Incorporation of a bioactive reverse-turn heterocycle into a peptide template using solid-phase synthesis to probe melanocortin receptor selectivity and ligand conformations by 2D 1H NMR

Article abstract of DOI:10.1021/jm101425m

By use of a solid-phase synthetic approach, a bioactive reverse turn heterocycle was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R-MC5R) are G-protein-coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nanomolar potency. Biophysical structural analysis [2D ¹H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families compared to the reference peptide and result in a novel template for further structure-function studies.

Full text of DOI:10.1021/jm101425m

ARTICLE
pubs.acs.org/jmc
Incorporation of a Bioactive Reverse-Turn Heterocycle into a Peptide
Template Using Solid-Phase Synthesis To Probe Melanocortin
Receptor Selectivity and Ligand Conformations by 2D 1H NMR
Anamika Singh,† Andrzej Wilczynski,† Jerry R. Holder,† Rachel M. Witek,† Marvin L. Dirain,† Zhimin Xiang,†
Arthur S. Edison,‡ and Carrie Haskell-Luevano*,†
†Department of Pharmacodynamics and ‡Department of Biochemistry and Molecular Biology, University of Florida, Gainesville,
Florida 32610, United States
ABSTRACT: By use of a solid-phase synthetic approach, a bioactive reverse
turn heterocycle was incorporated into a cyclic peptide template to probe
melanocortin receptor potency and ligand structural conformations. The five
melanocortin receptor isoforms (MC1R-MC5R) are G-protein-coupled re-
ceptors (GPCRs) that are regulated by endogenous agonists and antagonists.
This pathway is involved in pigmentation, weight, and energy homeostasis.
Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide
template integrated with a small molecule moiety to probe the structural and
functional consequences of the core His-Phe-Arg-Trp peptide domain using a
reverse-turn heterocycle. A series of six compounds are reported that result in
inactive to full agonists with nanomolar potency. Biophysical structural analysis
[2D 1H NMR and computer-assisted molecular modeling (CAMM)] were
performed on selected analogues, resulting in the identification that these
peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families compared to the reference
peptide and result in a novel template for further structure-function studies.
’ INTRODUCTION
Table 1. Amino Acid Sequence of Endogenous Melanocortin
Agonists
The melanocortin pathway has been implicated in a variety of
physiological functions including the regulation of pigmentation,1
blood pressure and heart rate,2,3 erectile function,4,5 food intake,6
and obesity.7 The endogenous agonists for the five melanocortin
receptors (MC1R-MC5R),8-14 include R-, β-, γ-melanocyte sti-
mulating hormones (MSH) and adrenocorticotropin (ACTH)
that are derived from the post-translational modification of pro-
opiomelanocortin (POMC).15,16 Endogenous antagonists for the
melanocortin receptors have also been identified as agouti17 and
agouti-related protein (AGRP).18 The R-MSH peptide is
13 amino acids in length with an acetylated N-terminus and
amidated C-terminus. All of the melanocortin hormones have a
common core domain consisting of the His-Phe-Arg-Trp residues
postulated to be important for receptor molecular recognition and
stimulation (Table 1).19-21 The melanocortin receptors (MCRs)
belong to the family of seven transmembrane (TM) spanning
G-protein-coupled receptors (GPCRs) that stimulate the cAMP
signal transduction pathway. The MC1R is expressed in skin and is
involved in tanning and pigmentation.8,9 The MC2R only re-
sponds to stimulation by ACTH, is expressed in the adrenal gland,
and is involved in steroidogenesis.9 The MC3R is expressed in a
variety of tissues including the brain, placenta, heart, and gut and
participates in energy balance via a mechanism that remains to be
identified.10-12,22,23 The MC4R is expressed primarily in the brain
and regulates obesity, feeding behavior, and satiety,6,7 making it an
attractive target for antiobesity drugs. The MC5R is expressed in a
sequence
ACTH (1-24)
R-MSH
SYSMEHFRWGKPVGKKRRPVKVYP
Ac-SYSMEHFRWGKPV-NH2
β-MSH
AEKKDEGPYRMEHFRWGSPPKD-OH
YVMGHFRWDRFG-OH
γ2-MSH
wide variety of tissues and has been identified to be important for
exocrine gland function in mice,24 with its other physiological roles
remaining to be identified. The endogenous melanocortin recep-
tor antagonists agouti-signaling protein (ASP)17 and agouti-re-
lated protein (AGRP)18 are competitive antagonists at distinct
melanocortin receptors (Figure 1). AGRP also functions as an
MC4R inverse agonist.25,26
The rational design of peptide ligands toward the understand-
ing of bioactive structures and ligand pharmacophores important
for GPCR subtype potency and selectivity has been an ongoing
theme for medicinal chemists for decades. These data are impor-
tant toward understanding the molecular interactions of ligand-
effector proteins as well as for the rational design of new ligands
with desirable biological properties. These ligands can be used to
probe the mechanism of human disease states and as potential
Received: November 5, 2010
Published: February 09, 2011
r
2011 American Chemical Society
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therapeutic moieties. However, rational design of potent selec-
tive agonist and antagonist ligands for some of the melanocortin
receptors is challenging. Synthetic peptide strategies used in
recent years include introduction of cyclic constraints,27-31 N-
methylation,32,33 D-amino acids,34 constrained amino acids,31,35,36
aza peptides,37 and peptoids38 in the bioactive peptides. These
strategies can restrict peptide conformations, aid in conformational
analysis, and often result in selective and potent ligands with the
common underlying theme of stabilizing secondary structure(s).
Herein, we have utilized a rational structure-based design approach
that is based upon the unique and potent chimeric AGRP-melanocortin
peptide template 1 (AMW3-130) Tyr-c[Cys-His-DPhe-Arg-Trp-
Asn-Ala-Phe-Cys]-Tyr-NH239,40 incorporating a reverse-turn cyclic
thioether based heterocyclic scaffold 241 (Figure 2). As an indivi-
dual structural moiety, this 10-membered heterocyclic template 2
possessed 160 and 650 nM full agonist potency at the mMC1R and
mMC4R, respectively.41 The strategy presented herein is based
upon the long-standing hypothesis that incorporation of semirigid
or rigid moieties into a flexible peptide backbone should decrease
the topographical space accessible to the molecule in this domain
and, with the aid of biophysical studies such as 2D 1H NMR and
computer assisted computational molecular modeling, identify
putative bioactive peptide conformations and important pharma-
cophores. Our approach is hypothesized to bridge a conceptual gap
between peptides and small molecules by incorporating a bioactive
peptidomimetic scaffold into a highly potent cyclic peptide tem-
plate. This experimental approach attempts to structurally identify
the melanocortin agonist pharmacophore side chains important for
receptor stimulation and potency as well as to probe the side chain
orientation of the core His-Phe-Arg-Trp residues. This design stra-
tegy incorporates the advantages of peptide derived potency and
receptor targeting with the inclusion of a bioactive small molecule
peptidomimetic moiety. Additionally, an objective of this study was
to develop a synthetic strategy so that all the chemistry associated
with the assembly of these modified heterocyclic-peptide analogues
could be performed using solid phase synthesis. To the best of
our knowledge, this is the first study incorporating bioactive
small molecule peptidomimetics into a potent melanocortin re-
ceptor peptide template to probe melanocortin ligand structure-
activity relationships.
’ DESIGN OF THE REVERSE TURN HETEROCYCLIC-
PEPTIDE HYBRID TEMPLATE
This study utilized the chimeric peptide template 1 (Figure 2)
we have previously identified as a potent and novel agonist
peptide template for the melanocortin receptors.39,40 This tem-
plate is derived from the antagonist hAGRP(109-118) deca-
peptide sequence with amino acid residues His-Phe-Arg-Trp
from the melanocortin agonists incorporated in the place of the
antagonist Arg-Phe-Phe amino acids39,40 and contains a single
disulfide bridge between two Cys side chains (Figure 2). The
hAGRP pharmacophore Arg-Phe-Phe42 amino acids have been
postulated to mimic putative ligand-receptor interactions43 of
the melanocortin agonist core domain and, upon substitution
with the His-DPhe-Arg-Trp residues, convert the functionality of
the peptide from an antagonist to an agonist.39,44 This chimeric
Figure 1. Sequence of the (A) human agouti related protein (hAGRP)
and (B) human agouti signaling protein (hASP).
Figure 2. Illustration of the chimeric hAGRP-melanocortin peptide template 1 and the bioactive heterocyclic molecule (2) utilized in this study. The
side chain of His is depicted in green, DPhe in orange, Arg in blue, the heterocycle moiety of 2 in magenta, and the disulfide bridge circled in yellow.
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template 1 was reported by our laboratory to exhibit subnanomolar
agonist potency at the hMC4R and results in the ability to
functionally rescue nanomolar potency at human MC4R poly-
morphisms (identified in morbidly obese humans) that do
not pharmacologically respond normally to the endogenous
agonists.45-47 A similar template incorporating a lactam bridge
instead of a disulfide bridge Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-
Ala-Phe-Dpr]-Tyr-NH2 has been studied by our laboratory and
resulted in a full agonist at the melanocortin receptors that is as
potent as the endogenous R-MSH agonist.40 Biophysical studies
(NMR and CAMM) of those chimeric melanocortin-AGRP
peptides identified the presence of a β-turn spanning different
amino acid residues, as well as unique structures postu-
lated to differentiate MC3R antagonist versus MC4R agonist
pharmacology.40
The endogenous melanocortin agonists are linear flexible
molecules that are potent at the melanocortin receptors but are
generally not selective at any receptor subtype, albeit with
species-specific variances and with the exception of the MC2R
which only responds to ACTH. All of the endogenous melano-
cortin agonists have aromatic and basic side chains in the core
His-Phe-Arg-Trp pharmacophore region and upon incorpora-
tion of the DPhe7 residue (R-MSH numbering) are postulated to
adopt a β-turn structure.48 One possible approach to stabilize a
turn structure in globally constrained peptides is to introduce a
ring structure in the ligand pharmacophore domain. A cyclic
thioether based mimetic (2) has been reported41 that can be
synthesized using solid-phase chemistry and with side chain
functionality that can be chemically modified. The advantageous
feature of this solid-phase synthetic protocol is that a mild on-
resin cyclization condition leaves intermediates attached to the
solid support for further synthetic chemistry. The side chain
functionality was introduced by commercially available Fmoc-
amino acids and can be readily diversified. Herein, we have
designed, synthesized, and analytically characterized a series of
compounds, incorporating a bioactive peptidomimetic reverse
turn heterocycle into a monocyclic peptide backbone. These
molecules were synthesized using solid-phase chemical strategies
and were pharmacologically characterized at the mouse melano-
cortin receptor isoforms for potency and selectivity. To probe
structure-function relationships, we designed this study to
examine the effect of insertion of the heterocyclic small molecule
into different regions of the peptide template backbone. This
approach modifies both side chain orientation and backbone
flexibility toward improving receptor selectivity and structural
determination of the role of the Arg residue for receptor potency.
The position of the heterocyclic structure (Figure 3) in the
chimeric AGRP-melanocortin peptide template 1 was position-
ally “walked” through the pharmacophore region of 1 to deter-
mine the importance of the His-DPhe-Arg elements for receptor
potency and selectivity. Both structural elements (heterocycle
and peptide template) used in this study have shown biological
activity at the melanocortin receptors individually but without
selectivity.
’ RESULTS
Table 2 summarizes the six peptide-heterocyclic compounds
synthesized in this study (structures of the compounds are shown
in Figure 3) and pharmacologically characterized at the mouse
MC1 and MC3-5 receptors. Chemistry was optimized on solid
support using the Rink amide MBHA linker resin. The C-term-
inal ligand domain was synthesized by coupling Fmoc-protected
amino acids, using standard solid phase peptide synthesis
procedures49,50 (Scheme 1). The ring structure was introduced
through a known procedure41 including reductive alkylation (using
NaBH3CN) together with the Fmoc-protected aminoaldehyde51
(prepared by LiAlH4 reduction of the corresponding Weinreb
Figure 3. Illustration of the peptide heterocyclic template (2) incor-
porating L- or D-Cys residue during synthesis to induce the R or S
stereocenter incorporated into the peptide template 1. A positional
“walking” approach was used to place the bolded amino acid side chain
into the AA position indicated within the peptide template.
Table 2. Mouse Melanocortin Receptor Agonist EC50 (nM) Values of the Peptide-Heterocyclic Molecules and Reference
Compoundsa
EC50 (nM)
compd
mMC1R
mMC3R
mMC4R
mMC5R
NDP-MSH
heterocycle
0.018 ( 0.003
0.35 ( 0.17
164 ( 22
0.14 ( 0.03
2.00 ( 0.45
7600 ( 1890
3900 ( 1380
>100000
0.20 ( 0.03
0.27 ( 0.09
650 ( 126
350 ( 76
0.03 ( 0.05
2.3 ( 0.57
335 ( 106
305 ( 106
390 ( 24
1
2b
3
4
5
6
7
8
190 ( 76
1180 ( 155
540 ( 290
240 ( 48
3060 ( 2330
2730 ( 310
3210 ( 1470
85 ( 13
>100000
1595 ( 960
990 ( 440
72 ( 16
3200 ( 610
495 ( 215
4620 ( 1060
33 ( 9
495 ( 180
780 ( 340
925 ( 360
a The indicated error represent the standard error of the mean determined from at least three independent experiments. >100000 denotes that some
stimulatory activity was observed at 100 μM but not enough to determine an EC50 value, since the curve did not plateau. b The heterocycle starting
template has been previously reported in ref 41 as the 1n compound.
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Scheme 1
amide).52-54 The N-fluorenylmethyloxycarbonyl (Fmoc)-R-ami-
noaldehydes for amino acids Fmoc-His(Trt)-OH, Fmoc-Phe-OH,
and Fmoc-Arg(Boc)2-OH were synthesized by a known two-step
procedure involving the transformation of N-Fmoc-R-amino acid
to the corresponding Weinreb amide, followed by reduction with
LiAlH4. The N-Fmoc-R-amino acid was first coupled with N,O-
dimethylhydroxylamine to give the resulting amide, followed by
LiAlH4 reduction at 0 °C to yield the aldehyde. The synthesized N-
Fmoc-aminoaldehydes were used for the reductive alkylation to
give monoalkylated product 9 at room temperature in 3 h, as
indicated by negative Kaiser test.55 Following reductive alkylation,
Fmoc-Cys (StBu)-OH was coupled with free amine using HBTU
activation to give 10. Acylation on the secondary amine using
chloroacetic anhydride was achieved in 30 min, as indicated by
negativechloraniltest forsecondaryamines.56 To avoid acylation of
the unprotected nitrogen of the guanidino group, Arg(Boc)2 was
used instead of Arg(Pmc) or Arg(Pbf). Deprotection of StBu from
the cysteine group with Bu3P resulted in the free thiol and served as
the cyclization precursor. Formation of the thioether bond was
achieved by addition of N-ethylmorpholine in DMF and heating
the resulting solution at 60 °C for 10 h to yield 11. After
deprotection of the Fmoc group, the remaining amino acids were
coupled using standard Fmoc synthetic strategies to give 12. The
analogues were cleaved from the resin by a 3 h treatment with a
cleavage cocktail (TFA/triisopropylsilane/EDT/water 91:3:3:3)
to give the crude compounds (13, Scheme 1) The disulfide bond
was formed by dissolving crude product in 20% DMSO/water and
stirred at room temperature. Progress of the disulfide cyclization
was monitored by UV-HPLC (λ = 214 nm) and was generally
completed in 24-36 h. After disulfide bond formation, the
molecules were purified to homogeneity using semipreparative
reversed-phase high-pressure liquid chromatography (RP-HPLC).
The peptides possessed the correct molecular weights, as deter-
mined by mass spectrometry, and the purity of the molecules
(>95%) was assessed by analytical RP-HPLC (at a wavelength of
214 nm) in two diverse solvent systems (Table 3).
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Table 3. Analytical Data of Synthesized Compoundsa
k0
peptide
MeCN
MeOH
purity, %
HRMS, m/z
(1) Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
4.5
5.7
6.2
5.4
5.9
6.1
5.5
8.0
9.3
>99
>96
>98
>99
>95
>96
>95
1506.6
1635.0
1636.8
1636.4
1635.3
1637.2
1636.2
(3) Tyr-c[Cys-(R)-MIM(His)-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
(4) Tyr-c[Cys-(S)-MIM(His)-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
(5) Tyr-c[Cys-His-(R)-MIM(DPhe)-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
(6) Tyr-c[Cys-His-(S)-MIM(DPhe)-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
(7) Tyr-c[Cys-His-DPhe-(R)-MIM(Arg)-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
(8) Tyr-c[Cys-His-DPhe-(S)-MIM(Arg)-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2
9.2
10.2
9.3
11.0
10.2
a The HPLC k0 value equals [(peptide retention time - solvent retention time)/solvent retention time]. Two different solvent systems were used.
Solvent system 1 is 10% acetonitrile in 0.1% trifluoroacetic acid/H2O with a gradient to 90% acetonitrile over 35 min. Solvent system 2 is 10% methanol
in 0.1% trifluoroacetic acid/H2O with a gradient to 90% methanol over 35 min. An analytical Vydac C18 column (Vydac 218TP104) with a flow rate of
1.5 mL/min was used for analytical characterization. The peptide purity was determined by HPLC in both solvent systems at a wavelength of 214 nm.
Melanocortin Receptor Pharmacology. All the compounds
were tested for agonist activity at the mouse melanocortin
receptors using a cAMP based β-galactosidase reporter gene
bioassay.57 The MC2R is only stimulated by the ACTH agonist
and was excluded from the present study. The heterocyclic temp-
late 2 pharmacology previously reported by our laboratory41 is
included as a control in addition to the standard NDP-MSH (Ac-
Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2)34
reference peptide.
Incorporation of the His6 (R-MSH numbering) side chain into
the heterocyclic-peptide template at the AA position (Figure 3)
in the R-configuration 3 resulted in essentially an equipotent me-
lanocortin receptor profile (Table 2) as the heterocyclic template
(incorporates the Arg side chain at the AA position) upon in-
corporation into template 1. Comparison of 3 with the peptide 1
results in 130- to 1950-fold decreased melanocortin receptor
agonist potency at the different isoforms. Compound 4, with
incorporation of the His6 (R-MSH numbering) side chain into
the heterocyclic template at the AA position (Figure 3) in the S-
configuration, resulted in the inability to stimulate the mMC3R
at up to 100 μM. However, at the mMC5R, 4 possessed a full ago-
nist potency of 390 nM, ∼170-fold less than 1, but the same potency
as the R-configured molecule 3. Compound 4 possessed 3370- and
11330-fold decreased full agonist potency compared to the flexible
peptide 1at the mMC1R and mMC4R, respectively. Incorporation of
the Phe7 (R-MSH numbering) side chain into the heterocyclic-
peptide template at the AA position (Figure 3) in the R-configuration
5 resulted in a loss of agonist activity at the mMC3R at up to 100 μM,
decreased potency at the mMC1R, mMC4R, and mMC5R compared
to the heterocyclic template 2, and 690- to 10111-fold decreased
potency at the same receptors relative to the flexible template 1.
Inversion of stereochemistry of the S-configured Phe7 containing
heterocyclic-peptide 6, similar to 5, possessing mMC1R, mMC4R,
and mMC5R decreased full agonist potency compared to 2. However,
6 functioned as a full micromolar agonist at the mMC3R while the
R-configured 5 was devoid of stimulatory activity.
The peptide-heterocyclic hybrid compounds 7 and 8 that in-
Figure 4. Histogram of the difference between the random coil
corporate the Arg side chain moiety at the AA position (Figure 3)
result in full agonists at all the melanocortin receptor isoforms
examined in this study. Molecule 7 containing the R-configuration
possessed nanomolar full agonist pharmacology at the mMC1R,
mMC4R, and mMC5R that was more potent than the correspond-
ing heterocyclic template 2 alone but is 30- to 310-fold less potent
than peptide 1. Compound 8 containing the S-configuration, while
(water solvent) and experimental chemical shifts of (A) peptide 1,
(B) analogue 7, and (C) analogue 8 (acetonitrile/water solvent). The
black bars indicate the amide protons, and the R protons are re-
presented by white bars.
possessing full agonist activity at all the melanocortin receptors
examined, was less potent compared to the R-configured 7. These
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1
1
Table 4. H NMR and Chemical Shift Assignment (ppm) for
Table 5. H NMR and Chemical Shifts Assignment for
Analogues 7 and 8
Reference Peptide 1a
amino acid
HN
HR
Hβ
others
Peptide 1
amino acid
HN
HR
Hβ
Analogue 7
Tyr1
Cys2
His3
DPhe4
Arg5
Trp6
Asn7
Ala8
-
-
-
-
Tyr1
Cys2
His3
DPhe4
Cys5*
Arg6
Trp7
Asn8
Ala9
4.63 3.07, 2.91 6.89, 7.28
8.04 4.9 2.78, 2.90
-
-
8.28
4.66
2.98, 3.11
2.92, 3.00
1.36, 1.54
3.19, 3.27
2.7
8.46 4.68 2.88, 3.01 6.93, 8.27
8.21 5.07 2.88, 3.01 7.23
8.06 4.74 2.71, 3.15
8.16
7.86
8.03
7.52
7.84
7.92
7.70
7.68
4.58
4.07
4.50
4.44
3.96
4.44
4.50
4.45
7.91 3.94 1.26, 1.26 1.05, 1.05; 2.91, 2.91; 6.89
4.14 3.33, 3.48 7.09, 7.43; 9.93
1.13
8.3
4.68 2.88, 2.88
Phe9
Cys10
Tyr11
2.97, 3.18
-
8.11 4.49 1.18
Phe10
Cys11
Tyr12
8.21 4.63 2.88, 3.01 7.29
8.02 4.88 2.78, 2.90
7.91 4.51 2.82, 3.01 6.86, 7.36
2.33, 3.01
2.88, 3.03
a The dash (-) indicates that the chemical shift values could not be
determined because of overlapping peaks or chemical exchange.
ring CH2 Arg
ring CH2 Acetyl
3.36, 3.43
Analogue 8
Tyr1
Cys2
7.96 4.54 2.78, 2.95
His3
DPhe4
Cys5*
8.32 4.65 2.87, 2.99 6.19, 8.30
8.03 4.68 2.80, 3.04 7.27
7.83 4.37 2.67, 2.90
Arg6
Trp7
Asn8
7.64 4.51 1.40, 1.40 1.24, 1.24; 3.01, 3.01; 6.96
4.16 3.41, 3.51 7.15, 7.53; NH 10.04
8.12 4.77 2.83, 2.90
Ala9
8
4.16 1.4
Phe10
Cys11
7.94 4.56 2.97, 3.06 7.34
7.94 4.51 2.78, 2.95
Tyr12
7.7
4.45 2.83, 3.01 6.80, 7.26
2.36, 2.71
ring CH2 Arg
ring CH2 Acetyl
3.41, 3.53
1
peptide structure, we performed 2D H NMR and CAMM
studies of the most potent derivative 7 at the melanocortin re-
ceptors. This compound contains the Arg side chain at the i þ 2
position in the ring AA position (Figure 3) and was compared
with the less potent analogue 8. Analogues 7 and 8 are sequentially
identical and differ configurationally at only one stereocenter. We
therefore wanted to probe the conformational differences between
these two compounds and compare them with the reference
peptide 1 to aid in understanding the effect of insertion of the
heterocyclic small molecule into the peptide backbone template
and correlate the structure with function.
NMR chemical shifts are extremely sensitive to the electronic
and chemical environment, therefore providing useful informa-
tion about molecular structure. However, in the case of flexible
peptides, they may not be as singularly informative because they
represent a population-weighted average of rapidly inter-
converting structures with different averaged chemical shifts.
Comparative analyses of peptide chemical shift values are
generally derived from the use of aqueous solvents and not a
mixed solvent system (acetonitrile and water), as used herein.
Nonetheless, by subtracting a common reference value from
each chemical shift, a qualitative comparison of the analogues in
this study can be made. Figure 4 shows the differences between
Figure 5. Summary of the NOE intensities from 400 ms NOESY data
observed for molecules (A) 1, (B) 7, and (C) 8. The height of the bar
indicates the strength of the NOE. The NOE volumes were categorized
as strong (1.8-3.0 Å), medium (1.8-3.5 Å), or weak (1.8-5.0 Å).
data led us to study the structural differences between the R- and S-
configured heterocycle-peptide moieties that incorporate the Arg
side chain at the AA position (Figure 3).
1
Biophysical Studies: 2D H NMR and Computer Assisted
Molecular Modeling (CAMM) Structures. In an attempt to
correlate the melanocortin receptor functional studies with
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Figure 6. Stereoview illustration of the “sausage” representations of the backbone superposition of the major family members for (A) reference peptide
1, (B) analogue 7, and (C) analogue 8. 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.
our mixed experimental solvent (acetonitrile and water) and
literature reported aqueous random coil amide and R proton
chemical shift values58 for the conserved His-DPhe-Arg-Trp-
Asn-Ala region of the three compounds examined. A noticeable
feature of Figure 4 is that each of the compounds looks differ-
ent by NMR. Both 7 and 8 are different from the reference
compound 1. Comparisons of the chemical shift values are pres-
ented in Tables 4 and 5.
The nuclear Overhauser effect (NOE) measures the distance
between atoms in space and is useful to characterize peptide
secondary structure features like reverse turns. Two-dimensional
NOESY experiments can be utilized to identify cross-peaks and
interactions between protons closer than 5 Å in space. Different
patterns of NOEs indicate different molecular secondary struc-
tures and can be used as a tool to identify regular structure in
molecules and/or to distinguish structural differences between
analogues. Figure 5 summarizes the NOE intensities observed
for these peptide analogues. Consistent with chemical shifts
above, compounds 7 and 8 have different NOE patterns. As
illustrated in Figure 5, compound 7 has more NOE interactions
in the His-Phe-Arg-Trp domain than 8. Moreover, compound 7
has regular and strong HR(i) to N(iþ1) NOEs across the entire
sequence.
Computer-Assisted Molecular Modeling (CAMM). Relative
proton distances derived from the 2D NOESY NMR spectra were
used as constraints for conformational studies using CAMM for
the reference peptide 1 and analogues7 and 8. In attempts toavoid
the risk of becoming “trapped” in local energy minima induced by
the formation of the disulfide bond, we performed restrained
molecular dynamics (RMD) simulations with unambiguous NMR
distance restraints before we covalently formed this cyclic con-
straint. At the end of the initial NOE-based RMD simulations,
the Cys amino acid side chains were identified to be located in a
favorable low energy orientation to allow for the formation of
the disulfide bond. Following the formation of the disulfide bond,
the analogues were allowed to fully relax by energy minimization
before performing a more robust RMD simulation using the cyc-
lized compounds and incorporating all the experimentally identified
NMR distance restraints (Figure 5). Conformational families of
structures were identified by energy-minimizing200 evenly spaced
points along a 10 ns RMD trajectory. The identical proce-
dure was followed for all three compounds studied herein.
Molecular dynamics simulation experiments of the reference
peptide 1 resulted in three major conformational families, with the
largest family possessing ∼60% of the sampled conformations. A
sausage diagram of the superimposed members of this family is
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’ DISCUSSION
The use of peptidomimetics has emerged as a powerful tool to
overcome the inherent limitations of the peptides and can im-
prove a ligands therapeutic potential.60 Linear peptides in their
native form can exist in multiple solution phase conformations in
equilibrium and consequently possess the potential to interact
with different shapes at the site of the target receptor. It has been
widely recognized that by incorporation of structural restrictions
(i.e., cyclizations, N-methylation, modified amino acid side chains,
and modified backbone amide bonds) into peptides, decreased
peptide conformational flexibility can result in potent and selective
ligands. By use of biophysical studies such as 2D NMR and CAMM,
“bioactive” conformations of these structurally restricted molecules
can be deduced. The approach presented herein involves the inte-
gration of a bioactive heterocyclic peptidomimetic moiety (2)41
into a highly potent and nonselective peptide agonist 146 with the
intention to improve the compound potency and receptor selec-
tivity and to perform structure-activity relationship studies. It was
also a goal to develop a synthetic strategy so that all the building
block assembly chemistry associated with the synthesis of these
modified heterocyclic-peptide analogues could be performed using
solid phase synthesis. Both structural elements in this novel temp-
late possess biological activity at the melanocortin receptors indivi-
dually but without selectivity (Table 2).
It was hypothesized that by insertion of a 10-membered
heterocyclic moiety into the peptide amide bond region, the re-
sulting compound could be used as a tool to probe the “bioactive”
peptide backbone conformation as well as the amino acid ori-
entation resulting from positioning of the different side chain
moieties along the heterocycle (Figure 3). The use of solid phase
chemistry for the synthesis in addition to the nanomolar agonist
potency at the melanocortin receptors made this peptidomimetic
scaffold (2)41 an attractive choice to use as a template. The 10-
membered thioether cyclized scaffold was introduced in the pep-
tide template using mild on-resin cyclization method as previously
reported.41 Unexpectedly, the heterocycle moiety made the entire
compound more flexible than the parent peptide scaffold alone.
The peptide-small molecule hybrid analogue 7 resulted in
the most potent full agonist at the melanocortin receptors and
possessed a modest 5-fold MC4R versus MC3R selectivity
(Table 2). Compounds 7 and 8 differ in the configuration at
one stereocenter of the 10-membered ring mimetic (Figure 3)
and variances modify receptor potency as anticipated. Reversing
the orientation of the Cys residue involved in the ring formation
allowed us to make subtle changes in the orientation of the Arg
moiety. The Arg side chain has been established to play an
important role in ligand binding and stimulation of the melano-
cortin receptors.65 The replacement of Arg with Ala in the MTII
cyclic template (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2)61
has been reported to cause at least a 100-fold drop in affinity at
the MC3, MC4, and MC5 receptors due to a lack of interactions
between the guanidinyl functionality and melanocortin receptor
residues.62 In another example, replacement of Arg with Ala in
the linear Ac-His-DPhe-Arg-Trp-NH2 tetrapeptide resulted in
decreased potency ranging from 170- to 1740-fold at the mouse
MC1R and MC3R-MC5R.63 Receptor mutagenesis studies have
postulated interactions between positively charged basic residues
of melanocortin peptides and negatively charged acidic residues in
the TM2 and TM3 region of melanocortin receptors.43,64
Figure 7. CAMM based φ-ψ angle distribution for the (A) reference
peptide 1, (B) analogue 7, and (C) analogue 8 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.
shown in Figure 6. This representative conformational family of 1
has a β-turn structure containing the Arg and Trp residues at i þ 1
and i þ 2 positions, respectively, and possesses a β-hairpin-like
structure. The φ and ψ values of the most highly populated 1
representative structure are φ(iþ1) ≈ 52, ψ(iþ1) ≈ 31, φ(iþ2)
≈ 95, ψ(iþ2) ≈ 106, which may be classified as a type I0 reverse
turn [idealized values for this type of turn have been reported as
φ(iþ1) = 60, ψ(iþ1) = 30, φ(iþ2) = 90, ψ(iþ2) = 0].59 Figure 7
represents the φ-ψ distribution of all the residues in the sequence
with the exception of the terminal Cys and Tyr residues. Comparisons
of φ-ψ distributions of analogues 7 and 8 reveal that while con-
sistent patterns are observed for the His, DPhe, and Phe residues,
notable differences are observed for the other residues within the
two analogues.
The CAMM structures of analogues 7 and 8 resulted in multi-
ple conformational families, all with very low populations. There-
fore, a cluster analysis was performed that grouped the structures
by backbone (j, ψ) dihedral angles in the region corresponding
to His-DPhe-Arg-Trp residues. Analogue 7 possessed six major
families with the largest family having only 9% of the sampled
conformations. The total number of conformers of the subfami-
lies is very small and only represents 36% of the total number of
total identified conformers. Analogue 8 possessed four major
families, with the most highly populated having only ∼11% of the
total sampled population. In summary, both 7 and 8 are highly
flexible in comparison to 1.
Orientation of the heterocycle stereocenter in compounds 7
and 8 changes the pharmacology, the NMR spectral parameters,
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Journal of Medicinal Chemistry
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and the resultingconformational families of these two compounds.
Contrarytoourexpectations, incorporation ofthe heterocycle into
the peptide template made both 7 and 8 very flexible. Given the
extremely low populations of structures in either compound, it is
difficult to perform a direct structural comparison. However, the
NMR data that are most distinct between 7 and 8 are for the chem-
icalshift valuesofarginine. Wecan speculate thattheorientation of
the arginine side chain is more important for the potency than the
selectivity at the melanocortin receptors, since there is at least a 10-
fold decreased potency for 8 than 7, but the selectivity profile is the
same for both the compounds.
The reference peptide 1 possessed a relatively constrained
structure in the molecular dynamics studies, compared to 7 and
8. Superposition of conformers of the major family of 1 was
relatively good (backbone rmsd = 1.80 ( 0.54 Å; in the H, F, R, W
region rmsd = 0.66 ( 0.30 A). A β-hairpin-like structure was
identified in 1, which was consistent with the structure obtained
for similar peptides having a lactam bridge instead of a disulfide
bridge.42 A putative type I0 β-turn was identified based on φ-ψ
dihedral angles involving Arg and Trp residues.
In conclusion, to our knowledge, this is the first report of inte-
grating a bioactive heterocyclic turn moiety into a highly potent
but nonselective melanocortin receptor peptide agonist. The synthe-
sis of six analogues and the assessment of their efficacy in stimulat-
ing melanocortin receptors resulted in analogues with nanomolar
potency to inactive molecules. This study has resulted in the
identification of a novel lead compound (7) for further structure
-activity studies, from an in vitro ligand-receptor standpoint, and
provided novel tools to study the in vivo melanocortin pathways.
reversed-phase HPLC using a Shimadzu chromatography system with a
photodiode array detector and a semi preparative RP-HPLC C18 bonded
silica column (Vydac 218TP1010, 1.0 cm ꢀ 25 cm).
Reactions at elevated temperatures were conducted in an automated
synthesizer (Advanced ChemTech 440MOS, Louisville, KY). MALDI-
TOF spectra were recorded on a Voyager MALDI-TOF MS, using R-
cyano-4-hydroxycinnamic acid as matrix. TLC plates used were Merck
silica gel 60F254 on aluminum. Visualization was achieved with UV light.
General Procedure for Preparation of Weinreb Amides.
To a dried flask flushed with N2 gas, the Fmoc-protected amino acid
(3 mmol), BOP (3.3 mmol), N,N0-diisoproplethylamine (DIEA, 4.4
mmol), and 6 mL of DMF were added. After the mixture was stirred for
30 min at room temperature, N,O-dimethylhydroxylamine (3.3 mmol)
and DIEA (3.3 mmol) and an additional 4 mL of DMF were added and
mixed for 3 h. Subsequently, 40 mL of ethyl acetate was added to the
reaction mixture and washed with saturated sodium bicarbonate solu-
tion, a 1 M potassium hydrogen sulfate (KHSO4) solution, and brine.
The organic layer was dried over MgSO4, concentrated, and used in the
next step without further purification.
General Procedure for Reduction of Weinreb Amides. The
Weinreb amide was dissolved in dry THF (20 mL) and added dropwise
to a suspension of LiAlH4 (2 equiv) in THF (30 mL) at -78 °C. The
suspension was stirred at -78 °C for 1-2 h or when judged to be com-
plete by TLC. The reaction was quenched at -78 °C with water (3 mL).
Subsequently, MgSO4 was added and the solution was filtered. The solvent
was removed in vacuo to give an oil or a sticky solid. Titration with diethyl
ether usually resulted in a white solid, which was essentially pure and could
be used directly without further purification in the next step.
Peptide Synthesis. The peptides were assembled on Rink amide
MBHA resin purchased from Peptides International (Louisville, KY).
The synthesis (0.2 mmol scale) was performed using a manual synthesis
reaction vessel. Each synthetic cycle consisted of the following steps: (i)
removal of the NR-Fmoc group by 20% piperidine in DMF (1 ꢀ 2 min,
1 ꢀ 20 min) and (ii) single 2 h coupling of Fmoc-amino acid (3 equiv)
using BOP (3 equiv), HOBt (3 equiv), and DIEA (6 equiv) in DMF and
repeated until the peptide synthesis was complete. The presence or ab-
sence of the NR-free amino group was monitored using the Kaiser test.55
Disulfide Bridge Formation in Solution. The linear peptides
were cleaved from the resin and fully deprotected using a cleavage
cocktail consisting of 91% TFA, 3% TIS, 3% EDT, and 3% H2O 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 of the disulfide cyclization was monitored by
UV-HPLC that generally was completed in 24-36 h. The resulting
solution was lyophilized to give the crude cyclic peptide and purified by
reversed-phase HPLC using a Shimadzu chromatography system with a
photodiode array detector and a semipreparative RP-HPLC C18 bond-
ed silica column (Vydac 218TP1010, 1.0 cm ꢀ 25 cm). The purified
peptides were at least >95% pure as determined by RP-HPLC (at a
wavelength of 214 nm) in two diverse solvent systems and had the correct
molecular mass (University of Florida Protein Core Facility) (Table 3).
Cell Culture and Transfection. The HEK-293 cells were main-
tained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal
calf serum and seeded 1 day prior to transfection at (1-2) ꢀ 106 cell per
100 mm dish. Melanocortin receptor DNA in the pCDNA3 expression
vector (20 μg) was transfected using the calcium phosphate method.65
Stable receptor populations were generated using G418 selection
(1 mg/mL) for subsequent bioassay analysis.
’ EXPERIMENTAL SECTION
Reagents and General Methods. p-Methylbenzhydrylamine
resin (p-MBHA resin, 0.47 mequiv/g substitution), NR-9-fluorenylmethox-
ycarbonyl (Fmoc) protected amino acids Cys(Trt), Tyr(tBu), Arg(Pbf),
His(Trt), Phe, Asn(Trt), Ala, and the coupling reagents benzotriazol-1-
yl-N-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP)
and 1-hydroxybenzotriazole (HOBt) were purchased from Peptides Inter-
national (Louisville, KY). Fmoc-Arg(Boc)2-OH was obtained from the
Bachem (Torrance, CA). Glacial acetic acid (HOAc), dichloromethane
(DCM), methanol (MeOH), acetonitrile (ACN), and anhydrous ethyl
ether were purchased from Fisher (Fair Lawn, NJ). N,N-Dimethylforma-
mide (DMF) was purchased from Burdick and Jackson (McGaw Park, IL).
Trifluoroacetic acid (TFA), 1,3-diisopropylcarbodiimide (DIC), and piper-
idine were purchased from Sigma (St. Louis, MO). N,N-Diisopropylethy-
lamine (DIEA) was purchased from Aldrich (Milwaukee, WI). All rea-
gents and chemicals were ACS grade or better and were used without
further purification.
Solid-phase reactions were performed using a manual synthesis reaction
vessel (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). Amino acid couplings consisted of the
following steps: (i) removal of the NR-Fmoc group by 20% piperidine in
DMF (1 ꢀ 2 min, 1 ꢀ 18 min) and (ii) 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 NR free amino group was
monitored using the Kaiser test. After the completed synthesis and final
Fmoc deprotection, the peptides were cleaved from the resin and
deprotected using a cleavage cocktail consisting of 91% TFA, 3% TIS,
3% EDT, and 3% H2O 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 subjected to cyclization and purified by
Functional Bioassay. The HEK-293 cells stably expressing the
mouse melanocortin receptors were transfected with 4 μg of CRE/β-
galactosidase reporter gene as previously described.39,43,57 Briefly, 5000-
15000 post-transfection cells were plated into collagen treated 96-well
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ARTICLE
plates (Nunc) and incubated overnight. Forty-eight hours post-transfec-
tion the cells were stimulated with 100 μL of peptide (10-4 to 10-12 M)
or forskolin (10-4 M) control in assay medium (DMEM containing 0.1
mg/mL BSA and 0.1 mM isobutylmethylxanthine) for 6 h. The assay
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 thecell lysates were thawed the following
day. Aliquots of 10 μL were taken from each well and transferred to
another 96-well plate for relative protein determination. To the cell lysate
plates, 40 μL of phosphate-buffered saline with 0.5% BSA was added
to each well. Subsequently, 150 μL of substrate buffer (60 mM sodium
phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM β-mercaptoethanol,
2 mg/mL 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). The relative protein was determined
by adding 200 μL of 1:5 dilution Bio Rad G250 protein dye/water to the
10 μL cell lysate sample taken previously, and the OD595 was measured on
a 96-well plate reader (Molecular Devices). Data points were normalized
both to the relative protein content and nonreceptor dependent forskolin
stimulation. Maximal efficacy was compared to that observed for the
NDP-MSH control peptide tested simultaneously on each 96-well plate.
All compounds were observed to result in full agonists with the same
maximal efficacy as observed for the NDP-MSH control.
Data Analysis. The EC50 values represent the mean of duplicate wells
performed in quadruplet or more independent experiments. EC50 value
estimates, and their associated standard errors were determined by fitting the
data to a nonlinear least-squares analysis using the PRISM program (version
4.0, GraphPad Inc.). The results are not corrected for peptide content.
NMR Spectroscopy. Peptide NMR samples were prepared by
dissolving 1.0-2.0 mg of peptide in a 750 μL solution containing 400 μL
of CD3CN and 350 μL of H2O and adding DSS as an internal standard
(0.0 ppm). The NMR data were collected using Bruker Avance spectrom-
eter operating at 500 MHz at the Advanced Magnetic Resonance
Imaging and Spectroscopy (AMRIS) facility at the University of Florida.
Standard proton TOCSY and NOESY 2D NMR data were collected,
processed, and analyzed as described previously.66 NOESY data were
collected at both 250 and 400 ms mixing times, and proton-proton
distances were obtained from the 400 ms data set. The chemical shifts
of each of the peptides in this study were assigned using standard TOCSY
and NOESY 1H-based strategies.67 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 R and/or
β protons of residue i with the amide proton of residue i þ 1.
backbone j and ψ dihedral angles in the region corresponding to His-
DPhe-Arg-Trp residues.
’ AUTHOR INFORMATION
Corresponding Author
*Before June 2011: (phone) (352) 273-7717; (e-mail) Carrie@
cop.ufl.edu. After July 2011: (address) Department of Medicinal
Chemistry, University of Minnesota, 8-101 Weaver-Densford
Hall, 308 Harvard Street, SE, Minneapolis, MN, 55455; (e-mail)
email:chaskell@umn.edu; phone and fax unknown as of January
2011.
’ ACKNOWLEDGMENT
This work was supported by NIH Grant DK064250.
’ ABBREVIATIONS USED
ACTH, adrenocorticotropin hormone; AGRP, agouti-related
protein; ASIP, agouti-signaling protein; BAL, backbone amide
linker resin; CAMM, computer-assisted molecular modeling;
cAMP, cyclic 50-adenosine monophosphate; DCM, dichloro-
methane; DMF, N,N-dimethylformamide; DMS, dimethyl
sulfide; Fmoc, NR-9-fluorenylmethoxycarbonyl; GPCR, G-protein-
coupled receptor; MC1R, melanocortin-1 receptor; MC2R, mel-
anocortin-2 receptor; MC3R, melanocortin-3 receptor; MC4R,
melanocortin-4 receptor; MC5R, melanocortin-5 receptor;
MCR, melanocortin receptor; MeOH, methanol; MSH, melano-
cyte stimulating hormone; nM, nanomolar; NOE, nuclear
Overhauser effect; POMC, pro-opiomelanocortin; SAR, structure-
activity relationship; SEM, standard error of the mean; TFA,
trifluoroacetic acid; TM, transmembrane; R-MSH, R-melano-
cyte stimulating hormone; β-MSH, β-melanocyte stimulating
hormone; γ-MSH, γ-melanocyte stimulating hormone; μM,
micromolar; RMD, restrained molecular dynamics; NDP-
MSH, Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-
Val-NH2; AMW3-130 (1), Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-
Phe-Cys]-Tyr-NH2
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