Melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-N?b modified at the para position of the benzyl side chain (DPhe): Importance for mouse melanocortin-3 receptor agonist versus antagonist activity

Article abstract of DOI:10.1021/jm800291b

The melanocortin-3 and -4 receptors (MC3R, MC4R) have been implicated in energy homeostasis and obesity. Whereas the physiological role of the MC4R is extensively studied, little is known about the MC3R. One caveat is the limited availability of ligands that are selective for the MC3R. Previous studies identified Ac-His-DPhe(p-I)-Arg-Trp-NH₂, which possessed partial agonist/antagonist pharmacology at the mMC3R while retaining full nanomolar agonist pharmacology at the mMC4R. These data allowed for the hypothesis that the DPhe position in melanocortin tetrapeptides can be used to examine ligand side-chain determinants important for differentiation of mMC3R agonist versus antagonist activity. A series of 15 DPhe⁷ modified Ac-His-DPhe ⁷-Arg-Trp-NH₂ tetrapeptides has been synthesized and pharmacologically characterized. Most notable results include the identification of modifications that resulted in potent antagonists/partial agonists at the mMC3R and full, potent agonists at the mMC4R. These SAR studies provide experimental evidence that the molecular mechanism of antagonism at the mMC3R differentiates this subtype from the mMC4R.

Full text of DOI:10.1021/jm800291b

J. Med. Chem. 2008, 51, 5585–5593
5585
Melanocortin Tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 Modified at the Para Position of the
Benzyl Side Chain (DPhe): Importance for Mouse Melanocortin-3 Receptor Agonist versus
Antagonist Activity
Bettina Proneth,† Irina D. Pogozheva,§ Federico P. Portillo,† Henry I. Mosberg,§ and Carrie Haskell-Luevano*,†
Department of Pharmacodynamics, UniVersity of Florida, GainesVille, Florida 32610, and Department of Medicinal Chemistry, UniVersity of
Michigan, Ann Arbor, Michigan 48109
ReceiVed March 16, 2008
The melanocortin-3 and -4 receptors (MC3R, MC4R) have been implicated in energy homeostasis and
obesity. Whereas the physiological role of the MC4R is extensively studied, little is known about the MC3R.
One caveat is the limited availability of ligands that are selective for the MC3R. Previous studies identified
Ac-His-DPhe(p-I)-Arg-Trp-NH2, which possessed partial agonist/antagonist pharmacology at the mMC3R
while retaining full nanomolar agonist pharmacology at the mMC4R. These data allowed for the hypothesis
that the DPhe position in melanocortin tetrapeptides can be used to examine ligand side-chain determinants
important for differentiation of mMC3R agonist versus antagonist activity. A series of 15 DPhe7 modified
Ac-His-DPhe7-Arg-Trp-NH2 tetrapeptides has been synthesized and pharmacologically characterized. Most
notable results include the identification of modifications that resulted in potent antagonists/partial agonists
at the mMC3R and full, potent agonists at the mMC4R. These SAR studies provide experimental evidence
that the molecular mechanism of antagonism at the mMC3R differentiates this subtype from the mMC4R.
Introduction
decreased lean body mass.11,20,21 Extensive studies have been
carried out to understand the physiological role of the MC4R,
but little is known about the MC3R. To date, only a limited
number of ligands are available that are 100-200-fold selective
for the MC3R over the MC4R subtype.22-24 However, for the
use of compounds as suitable in vivo tools, a higher difference
in potency (>500-fold) between the receptor subtypes is
desirable. The discovery of new selective MC3R ligands will
ultimately help in investigating the vast array of physiological
functions of this receptor subtype and contribute to the discovery
of new antiobesity agents.
The melanocortin receptors have been implicated in various
physiological functions, including pigmentation,1-4 inflamma-
tion,5 adrenal function,2 cardiovascular function,6-8 and energy
homeostasis in humans and rodents.9-11 To date, five melano-
cortin receptors (MC1R-MC5R) have been cloned that belong
to the super family of G protein coupled receptors (GPCRs)
and stimulate the cyclic AMP (cAMP) signal transduction
pathway. The peptides, R-melanocyte-stimulating hormone (R-
MSH),a ꢀ-MSH, γ-MSH, and ACTH are the endogenous
agonists of these receptors. These ligands are derived through
posttranslational processing of the precursor hormone pro-
opiomelanocortin (POMC)12,13 and contain a common core
sequence, His-Phe-Arg-Trp (residues 6-9 in R-MSH), postu-
lated to be important for melanocortin ligand-receptor recogni-
tion and stimulation.14,15
The centrally located melanocortin-3 and -4 receptors (MC3R
and MC4R)16-19 have been identified in knockout mice to be
involved in energy homeostasis, obesity,and metabolism11,20,21
and are potential drug targets for the treatment of obesity and
related diseases. Whereas the MC4R knockout mice are
extremely obese and hyperphagic, the MC3R knockout mice
possess normal body weight with increased fat mass and
Previous structure-activity relationship (SAR) studies, modi-
fying the DPhe7 of melanocortin ligands (R-MSH numbering)
such as 1 (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2),25
identified the MC3R/MC4R antagonists 2 (SHU9119, Ac-Nle-
c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2),26 3 (SHU8914, Ac-
Nle-c[Asp-His-DPhe(p-I)-Arg-Trp-Lys]-NH2),26 and 4 [SHU9005,
Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe(p-I)-Arg-Trp-Gly-Lys-Pro-
Val-NH2).27 This led to the hypothesis that bulky substituents
at the ligand DPhe7 position may be responsible for antagonism
at the MC3 and MC4 receptors.26 Further SAR studies of the
tetrapeptide template Ac-His-DPhe-Arg-Trp-NH2 led to the
discovery of Ac-His-DNal(2′)-Arg-Trp-NH2 that, as anticipated,
resulted in a melanocortin receptor pharmacological profile
similar to that of 2, albeit with decreased potency.28 Surprisingly,
modification at the DPhe position with pI, similar to 4, resulted
in the tetrapeptide Ac-His-DPhe(p-I)-Arg-Trp-NH2 that pos-
sessed “mixed pharmacology” with antagonist/partial agonist
activity at the mMC3R while retaining full nanomolar agonist
potency and efficacy at the mMC4R.28 The difference in
pharmacology of the DNal(2′)7- and DPhe(p-I)7-containing
tetrapeptide ligands suggests that (i) these compounds may cause
mMC3R antagonism by distinct mechanisms and (ii) these
ligands may be used to investigate the determinants that
discriminate between the MC3 and MC4 receptor subtypes.
* Author to whom correspondence should be addressed [telephone (352)
273 7717; fax (352) 273-7723; e-mail carrie@cop.ufl.edu].
†
University of Florida.
University of Michigan.
§
a Abbreviations: ACTH, adrenocorticotropic hormone; AGRP, Agouti-
related protein; BAL, backbone amide linker resin; cAMP, cyclic 5′-
adenosine monophosphate; DCM, dichloromethane; DMF, N,N-dimethyl-
formamide; DMS, dimethyl sulfide; GPCR, G protein coupled receptor;
MC1R, melanocortin-1 receptor; MC2R, melanocortin-2 receptor; MC3R,
melanocortin-3 receptor; MC4R, melanocortin-4 receptor; MC5R, melano-
cortin-5 receptor; MCR, melanocortin receptor; MeOH, methanol; MSH,
melanocyte-stimulating hormone; nM, nanomolar; POMC, proopiomelano-
cortin; SAR, structure-activity relationship; SEM, standard error of the
mean; TFA, trifluoroacetic acid; R-MSH, R-melanocyte-stimulating hor-
mone; ꢀ-MSH, ꢀ-melanocyte-stimulating hormone; γ-MSH, γ-melanocyte-
stimulating hormone; µM, micromolar.
The study presented herein employs ligand-receptor SAR
studies in combination with computational methods to inves-
10.1021/jm800291b CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

5586 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Proneth et al.
Figure 1. Structure of amino acid building blocks used in this study to replace DPhe in the peptide template Ac-His-Xaa-Arg-Trp-NH2.
tigate (i) the molecular mechanism by which substitution of the
DPhe phenyl ring at the para position with iodine causes this
unique mixed mouse MC3R/MC4R pharmacological profile and
(ii) the substructural determinants underlying receptor selectivity
and differentiation of agonist versus antagonist activity at the
mMC3R.
hydrogen-bond accepting groups (p-CN in peptide 11 or p-NO2
in peptide 12) results in similar or moderately less (10-fold)
reduced potency. On the other hand, incorporation of the small
hydrophobic p-CH3 group in peptide 8 is well tolerated by all
receptor subtypes, whereas introduction of bulky hydrophobic
groups (p-tBu in peptide 9 and p-Bz in peptide 12) decreases
ligand potency for all receptor subtypes, except mMC1R. The
presence of p-tBu group is much more detrimental, especially
for activation of mMC3R, than p-Bz.
Results
Peptide SAR Studies. The amino acids that were used for
substitution at the DPhe position in the tetrapeptide template
Ac-His-DPhe-Arg-Trp-NH2 are illustrated in Figure 1. These
substituents span a diverse range of electronic and polarity
properties and sizes to allow a thorough assessment of the effect
of the substituent physicochemical properties on melanocortin
receptor functional activity.
Effects of halogens included in the tetrapeptide ligands are
more interesting. The incorporation of the smaller fluorine,
chlorine, and bromine substituents at the para position of DPhe
slightly improved ligand potency for mMC1R, mMC4R, and
mMC5R (p-F in peptide 13, p-Cl in peptide 14 or p-Br in peptide
15), whereas the larger p-I substituent in peptide 16 and the
p-CF3 in peptide 17 or a chlorine substituent in the meta position
(3,4-diCl in peptide 19 and m-Cl in peptide 20) slightly
decreases ligand potency for these receptor subtypes. The effect
on functional activity of the mMC3R by halogen-substituted
tetrapeptide ligands is quite different. Whereas the peptides with
fluorine (peptide 13) and chlorine (peptide 14) substituents at
the para position of DPhe have slightly improved agonist
potency for mMC3R, the peptides with larger size halogens or
-CF3 at the para position (peptide 15-17) or with chlorine in
the meta position (peptide 19) function as partial agonist/
antagonists or as very poor agonists [peptide 20 with DPhe(m-
Cl)]. As shown in Figure 2, both the bromine-containing peptide
15 and the iodine-containing peptide 16 possess partial agonist
activity in addition to competitive antagonism at the mMC3R.
Comparison of halogen-containing tetrapeptides revealed that
peptide 13 with DPhe(p-F) was the most potent tetrapeptide
agonist for mMC3R (EC50 at 53 nM), whereas peptides 16 with
DPhe(p-I) and 19 with DPhe(3,4-diCl) were the most potent
tetrapeptide antagonists for the mMC3R with Ki ) 260 nM
(Table 1; Figure 2).
As summarized in Table 1, compounds synthesized in this
study were pharmacologically characterized at the mouse MC1
(mMC1R) and MC3-5 (mMC3R-mMC5R) receptors because
it was hypothesized that different substituents at the DPhe phenyl
ring might lead to distinct potency and/or functional activity at
the different receptor subtypes. The mMC2R was not included
because it can be stimulated only by ACTH and not by the
tetrapeptides examined in this study.2 The tetrapeptides Ac-His-
DPhe-Arg-Trp-NH2 (5), Ac-His-DTyr-Arg-Trp-NH2 (6), Ac-
His-DPhe(p-I)-Arg-Trp-NH2 (16), and 18 [JRH887-12, Ac-His-
DNal(2′)-Arg-Trp-NH2) have been previously reported28 and
have been included in this study as control compounds and to
complement SAR trends. Tetrapeptide 19 was designed to mimic
the para and meta DPhe substitution of the DNal(2′) group of
18, whereas analogue 20 was included to investigate single
substitution at the meta position. The table found in the
Supporting Information summarizes the analytical characteriza-
tion results for the tetrapeptides examined in this study.
As seen in Table 1, all new tetrapeptides described here are
1-2 orders of magnitude more potent at the mMC4R and the
mMC5R as compared to at the mMC1R and mMC3R. Thus,
although none of the new analogues is the desired highly potent
mMC3R-selective agonist, comparison of the functional proper-
ties of these DPhe-substituted tetrapeptides at different receptor
subtypes reveals several trends that suggest structural features
that underlie agonist versus antagonist activity at the mMC3R.
Incorporation of hydrophilic hydrogen-bond donating groups
(p-OH in peptide 6 and p-NH2 in peptide 7) results in decreases
of ligand potency of about 20-fold for mMC1R and ap-
proximately 100-fold for mMC3-5R receptor subtypes. In
contrast, the presence of polar electron-withdrawing and possible
To better characterize the antagonistic properties of halogen-
containing ligands, we compared them with the previously
reported tetrapeptide 18 containing a DNal(2′) substitution for
the DPhe residue. Peptide 18 displayed full agonist activity at
the mMC1R and mMC5R receptors with equipotency at the
mMC1R and 7-fold decreased potency at the mMC5R compared
to peptide 5, whereas it had partial agonist/antagonist activity
at the mMC3R (pA2 ) 6.7) and antagonist activity at the
mMC4R (pA2 ) 8.3).28 However, 18 was a much less potent

Melanocortin Tetrapeptide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5587

5588 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Proneth et al.
Figure 2. Illustration of melanocortin tetrapeptides displaying partial agonist and antagonist activity at the mouse MC3R and full agonist activity
at the mouse MC4R.
antagonist at the mMC3R than the halogen-containing tetrapep-
tides 15, 16, and 19 (Table 1; Figure 2).
hypothesis that the DPhe position can be exploited for improve-
ment of receptor selectivity for the mMC3R versus the mMC4R,
and for the differentiation of agonist versus antagonist activity
at the mMC3R. For this purpose, a series of functional groups
were chosen for substitution at the DPhe phenyl ring at the para
and meta positions. The resulting tetrapeptides were pharma-
cologically characterized at the cloned mouse melanocortin
MC1, MC3, MC4, and MC5 receptors (Table 1), the results of
Discussion
The study reported herein extends structure-activity relation-
ship studies of the tetrapeptide template Ac-His-DPhe-Arg-Trp-
NH2, in which DPhe was substituted by a series of natural and
unnatural aromatic amino acids.28 We continued to test the

Melanocortin Tetrapeptide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5589
which provide insight into aspects of ligand-receptor interaction
that result in either mMC3R stimulation or competitive
antagonism.
freedom of the benzyl ring and renders it less rigid as compared
to tert-butyl substitution. Tetrapeptide 18, which contains the
bulky and lipophilic DNal(2′) group substituted for the DPhe
amino acid, has been described previously.28 This peptide
behaves as a partial agonist/antagonist at the mMC3R and as
an antagonist at the mMC4R but retains full agonist activity at
the mMC1 and mMC5 receptors (Table 1). These data are
consistent with the effect of DNal(2′) substitution for DPhe in
the cyclic heptapeptide 1 that led to the hypothesis that bulky
aromatic amino acid substitutions at the DPhe7 are responsible
for differentiating agonist versus antagonist activities of mel-
anocortin ligands at the MC3 and MC4 receptors.26
Substitution with Hydrophilic Groups. Incorporation of
polar substitutions with different physiochemical properties at
the DPhe residue resulted in distinct pharmacological profiles
at the mouse melanocortin receptors (Table 1). Tetrapeptides 6
and 7 containing hydrophilic and hydrogen-bond donating
groups, p-OH and p-NH2, respectively, displayed large potency
drops at all melanocortin receptors studied. However, tetrapep-
tides 11 and 12 with hydrophilic and H-bond accepting groups,
p-CN and p-NO2, respectively, were equipotent to the parent
peptide (5) at the mMC1R and had only slightly decreased
agonist potencies in mMC3-5 receptors. Receptor mutagenesis
of the mouse and human MC1R, MC3R, and MC4R, in
combination with GPCR homology modeling of MC1R and
MC4R,27,29-37 identified a putative hydrophobic receptor pocket
consisting of aliphatic, aromatic, and sulfur-containing amino
acid residues from transmembrane helices 3-6 of melanocortin
receptors that are hypothesized to interact with the L/DPhe7
residue of melanocortin peptide ligands. The decrease in agonist
potency observed for peptides 6 and 7 as well as the previously
reported recovery of potency by using methoxy and ethoxy
substituents30 support the hypothesis that the ligand DPhe7
residue is interacting with a hydrophobic environment in the
receptor binding pocket. Furthermore, these data suggest that
this receptor site is likely devoid of polar residues that may
accept hydrogens from hydroxy or amino groups of the
tetrapeptides and thus participate in hydrogen-bond formation
between ligands and receptors. On the other hand, the better
tolerance of p-CN and p-NO2 groups from peptides 11 and 12,
respectively, may be explained by the presence of cysteines from
transmembrane helices 3-5 in the receptor binding pockets that
may serve as hydrogen donors forming hydrogen bonds with
these groups. However, this mechanism remains speculative and
should be experimentally verified.
Incorporation of Halogen Substituents. The effect of
halogen substitutents has been previously studied in linear and
cyclic melanocortin peptides of different sizes. The incorporation
of DPhe(p-I) into the 1 cyclic heptapeptide template led to the
potent hMC3R and hMC4R partial agonist/antagonist 3, which
possesses full agonist activity at the hMC1R and partial agonist
activity at the hMC5R.26 Similarly, incorporation of the DPhe(p-
I)7 into the linear 13-amino acid peptide NDP-MSH resulted in
4 with partial agonist and antagonist activity at the mMC3R
and mMC4R.27 However, 1 with DPhe(p-F) and DPhe(p-Cl)
substitutions retained agonist activity at all cloned human
melanocortin receptors.26 Furthermore, substitution of the DPhe
residue with DPhe(p-Cl) and DPhe(p-Br) in the linear pen-
tapeptide template Bu-His-DPhe-Arg-Trp-Gly-NH2 resulted in
full agonist activity at the hMC1 and hMC4 with similar potency
as the parent pentapeptide.38 On the other hand, a pentapeptide
with a dichloro-DPhe substitution, Bu-His- DPhe(3,4-diCl)-Arg-
Trp-Gly-NH2, showed enhanced binding affinity, but was devoid
of any stimulatory activity at up to 50 µM concentration.30
The results obtained for the series of halogenated tetrapeptides
studied herein are generally consistent with the previously
reported observations. Substitution with either fluorine or
chlorine at the para position of the DPhe residue (peptides 13
and 14) resulted in full agonists at the mouse MC1, MC4, and
MC5 receptors with slightly increased potency, as compared to
the parent peptide 5 (Table 1). Interestingly, upon substitution
with bromine, iodine, trifluoromethyl, or 3,4-dichloro in tet-
rapeptides 15, 16, 17, and 19, respectively, the ligands were
converted into partial agonists/antagonists only at mMC3R while
retaining agonist potencies similar to peptide 5 at the other
mouse melanocortin receptor subtypes (Table 1; Figure 2). The
bromine- and iodine-containing peptides displayed notable
partial agonist activity (up to 50% maximal response) in addition
to competitive antagonism at the mMC3R.
Substitution with Hydrophobic Aliphatic and Aromatic
Groups. Peptide 8, containing the small hydrophobic p-CH3
group in DPhe, retained equal potency and full agonist activity
at all melanocortin receptors, as compared to peptide 5 (Table
1). This observation is consistent with the existence of a putative
hydrophobic receptor binding pocket described above, which
may be large enough to tolerate small hydrophobic modifications
in DPhe. Introduction of the bulky p-tBu substituent at DPhe
in peptide 9 was well tolerated only at the mMC1R and resulted
in significantly reduced potency at the mMC4R and mMC5R,
with complete loss of stimulatory activity at the mMC3R at up
to 100 µM concentrations. These results may indicate that the
DPhe7 interaction site of the ligand binding pocket might be
more constrained in mMC3R, therefore tolerating less bulk than
in the other melanocortin receptor subtypes. The putative
mMC1R binding pocket, however, seems to be more flexible
for all of the tetrapeptides examined in this study. This is in
agreement with previously reported results of substitutions in
the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 by various natural
and unnatural aromatic amino acids,28 where all DPhe modifica-
tions resulted in full agonists at mMC1R, unlike at the other
mouse melanocortin receptor isoforms. Tetrapeptide 10 with
DPhe(p-Bz) resulted in high potency at the mMC1R, slightly
reduced potency at the mMC3R, and 28- and 16-fold losses in
potency at the mMC4 and mMC5 receptors, respectively (Table
1). The smaller changes in agonist activity of peptide 10
compared to 9 are postulated to be attributed to the methylene
linker of the benzyl group, which allows increased rotational
In tetrapeptide 19, the DPhe phenyl ring was substituted
simultaneously at the meta and para positions with chlorine.
This compound was designed (i) to investigate if a second
chlorine substituent, which might enhance the lipophilicity of
the molecule and/or decrease electron density of the phenyl ring,
is needed to confer antagonist activity at the mMC3R and (ii)
to mimic the DNal(2′) substitution of 18, which is an antagonist
at both mMC3 and mMC4 receptors.28 Similar to peptides
15-17, tetrapeptide 19 demonstrated partial agonist/antagonist
activity only at the mMC3R subtype, while retaining full and
equipotent agonist activity at all other mouse melanocortin
receptor subtypes (Table 1; Figure 2). To examine if this
antagonist activity at the mMC3R is due to a substitution or a
steric effect at the phenyl meta position, the DPhe(m-Cl)-
containing tetrapeptide 20 was synthesized and characterized.
This peptide, however, resulted in a full agonist at all melano-
cortin receptors, with slightly decreased potency at the mMC3R
and mMC5R receptors and almost equipotency at the mMC1R

5590 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Proneth et al.
the delocalized π-electrons of peptide bonds of carboxy and
amide groups.39 The magnitude of these interactions increases
as Cl < Br < I, whereas fluorine is usually not involved.39
Therefore, the antagonism of the CF3-containing tetrapeptide
17 cannot be primarily attributed to charge transfer interactions,
but may be largely related to its lipophilic properties.
All of the halogen-containing compounds, including tetrapep-
tide 17, possess high lipophilicity, which increases as F < Cl
< Br < CF3 < I < 3,4-diCl. These parameters might be
important considering the fact that the DPhe phenyl ring
putatively interacts with aromatic and hydrophobic receptor
amino acids in the melanocortin receptor binding pocket. On
the other hand, it is commonly thought that van der Waals free
energies of molecules are proportional to the polarizabilities.40
It has been shown that the electronic polarizability is relatively
high for sulfur- and halogen-containing groups and increases
as F < Cl < S < Br< I.41 Thus, the increased hydrophobicity
of halogen-containing compounds together with stronger disper-
sion attractions between halogens (particularly -Br and -I) and
sulfur-containing receptor residues from transmembrane helices
3-5 may likely play the dominant role in the stabilization of
the tightly packed inactive receptor conformation. The unique
pharmacological profile of the mMC3R for halogen-containing
peptides may be related with tighter packing of residues within
the mMC3R binding pocket in comparison with other subtypes
of mouse melanocortin receptors. Indeed, peptide 9 containing
the bulky hydrophobic substituent p-tBu was devoid of stimula-
tion activity only at mMC3R, suggesting a more constrained
geometry of its ligand binding pocket.
The mechanism of antagonism of halogen-containing tet-
rapeptides at MC3R proposed here differs from the steric
hindrance mechanism previously suggested to explain the MC4R
antagonism of bulky DNal(2′)-containing analogues.25 This
steric mechanism was proposed on the basis of molecular
modeling and mutagenesis studies of hMC4R, which indicated
that DNal(2′) might hinder side-chain rotation of conserved
Trp258 from the transmembrane helix 6 and Leu133 from the
transmembrane helix 3 (hMC4R numbering), locking the
receptor in the inactive state.37,47
Figure 3. Electrostatic surface representation of tetrapeptide agonist
Ac-His-(pI)DPhe-Arg-Trp-NH2 analogue 14 (A) and antagonists tet-
rapeptides 15-19 (B-F) of MC3 receptor colored by calculated charge
from red (-15kT/e) to blue (+15kT/e) using the dielectric constant of
10. Poisson-Boltzmann electrostatic was calculated using the APBS
program implemented in PyMOL.
and mMC4R receptors. It can be inferred that double substitution
of the phenyl ring is necessary for antagonist activity at the
mMC3R for the chlorine-containing tetrapeptides.
It is of note that the p-I-substituted tetrapeptide Ac-His-
DPhe(pI)-Arg-Trp-NH2 (16) resulted in a new pharmacological
profile and possessed partial agonist/antagonist activity only at
the mMC3R while retaining full agonist activity at nanomolar
concentrations at the centrally expressed mMC4R as well as
the peripherally expressed mMC1R and mMC5R receptors.28
The difference in pharmacology of the tetrapeptide in compari-
son with 3 and 4 may be attributed to the additional amino acids
at the N and C termini in the larger peptides that may add
ligand-receptor contact points, modify the peptide secondary
structure, and/or constrain the ligand core sequence “His-Phe-
Arg-Trp” within the receptor binding pocket. Therefore, further
systematic modifications of a pharmacophore tetrapeptide are
essential to separate structural requirements imposed solely on
the core sequence of melanocortin peptides. This knowledge
would facilitate the development of small-molecule peptidomi-
metics with greater drug potential.
To understand whether the pharmacological properties of the
ligands are related to their electrostatic interactions with
receptors, we analyzed the distribution of atomic charges on
the ligand surface (Figure 3). Comparison of electrostatic
surfaces for the tetrapeptide agonists and antagonists indicates
that there is no direct correlation between the atomic charges
of halogen-containing compounds and their antagonistic proper-
ties [compare agonist 14 (Figure 3A) with antagonists 16 and
19 (Figure 3C,E)]. Thus, we suggest that antagonism of bromo-,
iodo-, dichloro- and trifluoromethyl-containing tetrapeptides at
mMC3R may be attributed to a combination of hydrophobicity
and van der Waals interactions with surrounding receptor atoms
from the putative hydrophobic ligand binding pocket. Other
mechanisms originating from electron-withdrawing properties
of halogens and their ability to form charge transfer complexes
may also contribute, but to a lesser extent. It is known that in
charge transfer complexes halogens serve as acceptors and
interact with a donor by transferring electronic charge.39 These
interactions may occur in biological systems between halogens
and the π-electron clouds of benzene rings as well as between
halogens and the lone pair of oxygen, nitrogen, and sulfur and
Melanocortin Receptor Selectivity. The study presented here
provides a better understanding that substitution at the ligand
DPhe phenyl ring is a major contributor in switching receptor
function of the mMC3R. Furthermore, modifications of DPhe
also result in slight changes in receptor subtype specificities.
The unsubstituted parent tetrapeptide 5 used in this study was
50-60-fold selective for the mMC4 and mMC5 receptors over
the mMC1 and mMC3 receptors (Table 1). The same pattern
of receptor selectivity was maintained for most of the ligands
characterized in this study (Table 1). Substitution with the
hydrophilic electron-donating groups (-OH and -NH2) did not
result in substantial selectivity changes between the receptor
subtypes. This observation may be consistent with the hypothesis
that all melanocortin receptors possess a common hydrophobic
ligand binding site that interacts with the DPhe residue of
peptide ligands.27,29-37
Interestingly, substitution with the hydrophilic electron-
withdrawing groups (-NO2 and -CN) resulted in increased
selectivity for mMC4R and mMC5R over the skin mMC1R. In
addition, incorporation of hydrophobic electron-withdrawing
halogens (-F and -Cl) at the para position of the DPhe phenyl
ring increased the selectivity of the peptides for the mMC4R
relative to the mMC1R and mMC3R by 200-fold. Substitution
with -Cl at the DPhe meta position increased selectivity by
140-fold only for the MC4R over the mMC3R. In summary,

Melanocortin Tetrapeptide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5591
substitutions at the DPhe phenyl ring led to slight changes in
receptor subtype specificities, and this knowledge is valuable
for further SAR studies of melanocortin ligands.
brought up to 5 mL using DMF. The coupling reaction was
mixed for 2 h at 450 rpm, followed by emptying of the reaction
block by vacuum. After the coupling cycle, the reaction block
was emptied and the NR-Fmoc-protected peptide resin was
washed with DMF (8 mL, three times). NR-Fmoc deprotection
was performed by the addition of 8 mL of 20% piperidine in
DMF and mixed for 2 min at 450 rpm followed by 18 min
deprotection at 450 rpm. The reaction well was washed with
DMF (8 mL, three times), and the next coupling cycle was
performed as described above. Following NR-Fmoc deprotection
of the final amino acid, acetylation of the NR amine was
performed by the addition of 4 mL of acetic anhydride and 2
mL of pyridine to the reaction block wells and mixed for 30
min at 450 rpm. The acetylated peptide resin was washed with
DCM (8 mL, five times) and dried thoroughly prior to cleavage
from the resin. Deprotection of the amino acid side chains and
cleavage of the acetylated-peptide-resin was performed with 8
mL of cleavage cocktail (95% TFA, 2.5% water, and 2.5% Tis)
for 2 h at 450 rpm. The cleavage product was emptied from
the reaction block into a cleavage block containing the collection
vials. The resin was washed with 1.5 mL of cleavage cocktail
for 5 min at 450 rpm and added to the previous cleavage
solution. The peptides was transferred to 50 mL conical tubes
and precipitated with cold (4 °C) anhydrous ethyl ether (up to
50 mL). The flocculent peptide was pelleted by centrifugation
(Sorval Super T21 high-speed centrifuge using the swinging
bucket rotor) at 4000 rpm for 5 min, the ether was decanted
off, and the peptide was washed one time with cold anhydrous
ethyl ether and again pelleted. The crude peptide was dried in
vacuo for 24 h. A 15-30 mg sample of crude peptide was
purified by RP-HPLC using a Shimadzu chromatography system
with a photodiode array detector and a semipreparative RP-
HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 cm
× 25 cm) and lyophilized. The purified peptides were analyti-
cally characterized by RP-HPLC using a two-solvent system
and mass spectrometry (University of Florida Protein Core
Facility). The Supporting Information shows the analytical data
of the tetrapeptides synthesized in this study.
Conclusions
By using a combination of tetrapeptide ligand SAR studies
and melanocortin receptor pharmacology, we have investigated
the requirements at the tetrapeptide DPhe7 position for the
differentiation of antagonist activity versus agonist activity at
the mMC3R. We propose the following hypothesis by which a
combination of van der Waals forces, charge transfer interac-
tions, and hydrophobicity might cause antagonism at the
mMC3R by halogen-containing tetrapeptides that appears to be
distinct from the DNal(2′)7-containing peptides. The results from
these studies highlight the differential receptor activation and
inactivation mechanism of the mMC3R versus the mMC4R and
may be valuable for future drug design aspects and ligand
substructural considerations for melanocortin receptor ligands.
Additionally, we report new compounds with mixed pharmacol-
ogy at the mouse MC3 and MC4 receptors (tetrapeptides 15,
17, and 19), which may be more metabolically stable and
therefore useful for in vivo studies to differentiate the physi-
ological roles of the MC3 and MC4 receptors.
Experimental Section
Peptide Synthesis. Peptide synthesis was performed using
standard Fmoc methodology42 on a semiautomated synthesizer
(LabTech, Louisville, KY). The amino acids Fmoc-Tyr(tBu),
Fmoc-His(Trt), Fmoc-DPhe, Fmoc-D-p-Cl-Phe-OH, Fmoc-D-
p-F-Phe-OH, Fmoc-D-p-Phe(NO2), and Fmoc-Trp(Boc) were
purchased from Peptides International (Louisville, KY). Fmoc-
p-amino-D-Phe(Boc)-OH, Fmoc-4-bromo-D-Phe-OH, Fmoc-p-
Me-D-Phe-OH, Fmoc-p-tBu-D-Phe-OH, Fmoc-4-cyano-D-Phe-
OH, Fmoc-p-Bz-D-Phe-OH, and Fmoc-(3,4diCl)DPhe-OH were
purchased from Bachem (Torrance, CA). Fmoc-4-iodo-D-
phenylalanine, Fmoc-D-3-Cl-Phe-OH, and Fmoc-D-4-trifluo-
romethylphenylalanine were purchased from Synthetech (Al-
bany, OR). The coupling reagents 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and
1-hydroxybenzotriazole (HOBt) were purchased from Peptides
International. Glacial acetic acid (HOAc), dichloromethane
(DCM), methanol (MeOH), acetonitrile (ACN), and anhydrous
ethyl ether were purchased from Fisher (Fair Lawn, NJ). N,N-
Dimethylformamide (DMF) was purchased from Burdick and
Jackson (McGaw Park, IL). Trifluoroacetic acid (TFA), pyridine,
piperidine, and acetic anhydride were purchased from Sigma
(St. Louis, MO). N,N-Diisopropylethylamine (DIEA) and tri-
isopropylsilane (Tis) were purchased from Aldrich (Milwaukee,
WI). All reagents and chemicals were of ACS grade or better
and were used without further purification.
The peptides were assembled on rink-amide-MBHA resin,
purchased from Peptides International. The synthesis was
performed using a 16-well Teflon reaction block. Approximately
200 mg of resin (0.1 mmol) was added to each reaction block
well. The resin was allowed to swell for 2 h in DCM and
deprotected using 20% piperidine in DMF for 2 min followed
by 18 min 20% piperidine incubation at 450 rpm. A positive
Kaiser test was performed indicating free amine groups on the
resin.43 The growing peptide chain was added to the amide-
resin using the general amino acid cycle as follows: a 3-fold
excess of amino acid (0.1 mmol scale) starting from the C
terminus was added, containing the coupling reagents HOBt
(0.1 mmol) and HBTU (0.1 mmol) in DMF. Subsequently, 90
µL of DIEA was added and the reaction well volume was
Functional Characterization at the Mouse Melanocortin
Receptors. For the cAMP response element (CRE)/ꢀ-galac-
tosidase reporter gene assays44 the mMC1 and MC3-5 receptor
sequences were cloned into the pCDNA3 mammalian expression
vector and stably expressed in HEK293 cells. Herby HEK293
cells were transfected with pCDNA3 expression vector contain-
ing the corresponding melanocortin receptor DNA (20 µg) using
the calcium phosphate method.45 Stable receptor populations
were generated using G418 selection method (1 mg/mL) for
the subsequent bioassay analysis. HEK293 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) with 10%
newborn calf serum and plated 1 day prior to transfection at
(1-2) × 10-6 cells/100 mm dish.
For the (CRE)/ꢀ-galactosidase reporter gene assay, HEK293
cells stably expressing the mouse MC1, or MC3-5 receptors
were transfected with 4 µg of CRE/ꢀ-galactosidase reporter gene
as previously described.28,44,46-48 Twenty-four hours after
transfection, 5000-15000 cells were plated into collagen-treated
96-well plates and incubated overnight. Forty-eight hours after
transfection, the cells were stimulated with 100 µL of peptide
synthesized (10-4-10-12 M) or forskolin (10-4 M) control in
assay medium (DMEM containing 0.1 mg/mL BSA and 0.1
mM isobutylmethylxanthine) for 6 h. The antagonistic properties
of these compounds were evaluated by the ability of these
ligands to competitively displace the 1 agonist in a dose-
dependent manner, at up to 10 µM concentrations. After

5592 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Proneth et al.
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stimulation, the assay medium 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. For
relative protein determination, aliquots of 10 µL were taken from
each well and transferred to another 96-well plate. 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,
and the OD595 was measured on a 96-well plate reader
(Molecular Devices). To the cell lysate plates was added 40
µL of phosphate-buffered saline with 0.5% BSA to each well.
Next, 150 µL of substrate buffer [60 mM sodium phosphate, 1
mM MgCl2, 10 mM KCl, 5 mM ꢀ-mercaptoethanol, 2 mg/mL
of o-nitrophenyl-ꢀ-D-galactopyranoside (ONPG)] was added to
each well, and the plates were incubated at 37 °C. The sample
absorbance, OD405, was measured using a 96-well plate reader
(Molecular Devices).
Data Analysis. Data points were normalized both to the
relative protein content and receptor independent forskolin
control values that indicate the maximal observable stimulation
levels in the different cell lines. The assays were performed
using duplicate data points and repeated in at least three
independent experiments. Data analysis, EC50 and pA2 estimates,
and their associated standard errors of the mean49 were
determined using the PRISM program (v4.0, GraphPad Inc.).
Antagonistic properties were determined by the ability of each
of those peptides to competitively displace the 1 agonist in a
dose-dependent manner. The pA2 values were generated using
the Schild analysis method.50
Visualization of Electrostatic Surfaces of Tetrapeptide
Ligands. Electrostatic properties of proteins and peptide ligands
can be visualized using the coloring of the solvent-accessible
molecularsurfacebyelectrostaticpotential.ThePoisson-Boltzmann
equation of electrostatics was calculated using the Adaptive
Poisson-Boltzmann Solver (APBS) algorithm implemented into
PyMOL.51 Partial atomic charges of unusual amino acids, such
as DPhe7 substitutents, which were not amenable to automatic
assignment by APBS algorithm, were assigned by QUANTA.
The dielectric constant ε ) 80 was used for the calculation of
the electrostatic potential of proteins, and the dielectric constant
ε ) 10 was used for the ligands.
Acknowledgment. This work was supported by NIH Grants
DK57080 (C.H.-L.) and DA03910 (H.I.M.) and an American
Diabetes Association Research Award (C.H.-L.).
Supporting Information Available: Table of analytical data for
the peptides synthesized in this study. This material is available
free of charge via the Internet at http://pubs.acs.org.
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