 resulted in equipotent ligand activity at

mMC3R. Compound 9, with the DNal(2′) modiﬁcation of the

DPhe side chain in the template used herein, resulted in a

MC3R antagonist, consistent with previous reports for this

substitution in other melanocortin-based peptide tem-

26

15-fold) MC4R versus MC3R selectivity. Unexpectedly,

substitution of DPhe with DNal(2′) in the template examined

herein resulted in an equipotent MC4R agonist. The same

modiﬁcation in the peptide template KAR, lacking the

heterocyclic moiety, resulted in a partial agonist/antagonist

2

6,77,78

plates.

This compound also had partial MC3R agonist

activity (Figure 3). The same Phe-DNal(2′) substitution in the

chimeric melanocortin-AGRP template KAR also resulted in an

antagonistic pharmacological proﬁle at the MC3R (Figure 7).

However, this substitution has a diﬀerent proﬁle at MC4R than

previously observed and will be discussed below. Surprisingly,

compound 10 with the addition of the para-I onto the phenyl

DPhe side chain resulted in a full agonist with ca. 6-fold

increased potency as compared to the control 2 at MC3R.

Compound 11, with the DBip residue in place of the DPhe,

resulted in mixed pharmacology. At the mMC3R it is an

antagonist with slight partial agonist activity and a full agonist at

the mMC4R (Figure 3). Additionally, 11 is 5800-fold selective

in potency for the mMC3R over the mMC4R (Table 1).

Melanocortin-4 Receptor. All substitutions made at the

His, DPhe, Arg, and Trp positions resulted in full agonist

activity at the mMC4R, in contrast to previous SAR studies in

37

pharmacological proﬁle at the mMC4R (Figure 8). In

previous studies involving linear or cyclic melanocortin peptide

templates, substitution of the Phe to the DNal(2′) resulted in

potent MC4R competitive antagonists devoid of any partial

7

agonist activity. Speciﬁcally, modiﬁcation of the DPhe of

melanocortin ligands (α-MSH numbering) such as MTII (Ac-

23,41

Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂),

resulted in the

discovery of the MC3R antagonist/partial agonist and MC4R

antagonist SHU9119 (Ac-Nle c[Asp-His-DNal(2′)-Arg-Trp-

26

Lys]-NH2). The postulated mechanism of MC4R antago-

nism by incorporation of bulky DNal(2′)-containing analogues

was proposed based upon previous reports of SAR, computa-

tional molecular modeling, and receptor mutagenesis studies of

2

6,80

the MC4R.

Receptor mutagenesis studies resulted in the

hypothesis that the DNal(2′) side chain might hinder side-

chain rotation of the conserved MC4R Trp258 amino acid in

transmembrane helix 6 and Leu133 in transmembrane helix 3

(hMC4R numbering), thus “locking” the receptor into the

3

7,52

the KAR and tetrapeptide templates (Figure 8).

Sub-

stitution with Ala (3), Phe (4), or Pro (5) at the His position,

DNal(1′) (8), DNal(2′) (9), DBip (11) at the DPhe, Lys (13)

at the Arg and Ala (16) or Nal(2′) (17) at the Trp position in

72

“inactive” state. Similarly, Chen et al. postulated that in a

I

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Figure 8. Histogram comparing the relative fold change in EC₅₀values of the indicated amino acid relative to the appropriate control ligand at the

‡

mMC4R. For the color coding scheme, refer back to the legend in Figure 6. Signiﬁes that this amino acid substitution was not reported in one of the

@

templates used for the comparative analysis in this study. *Indicates that the amino acid change in the template resulted in an antagonist. Indicates

that the modiﬁcation results in an increase in potency and is shown as bars pointing down.

similar manner, the hMC3R L165 in TM3 interaction upon

peptide ligand binding is critical for agonist or antagonist

mMC4R, consistent with previous studies of other peptide

templates (Figure 8), with the exception of 19.

4

8,72,84

8

2

selectivity. These hypotheses, that bulky substituents at the

Melanocortin-5 Receptor. The MC5R mRNA has been

identiﬁed to be expressed in peripheral and central locations

7

ligand DPhe (α-MSH numbering) position may be responsible

7

,85,86

26

and tissues.

Studies in knockout mice indicated that the

for antagonism at the MC3 and MC4 receptors, is applicable

in the present template for the MC3R as well. However, the

ligands examined herein result in MC4R pharmacological

proﬁles inconsistent with this originally proposed hypothesis

86

MC5R has a role in exocrine gland function, but other

physiological functions for this receptor in the body are not

fully elucidated. In the heterocyclic-peptide template studied

herein, replacement of the His side chain with Ala (3), Phe (4),

and Pro (5) resulted in 6−15-fold decreased activity at the

mMC5R, similar to the chimeric peptide template KAR (Figure

(

Figure 8). Compound 10, with addition of the p-Iodo moiety

to the DPhe amino acid side chain in the control 2 peptide-

heterocycle template resulted in a 9-fold more potent agonist at

the mMC4R as compared to control 2. Interestingly, in the

chimeric peptide template KAR, the linear NDP-MSH, and

cyclic MTII templates, this addition of the p-Iodo moiety to the

DPhe side chain resulted in mixed partial agonist and/or

37

9

). Notably however, a diﬀerence between the templates

exists for the His to Ala substitution, which is equipotent in the

peptide KAR template, but is 15-fold less potent for 3 at the

37

mMC5R compared to control 2. Studies of the tetrapeptide

melanocortin template with similar substitutions resulted in

greater reduction in potency (170-fold when His is replaced by

2

6,80

antagonist MC4R pharmacology,

while the tetrapeptide Ac-

5

2,53

His-(pI)DPhe-Arg-Trp-NH is a potent MC4R agonist.

50

2

Pro). These comparisons in pharmacological proﬁles at the

Thus, at the MC4R, compound 10 is consistent with the

tetrapeptide melanocortin template that incorporates (pI)DPhe

at the seven position (α-MSH numbering) (Figure 8).

Modiﬁcation at the Arg position of template 2 with Ala (12),

hArg (14), and Pro (15) resulted in 5−7-fold decreased

mMC4R potency. Replacement of the Arg side chain with Lys

mMC5R suggest that in the chimeric melanocortin-AGRP

template KAR, independent of the heterocyclic moiety, the His

residue might not be a major potency determining factor at the

mMC5R. Substitution of DPhe by Ala (7) resulted in ca. 170-

fold decreased activity at the mMC5R. Compounds 8;

DNal(1′), 9; DNal(2′), 10; (pI)DPhe and 11; (DBip) showed

equipotent response at the mMC5R compared with the control

(

13) resulted in an equipotent analogue for mMC4R.

Modiﬁcation at the Arg position in the present template

resulted in MC4R pharmacology diﬀerent than the previously

2

template. Substitution at the DPhe with Pro resulted in 24-

fold decrease activity at the mMC5R. Replacement of the Arg

side chain with Ala (12), Lys (13), hArg (14), and Pro (15)

resulted in 8−18-fold decreased activity at the mMC5R. In the

chimeric peptide template KAR, similar substitutions resulted

3

7,71,79,83

reported melanocortin based templates (Figure 8).

The functional role of the Trp indole side chain in the control

template 2 was explored by substitution with following amino

acids: Ala (16), Nal(2′) (17), DNal(2′) (18), Bip (19). All

these modiﬁcations resulted in nearly equipotent activity at the

37

in 6−8600-fold reduced activity (Figure 9). Although the

present template 2 and chimeric peptide template KAR have

J

dx.doi.org/10.1021/jm301253y | J. Med. Chem. XXXX, XXX, XXX−XXX

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Figure 9. Histogram comparing the relative fold change in EC₅₀values of the indicated amino acid relative to the appropriate control ligand at the

‡

mMC5R. For the color coding scheme, refer back to the legend in Figure 6. Signiﬁes that this amino acid substitution was not reported in one of the

@

templates used for the comparative analysis in this study. Indicates that the modiﬁcation results in an increase in potency and is shown as bars

pointing down.

similar mMC5R pharmacological patterns with similar amino

acid modiﬁcations at the Arg side chain, the peptide-heterocycle

template 2 has less dramatic changes in potency than the KAR

template, but appear to be distinct from other melanocortin

control 2 ligand were selected for further biophysical analysis

using 2D H NMR and computer-assisted molecular modeling

1

studies to attempt to correlate structure with function.

Compounds 6, 8, and 10 are full agonists at all four

melanocortin receptors MC1R and MC3−5R. Compounds 9

and 11 are mMC3R antagonists with partial agonistic activity

(Figure 3) and full mMC4R agonists. These compounds can be

compared with similar substitutions in other NMR based

structural studies of melanocortin ligands MTII and SHU9119.

The template examined herein, 2, consists of a 10-membered

heterocycle imbedded into a chimeric peptide 1 postulated to

possess a diﬀerent backbone structure in the pharmacophore

region, from the typical melanocortin based peptide template.

The objective of this study was to compare results of the

structural studies presented herein in the context of previous

reports to determine any overlapping and/or distinct structural

features in attempts to explain diﬀerences in ligand−receptor

SAR and MC3R/MC4R pharmacological proﬁles. In our

previous study, incorporation of the bioactive turn mimetic

into the chimeric AGRP-melanocortin peptide (1) was based

upon the hypothesis that incorporating the heterocycle into the

3

7,71,79

templates (Figure 9).

Speciﬁcally, the tetrapeptide Ac-

His-DPhe-Arg-Trp-NH resulted in 1740-, 11800-, and 80-fold

2

decreased activity upon substitution of Arg with Ala, Pro and

7

1

Lys respectively. Substitution of Trp amino acid side chain

with Ala (16), Nal(2′) (17), and DNal(2′) (18) resulted in

equipotent agonist potency at the mMC5R, as compared with

control template 2, whereas compound 19 is 18-fold less

potent. This template has a diﬀerent pharmacological proﬁle at

the mMC5R with Trp substituted by Ala in comparison to

chimeric template KAR, which resulted in a peptide with 135-

fold decreased mMC5R potency (Figure 9). Substitution of

Trp in the heterocyclic-peptide template examined herein with

Nal(1′) and DNal(2′) is consistent with similar substitutions at

the tetrapeptide template; however the Bip residue substitution

resulted in diﬀerent MC5R pharmacology in the tetrapeptide

7

1

template.

In summary, these SAR studies presented herein with the

peptide-heterocyclic template demonstrate that this is a unique

template that possesses distinct mouse melanocortin receptor

pharmacological proﬁles. Some of these ligands may be used to

investigate the structural determinants that discriminate

between the MC3 and MC4 receptor subtypes.

39

peptide backbone would increase structural rigidity. The

ligands reported in our previous study used a positional

scanning approach that examined both the location of the

heterocycle turn in relationship to the His-DPhe-Arg side

chains as well as a stereochemistry modiﬁcation of the

heterocycle. The control peptide 1 and the most potent ligand

identiﬁed in that study (referred to as 2 in this study) served as

the starting template for the SAR examined in this study.

Surprisingly, while the initial hypothesis that incorporating a

constrained heterocycle into the backbone template of 1 would

1

H NMR and Computer-Assisted Molecular Modeling

Based Structural Studies. Upon the basis of the distinct

mMC3R and mMC4R pharmacological proﬁles of the DPhe

ligand modiﬁcations in this heterocyclic-peptide template

(Table 1), the six DPhe substituted analogues (6−11) and

K

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1

produce a more structured biomolecule, 2D H NMR and

was characterized to be oriented diﬀerently than observed in

compounds 8 and 10 (Figure 4).

NMR and Modeling Based Structural Features of

mMC3R Mixed Pharmacology (Antagonist/Partial Ago-

nist) Ligands. Compound 11, containing the DBip amino acid

in place of the DPhe residue, resulted in mixed pharmacology

CAMM studies resulted in the observation that the ligand 2 was

actually more structurally ﬂexible as compared to 1.

3

9

39

Consistent with our previous study, the lead ligand 2 resulted

in multiple observed conformational families that were not

highly populated. The most populated family was observed to

possess 21% of the sampled conformers, whereas the other

three remaining families contained approximately 5% of the

sampled structures. In contrast, three of the ﬁve DPhe

substituted ligands (8, 10, and 11) were observed to possess

either a β- or a γ- reverse turn secondary structure. The ﬁve

analogues 6, 8, 9, 10, and 11 were observed to cluster into one

major structural family, in contrast to the control ligand 2.

These results are consistent with previous biophysical studies

on various potent melanocortin peptide ligands that identiﬁed a

reverse turn structure in His-DPhe-Arg-Trp region of the

with antagonist (pA = 9.9) and slight partial agonist activity at

2

the mMC3R, while retaining full agonist potency at the

mMC4R (∼700 nM) (Figure 3). This compound is more than

5800-fold selective at the MC3R versus the MC4R and was

characterized to possess an inverse γ-turn involving the residues

8

9

10

Asn , Ala , and Phe in this study (Figure 4). A γ-turn was

previously reported in the peptide KAR template and

postulated to explain the diﬀerence between the mMC3R and

37

mMC4R pharmacological proﬁles. In the chimeric KAR

peptide template, the Phe substitution for the His residue,

resulted in mMC3R antagonist/partial agonist activity and full

agonist activity at the mMC4R. Upon the basis of the NMR

biophysical studies, the Phe containing KAR derivative was

8

7−90

ligands.

NMR and Modeling Based Structural Features of

Agonist ligands. Compound 8, which contains a bulkier

DNal(1′) residue in place of DPhe, is a full agonist at mMC1R

and mMC3−5R and is equipotent to the control template 2 at

all four melanocortin receptor subtypes. This analogue was

characterized as possessing a β-turn involving the His and

DNal(1′) residues. The chimeric peptide KAR compound was

reported to possess a β-turn involving the Trp and Asn

residues. NMR studies of melanocortin ligands have shown

that a similar type of reverse β-turn may also present in the

MTII agonist ligand around the His-DPhe-Arg-Trp message

sequence. Compound 8 was identiﬁed to possess an

amphiphilic side chain orientation (consistent with observa-

tions for other melanocortin analogues), in which the

hydrophobic pharmacophore residues are oriented toward

one plane which is diﬀerent for the other hydrophilic amino

37

postulated to contain a γ-turn secondary structure. Since the

present template originates from the same chimeric template, it

can be postulated that the γ-turn secondary structure is an

important ligand structural feature that may diﬀerentiate

between mMC3R antagonist and mMC4R agonist pharmacol-

ogy. However, the second compound 9 with substitution of the

DNal(2′) side chain instead of the DPhe side chain also

resulted in a similar MC3R/MC4R pharmacological proﬁle but

did not result in the characterization of a “classical” turn in the

pharmacophore region from these biophysical experiments.

Both compounds 9 and 11 were observed to be generally more

ﬂexible than the other compounds from the biophysical

experiments and are full agonist at MC1R, MC3−5R,

3

4

6

7

3

7

8

37

consistent with the earlier study. The similar substitution in

chimeric KAR peptide template, lacking the heterocyclic ring,

resulted in a potent antagonist at the mMC3R and a partial

agonist and antagonist at mMC4R. The mixed pharmacology

was attributed to the equilibrium between two conformer

6

5,88

acids.

Compound 10 with (pI)DPhe in place of DPhe

resulted in ca. a 5-fold increased activity at all four of the

receptors and was characterized in this study to possesses a

classic γ-turn with the (pI)DPhe residue at the center. Classic γ-

turns have been found to generally occur at the end of β-hairpin

structure where a hydrogen bond is formed between the

backbone carbonyl oxygen of one residue (i) the backbone

amide NH of the residue two positions further along the chain

37

families. The solution structure of synthetic MC3R/MC4R

antagonist SHU9119 was reported to have type II β-turn, and

6

7

the orientation of the His and DNal(2′) residues resulted in

88

no observed aromatic stacking between these two side chains.

(

i + 2). The classic γ-turn, less common than the abundant

The reduced potency or antagonistic activity of the SHU9119

inverse γ-turn, and is diﬀerentiated only by inversion of the sign

of φ and ψ angles. For the classic γ-turn, the idealized values are

φ+1 ∼ 70.0 to 85.0, ψ+1 ∼ −60.0 to −70.0, whereas for an

inverse γ-turn they are φ+1 ∼ −70.0 to −85.0, ψ+1 ∼ 60.0 to

was postulated to be the diﬀerence in the side-chain

88

dispositions of His and DNal(2′). In the present template,

as anticipated, introduction of the 10-membered heterocyclic

ring around the ligand pharmacophore region changed the

pharmacology and structure of compound 9. Unexpectedly 9,

with the bulky aromatic DNal(2′) side chain in place of the

DPhe residue, resulted in a full agonist at the mMC4R.

Incorporation of the DNal(2′) residue at the DPhe position in

other templates resulted in competitive antagonists at the

MC4R. Thus, these unanticipated data for 9 demonstrate that it

has a diﬀerent molecular interaction with the mMC4R receptor

versus other templates possessing the DNal(2′) substitution at

the DPhe position. These ﬁndings support the previous

observation that the substitution of Phe by bulky aromatic

groups could be decisive in maintaining the MC4R selectivity

by enhancing hydrophobic interactions. The partial agonist/

antagonist proﬁle of 9 and 11 can be attributed to a more

ﬂexible backbone of these two compounds, as discussed above,

that can facilitate binding of the ligands more easily in the

mMC3R binding pocket as compared with other mMC3R

5

7

70.0.

Compound 6 with the Pro residue substituted for DPhe

resulted in 24−2000-fold decreased agonist potency at all the

melanocortin receptors examined in this study. Although the

backbone atoms around the pharmacophore (His-Pro-Arg-Trp)

region were observed to possess a “bend” conformation, the ϕ

−

ψ angles were not consistent with any conventional turn

structure. This result is consistent with a previous report where

the Pro amino acid was substituted for the His residue in an

7

9,91

MTII analogue.

The backbone heavy atoms for conformers

from the major family of 6 are similar, indicating a structured

domain, with the root-mean-square (RMS) superposition

deviations for these conformers of 0.78 Å. As a result of the

diﬀerent backbone conformations in all the three compounds

(6, 8, and 10), the putative side chains of the reference

sequence His-Phe-Arg-Trp were observed to be oriented

diﬀerently. Speciﬁcally, the Arg side chain of compound 6

88

agonist compounds.

L

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equiv of BOP and 4.4 equiv of DIEA for 30 min at rt. Then 3.3 equiv

of NH(Me)OMe and 3.3 equiv of DIEA were added and mixed at rt

CONCLUSIONS

■

This study reports SAR experiments of a hybrid heterocyclic-

peptide template by substituting the putative melanocortin

receptor peptide agonist pharmacophore (His-DPhe-Arg-Trp)

residues to modify the pharmacological proﬁle of a lead

compound. The systematic substitution of amino acids with

natural and unnatural amino acid residues (based on knowledge

of earlier studies) results in novel pharmacological proﬁle(s) at

the mouse melanocortin receptors. The most notable results

from this study include the identiﬁcation of 11, a 5800-fold

mMC3R antagonist/slight partial agonist versus the mMC4R

full agonist pharmacological proﬁle. This ligand is also a potent

nanomolar full agonist at the mMC1R that is expressed in the

skin. Biophysical studies identiﬁed the presence of an inverse γ-

turn in the ligand pharmacophore domain of ligand 11. This

SAR presented herein will increase the general knowledge base

of ligand-melanocortin receptor interactions as well as provide

novel molecules to test in in vivo models. The conformational

studies presented herein identify the structural diﬀerences

between agonist and antagonist activity at the MC3/MC4

receptors.

for 3 h. Subsequently, 2 equiv of LiAlH in THF was stirred at −78 °C

4

for 1−2 h until the reaction was complete, followed by quenching with

water. The thioether ring closure was performed at elevated

temperatures (55−60 °C) in an automated synthesizer (Advanced

ChemTech 440MOS, Louisville, KY). After the completed synthesis,

the peptides were cleaved from the resin and deprotected using a

cleavage cocktail consisting of 91% TFA, 3.0% H O, 3.0% EDT, and

2

3

.0% TIS for 3 h at room temperature. After cleavage and side chain

deprotection, the solution was concentrated and the peptide was

precipitated and washed using cold (4 °C) anhydrous diethyl ether.

The crude linear peptides were dissolved in 20% DMSO in water

(1.0 mg/mL) and stirred at room temperature. Progress for the

disulﬁde cyclization was monitored by UV-HPLC which was generally

completed within 24−36 h. The resulting solution was lyophilized to

yield the crude cyclic peptide and puriﬁed by reversed-phase HPLC

[ﬂow rate of 5 mL/min and gradients ranging (10−35 min) from 17−

4

5% acetonitrile/water 0.1% TFA] using a Shimadzu chromatography

system with a photodiode array detector and a semi preparative RP-

HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 × 25 cm).

The puriﬁed peptides were at least >95% pure as determined by RP-

HPLC in two diverse solvent systems and had the correct molecular

mass (Supporting Information). Matrix-assisted laser desorption/

ionization-time of ﬂight (MALDI-TOF) mass spectrometry spectra

were recorded on a Voyager instrument (University of Florida Protein

Core Facility), using α-cyano-4-hydroxycinnamic acid as the matrix.

Solid Phase Peptide Synthesis using microwave irradiation.

The Rink-amide MBHA resin was transferred into a 25 mL

polypropylene reaction vessel (CEM) and “swelled” in dichloro-

methane for 1 h. The bottom cap of the vessel was removed after the 1

h swelling process and the vessel was transferred onto a vacuum

ﬁltration manifold. Deprotection of the Fmoc group was achieved in

EXPERIMENTAL SECTION

Reagents and General Methods. The peptide analogue 1 was

synthesized using standard Fmoc methodology

reaction vessel. The amino acids Nα-9-ﬂuorenylmethoxycarbonyl

Fmoc)-Cys(Trt), Fmoc-Tyr(tBu), Fmoc-Arg(Pbf), Fmoc-His(Trt),

Fmoc-Phe, Fmoc-Asn(Trt), Fmoc-Ala, Fmoc-(1-naphthyl)-D-alanine

■

9

2,93

in a manual

(

[

DNal(1′)], Fmoc-(2-naphthyl)-D-alanine [DNal(2′)], Fmoc-(2-naph-

thyl)-L-alanine [Nal(2′)], and the coupling reagents: benzotriazol-1-yl-

2

0% piperidine in DMF by irradiating the sample at 75 °C, 30 W for 4

N-oxy-tris-dimethylamino)phosphonium-hexaﬂuoro-phosphate

(

BOP), O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexaﬂuoro-

min in the CEM Discover SPS instrument. After cooling and washing

94

phosphate (HBTU), and 1-hydroxybenzotriazole (HOBt) were

purchased from Peptides International (Louisville, KY). The amino

acids Fmoc-p-iodo-D-phenylalanine [(pI)DPhe], Fmoc-4-phenyl-D-

phenylalanine (DBip), Fmoc-4-phenyl−phenylalanine (Bip) were

purchased from Synthetech (Albany, OR). The amino acid Fmoc-

of the resin with DMF, a ninhydrin “Kaiser” test was performed. The

amino acid coupling step was performed under the same microwave

conditions (75 °C, 30 W) for 5 min using HBTU. The coupling of the

Cys and His amino acids was performed at lower temperatures (50 °C,

30 W) for 5 min. The heterocycle ring was assembled as previously

3

9

hArg(Boc) -OH was purchased from Advanced Chem Tech (Louis-

described. The reductive alkylation, acylation of chloroacetic

anhydride, and the deprotection of the thio tert-butyl group steps

were performed at the room temperature. The thioether bridge was

formed using N-ethylmorpholine in DMF and the microwave

conditions of (55 °C, 50 W) for 10 min. The reagents were mixed

by bubbling nitrogen gas into the reaction vessel. The iterative process

of the Fmoc deprotection/coupling cycle was performed to elongate

the peptide chain. After the ﬁnal deprotection, the peptide was cleaved

2

ville, KY) and Fmoc-Arg(Boc) -OH was purchased from Bachem

Torrance, CA). Rink amide p-methylbenzhydrylamine resin (p-

2

(

MBHA Resin, 0.43 mequiv/g substitution) was purchased from

Peptides International (Louisville, KY). Glacial acetic acid (HOAc),

dichloromethane (DCM), methanol (MeOH), acetonitrile (MeCN),

and anhydrous ethyl ether were purchased from Fisher (Fair Lawn,

NJ). N,N-Dimethylformamide (DMF) was purchased from Burdick

and Jackson (McGaw Park, IL). Triﬂuoroacetic acid (TFA),

piperidine, N,N-diisopropylethylamine (DIEA), N,O-dimethylhydrox-

ylamine, lithium aluminum hydride (LiAlH ), 1,2-ethanedithiol

EDT), triisopropylsilane (TIS) were purchased from Sigma-Aldrich

St. Louis, MO). All reagents and chemicals were ACS grade or better

from the resin using the cleavage cocktail (91% TFA, 3.0% H O, 3.0%

2

EDT, and 3.0% TIS) at room temperature for 3 h. Note, as the

peptides containing the heterocyclic moiety were synthesized to

completion on resin, if racemization occurred during the isolated

4

(

3

5

heterocycle formation as previously reported, it was not identiﬁed or

and were used without further puriﬁcation.

puriﬁed in the major product peak collected by RP-HPLC.

The modiﬁed peptides were synthesized using standard Fmoc-solid

phase methodology at room temperature using manual reaction vessel

Functional Bioassay. The HEK-293 cells stably expressing the

mouse melanocortin receptors were maintained in Dulbecco’s

modiﬁed Eagle’s medium (DMEM) with 10% fetal calf serum and

transfected with 4 μg of the CRE/β-galactosidase reporter gene as

(ﬂat-bottom polyethylene syringes equipped with sintered Teﬂon

ﬁlters, Teﬂon valves for ﬂow control, and suction to drain the syringes

from below) at room temperature or in a microwave (Scheme 1).

Manual amino acid couplings consisted of the following steps: (i)

removal of the Nα-Fmoc group by 20% piperidine in DMF (1 × 2

min, 1 × 18 min) and (ii) a single 2 h coupling of Fmoc-amino acid (3

equiv) using BOP (3 equiv), HOBt (3 equiv), and DIEA (6 equiv) in

DMF. The presence or absence of the Nα free amino group was

4

0

previously described. Brieﬂy, 5000−15000 post-transfection cells

were plated into collagen treated 96-well plates (Nunc) and incubated

overnight. Forty-eight hours post-transfection the cells were stimulated

−4

−12

−4

with 100 μL of peptide (10 to 10

M) or forskolin (10 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

94

monitored using the Kaiser test.

The thioether ring moiety was assembled on the resin according to

3

5,39

the general procedure previously reported.

The Fmoc-amino

aldehydes were used directly without further puriﬁcation in the next

39

step as previously reported. Brieﬂy the Fmoc-amino aldehydes were

prepared by using 3 equiv of the Fmoc amino acid reacted with 3.3

M

dx.doi.org/10.1021/jm301253y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

added to each well. Subsequently, 150 μL of substrate buﬀer (60 mM

ASSOCIATED CONTENT

■

sodium phosphate, 1 mM MgCl , 10 mM KCl, 5 mM β-

2

*

S

Supporting Information

mercaptoethanol, 2 mg/mL ortho-nitrophenyl-β-galactoside

1

Table S1. H NMR and chemical shifts assignment (ppm) for

the selected analogues. Table S2. Analytical data of the

compounds synthesized in this study. Figure S1. Summary of

the NOE intensities from 400 ms NOESY data observed for

compounds 2, 6, and 8−11. Figure S2. Illustration of the

compound deviation from random coil values. Table S3.

Backbone dihedral angles of the representative structures

shown in Figure 4. This material is available free of charge

via the Internet at http://pubs.acs.org.

[

3

ONPG]) was added to each well and the plates were incubated at

7 °C. 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 OD₅₉₅was

measured on a 96-well plate reader (Molecular Devices). Data points

were normalized both to the relative protein content and nonreceptor

dependent forskolin stimulation. Maximal eﬃcacy was compared to

that observed for the NDP-MSH control peptide tested simulta-

neously on each 96-well plate. The antagonistic properties of the

compounds examined in this study were evaluated by their ability to

competitively displace the MTII agonist (Bachem) in a dose-

dependent manner, at up to 10 μM concentrations. The antagonist

AUTHOR INFORMATION

■

Corresponding Author

E-mail: chaskell@umn.edu; phone: 612-626-9262; fax: 612-

26-3114.

*

4

3

pA values were generated using the Schild analysis method.

2

6

Data Analysis. The agonist EC₅₀and antagonist pA₂values

Notes

represent the mean of duplicate wells performed in three or more

independent experiments. The EC₅₀and pA value estimates, and their

The authors declare no competing ﬁnancial interest.

2

associated standard errors, were determined by ﬁtting the data to a

nonlinear least-squares analysis using the PRISM software program

ACKNOWLEDGMENTS

■

(

v4.0, GraphPad Inc.). The results are not corrected for peptide

content.

NMR Spectroscopy. The ligand NMR samples were prepared (∼1

This work has been supported in part by NIH Grants

RO1DK064250 and R01DK091906.

mM) by dissolving 1.0 mg of the puriﬁed peptides in a 500 μL solution

of 90% (v/v) acetonitrile-d and 10% H O, with an adjusted pH of

ABBREVIATIONS USED

■

3

2

ACTH, adrenocorticotropin hormone; AGRP, Agouti-related

protein; ASIP, Agouti-signaling protein; CAMM, computer-

assisted molecular modeling; cAMP, cyclic 5′-adenosine

monophosphate; DCM, dichloromethane; DMF, N,N-dime-

thylformamide; DMS, dimethylsulﬁde; Fmoc, Nα-9-ﬂuorenyl-

methoxycarbonyl; GPCR, G protein coupled receptor; MC1R,

melanocortin-1 receptor; MC2R, melanocortin-2 receptor;

MC3R, melanocortin-3 receptor; MC4R, melanocortin-4

receptor; MC5R, melanocortin-5 receptor; MCR, melanocortin

receptor; MeOH, methanol; MSH, melanocyte stimulating

hormone; NOE, nuclear Overhauser eﬀect; POMC, proopio-

melanocortin; ϕ, phi; ψ, psi; SAR, structure−activity relation-

ship; SEM, standard error of the mean; TFA, triﬂuoroacetic

acid; TM, transmembrane; α-MSH, alpha-melanocyte stimulat-

ing hormone; β-MSH, beta-melanocyte stimulating hormone;

γ-MSH, gamma-melanocyte stimulating hormone; μM, micro-

molar; RMD, restrained molecular dynamics; NDP-MSH (4-

norleucine-7-D-phenylalanine), Ac-Ser-Tyr-Ser-Nle-Glu-His-

∼

3.0. The NMR data were collected at 34 °C with a Bruker Avance II

spectrometer operating at 600 MHz (using a cryoprobe) at the

Advanced Magnetic Resonance and Imaging Spectroscopy (AMRIS)

University of Florida facility. Standard proton TOCSY and NOESY

1

2

D H NMR data were collected, processed, and analyzed as described

3

7,39,95

previously.

Acetonitrile was used as an internal standard (1.94

ppm). The chemical shifts of each of the peptides in this study were

1

96

assigned using standard TOCSY and NOESY H-based strategies.

This approach utilizes TOCSY spectra to identify resonances within a

given amino acid and NOESY spectra to correlate one amino acid with

the next through interactions of the α and/or β protons of residue i

with the amide proton of residue i + 1.

Computer-Assisted Molecular Modeling (CAMM). Proton−

proton distances were calibrated using the well resolved methylene

protons of the His residue based on the relationship r = r ^×⁽^ηref^/

ref

1/6

η) , where r is the distance between atoms, η is the NOESY cross-

^p^e^a^k^v^o^l^u^m^e^,^rref ⁱ^s^t^h^e^kⁿ^o^wⁿ^dⁱ^s^t^aⁿ^c^e^,^aⁿ^d^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

DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH ; control peptide 1

2

(

1.8−5.0 Å) (Supporting Information). All conformational molecular

AMW3−130, Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-

modeling experiments were performed using the SYBYL v7.0 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, at a temperature of 500 K, and using

Cys]-Tyr-NH ; MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-

2

NH ; KAR, Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-

2

Dpr]-Tyr-NH₂

̈

the Tripos force ﬁeld and Gastaiger-Huckel partial atomic charges. The

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and energy minimized without restraints. Finally, all the NMR based

NOE restraints were included, and 10 ns RMD trajectories were

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spaced points along the dynamics trajectory were energy minimized,

analyzed, and grouped into conformational families. Cluster analysis

was performed by comparing the backbone phi and psi angles of the

His-DPhe-Arg-Trp amino acid ligand domain. This process identiﬁed

representative structures (lowest energy conformers) of the conforma-

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dx.doi.org/10.1021/jm301253y | J. Med. Chem. XXXX, XXX, XXX−XXX

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Structure-activity relationships of peptides incorporating a bioactive reverse-turn heterocycle at the melanocortin receptors: Identification of a 5800-fold mouse melanocortin-3 receptor (mMC3R) selective antagonist/partial agonist versus the mouse melanocortin-4 receptor (mMC4R)

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