Solid-phase peptide head-to-side chain cyclodimerization: Discovery of C2-symmetric cyclic lactam hybrid α-melanocyte-stimulating hormone (MSH)/agouti-signaling protein (ASIP) analogues with potent activities at the human melanocortin receptors

Article abstract of DOI:10.1016/j.peptides.2010.06.026

A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the agouti-signaling protein (ASIP), an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide lactam cyclodimerization yielded nanomolar range (25-120 nM) hMC1R-selective full and partial agonists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the development of novel GPCR peptide ligands.

Full text of DOI:10.1016/j.peptides.2010.06.026

Peptides 31 (2010) 1894–1905
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
Solid-phase peptide head-to-side chain cyclodimerization: Discovery of
C2-symmetric cyclic lactam hybrid ␣-melanocyte-stimulating hormone
(MSH)/agouti-signaling protein (ASIP) analogues with potent activities
at the human melanocortin receptorsଝ
Alexander V. Mayorov1, Minying Cai, Erin S. Palmer, Zhihua Liu, James P. Cain,
Josef Vagner, Dev Trivedi, Victor J. Hruby∗
Department of Chemistry and Biochemistry, University of Arizona, 1306 E. University Blvd., Tucson, AZ 85721, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 April 2010
Received in revised form 23 June 2010
Accepted 23 June 2010
Available online 3 August 2010
A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the
agouti-signaling protein (ASIP), an endogenous melanocortin antagonist, and ␣-melanocyte-stimulating
hormone (␣-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore
was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series
yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide
lactam cyclodimerization yielded nanomolar range (25–120 nM) hMC1R-selective full and partial ago-
nists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts
at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore
hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin
receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the develop-
ment of novel GPCR peptide ligands.
Keywords:
Agouti-signaling protein
Melanocortin receptors
Cyclodimerization
C2-symmetry
Macrocyclic peptide
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
conformational freedom of the parent peptide and thus a better
defined secondary structure required for efficient receptor–ligand
Peptide cyclization is a well-established approach to improving
peptide biological activity [38,53,59], which stems from reduced
interaction, i.e., “bioactive conformation” [39,54]. It has been pre-
viously noted that the success of a peptide cyclization depends
strongly on the probability of juxtaposition of the reactive groups
of the linear peptide precursor, and is usually encumbered by side
reactions, most notably, oligomerizations and cyclooligomeriza-
tions [20,57]. These side reactions become prevalent in syntheses
of small strained cyclic peptides, such as N-unsubstituted tri- and
tetrapeptides, typically incurring formation of cyclic dimers and
trimers (Scheme 1) [57].
Although several peptide natural products, such as grami-
cidin S [27] and marine cyclodepsipeptide IB-01212 [19], are
known to possess C2-symmetry, there are few examples of pep-
tide cyclodimerizations pertinent to development of peptides with
enhanced biological activities, which include syntheses of cyclic
biphalin [70] and morphiceptin [96] analogues. Thus, practical
aspects of this area of peptide chemistry and its applications
to development of novel GPCR ligands remain relatively unex-
plored. Recent reports on preparative peptide cyclodimerizations
including syntheses of dimeric disulfides [76,80,91], depsipep-
tides [19], lactams [98], and peptide cyclodimerizations involving
azide-alkyne cycloaddition “click chemistry” [14,62,77] prompted
us to disclose our results on preparation of structurally unique C2-
Abbreviations: All, allyl; AgRP, Agouti-related protein; ASIP, agouti-signaling
protein; Boc, tert-butyloxycarbonyl; Fmoc, fluorenylmethoxycarbonyl; CH3CN,
acetonitrile; Cl-HOBt, 1-hydroxy-6-chlorobenzotriazole; DCM, dichloromethane;
DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DIC, diisopropyl
carbodiimide; HOBt, N-hydroxybenzotriazole; hMCR, human melanocortin recep-
tor; mMCR, mouse melanocortin receptor; MSH, melanocyte-stimulating hor-
mone; Nal(2ꢀ), 2ꢀ-naphthylalanine; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-
5-sulfonyl; TFA, trifluoroacetic acid; SPPS, solid-phase peptide synthesis;
RP-HPLC, reverse-phase high performance liquid chromatography; hMC3R,
human melanocortin-3 receptor; ␣-MSH, ␣-melanocyte-stimulating hormone
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2; NDP-␣-MSH, Ac-
Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2; hASIP (116–123),
human agouti-signaling protein c [Cys-Arg-Phe-Phe-Arg-Ser-Ala-Cys]; MT-II, Ac-
Nle-c [Asp-His-D-Phe-Arg-Trp-Lys]-NH2.
ଝ
Abbreviations used for amino acids and designation of peptides follow the rules
of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972,
247, 977–983.
∗
Corresponding author. Tel.: +1 520 621 6332; fax: +1 520 621 8407.
E-mail address: hruby@u.arizona.edu (V.J. Hruby).
Current address: Department of Chemistry, The Scripps Research Institute, La
1
Jolla, CA 92037, USA.
0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2010.06.026

A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
1895
Scheme 1. Peptide cyclization and cyclodimerization.
symmetrical hybrid analogues of ␣-MSH/agouti-signaling protein
via solid-phase head-to-side chain lactam cyclodimerization, and
their biological evaluation at the human melanocortin receptors.
The melanocortin system [16,17,25] remains a challenging
target for rational peptide and peptidomimetic design since the 3D-
topographical requirements for the specific melanocortin receptor
subtype recognition have not been fully elucidated [28,33,100].
At the same time, the numerous multifaceted physiological func-
tions of the five known subtypes of human melanocortin receptors
(hMC1-5R), including skin pigmentation [15,32], control of the
immune system [15,32], erectile function [25,55,99,100], blood
pressure and heart rate [60,74], control of feeding behavior
and energy homeostasis [6,16,22,23,94,95,100,102], modulation
of aggressive/defensive behavior [71,72], and mediation of pain
[50,69,97], continue to provide a strong stimulus for development
of potent and selective melanocortin agonists and antagonists.
Several general approaches to development of such compounds
have been described in literature [9,24,40,41], and include: (a)
D-amino acid scan/unnatural amino acid substitutions in linear
␣-, ␤- and ␥-MSH-derived sequences [29,83]; (b) hybridiza-
tions of the native MSH sequences with each other and with
sequences of other bioactive peptides [4,8,34]; (c) implementa-
tion of various global and local conformational constraints via
peptide cyclizations and employment of constrained amino acids
[1,5,7,30,31,65,66,82]; and (d) manipulation of steric factors that
influence receptor–ligand interactions [3,64,65]; as well as (e) con-
struction of small molecules based on ␤-turn peptidomimetics
and “privileged structure” scaffolds [10,73,79,85,89]. Until recently,
this work had primarily been focusing on the hMC4R due to its
direct involvement in the regulation of feeding behavior and energy
homeostasis [6,16,22,23,94,95,100,102], as well as sexual behavior
[25,55,93,99,100]. The hMC3 receptor has been acknowledged to
play a complementary role in weight control [6,21,102], and current
reports suggest that hMC3R is an inhibitory autoreceptor on POMC
neurons [16] based on the observed stimulation of food intake by
peripheral administration of an MC3R-selective agonist [63], and
MC3R agonist-induced inhibition of spontaneous action of POMC
neurons [18], although the full scope of physiological functions
of this receptor is still poorly understood. Development of selec-
tive ligands for the hMC1 and hMC5 receptors is also receiving
some attention lately due to the roles of these receptors in reg-
ulation of skin pigmentation [15,32] and control of the immune
system [15,32] (hMC1R), and in regulating exocrine gland function
[13], and coordinating central and peripheral signals for aggression
(hMC5R) [71,72].
Agouti-signaling protein (ASIP) [61] and agouti-related pro-
tein (AgRP) [75] (Fig. 1) were originally described as endogenous
antagonists for the melanocortin-1 and melanocortin-3/-4 recep-
tors, respectively, although recent reports ascribe inverse agonist
activity to both proteins [11,36]. There is some evidence that
AgRP mediates orexigenic signaling of ghrelin [12], which
makes it an important regulator of feeding behavior [44]. Ini-
tial structure–function relationship studies have attributed the
melanocortin receptor antagonist activities to the Arg-Phe-Phe
tripeptide pharmacophore, which is a part of the central loop
within the inhibitor cystine knot (ICK) motif in both agouti pro-
teins [68], as displacement of these three key amino acids with
alanine within the AgRP/ASIP pharmacophore sequence results
Fig. 1. Sequences of some endogenous and synthetic melanotropin peptides.

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A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
in a loss of activity [101]. Truncation of both ASIP and AgRP
sequences results in substantial loss of both binding affinities
and antagonist/inverse agonist potencies [47,90], and Ac-mini-
AGRP(87-120, C105A)-NH2 has been reported to be the minimal
AgRP sequence equipotent to the full-length AgRP [45], indicat-
ing possible significance of N- and C-terminal sequences of these
proteins in receptor–ligand interactions. Replacement of the Arg-
Phe-Phe tripeptide sequence with the His-D-Phe-Arg-Trp MSH
tetrapeptide pharmacophore, quite predictably, led to conver-
sion of AgRP/ASIP-derived melanocortin antagonists to relatively
potent agonists [46,101], since the His-D-Phe-Arg-Trp tetrapep-
tide sequence is known to induce melanocortin agonist activity in a
wide variety of linear and cyclic peptide templates [9,24,37,43]. In
other instances, the agouti Arg-Phe-Phe tripeptide sequence was
embedded into the linear and cyclic ␣-MSH templates to yield
nanomolar range mMC1R agonists, although the agonist poten-
cies of these peptides were reported to be >300-fold below that
of the super-agonist MT-II as determined by the mMCR CRE/␤-
galactosidase assay [48].
(4× 15 mL). The first N␣-Fmoc amino acid was coupled using pre-
activated ester (3 equiv. of N␣-Fmoc amino acid, 3 equiv. of HOBt,
and 3 equiv. of DIC) in DMF. The coupling mixture was transferred
into the syringe with the resin and shaken for 90 min, at which
point the blue color of the resin changed to yellow, indicating com-
plete coupling. The resin was washed with DMF (3× 15 mL), and
thrice with DCM (3× 15 mL), and the unreacted amino groups were
capped using acetic anhydride (2 mL) and pyridine (2 mL) in DCM
(15 mL) for 30 min, and the resin was once again washed with DMF
(6× 15 mL). The peptide sequences were completed by consecu-
tively coupling the appropriate amino acids using the procedure
described above.
2.3. Disulfide cyclizations
Upon completion of the peptide sequences, the resin was
washed with DMF (5× 15 mL), DCM (3× 15 mL), methanol (5×
15 mL), and diethyl ether (5× 15 mL), and dried under reduced pres-
sure (16 h). The peptides were cleaved off the solid support with
95% (v/v) TFA, 2.5% water, and 2.5% triisopropylsilane (5 mL, 3 h),
and the crude peptides were precipitated out by the addition of a
chilled 3:1 mixture of diethyl ether and petroleum ether (50 mL)
to give white precipitates. The resulting peptide suspensions were
centrifuged for 10 min at 6,500 rpm, and the liquid was decanted.
The crude peptides were washed with diethyl ether (4× 50 mL),
and after the final centrifugation, the peptides were dried under
vacuum (2 h). The resulting white residues were dissolved in 2 M
acetic acid, the insoluble impurities were removed by passing the
solutions through Gelman Laboratory Acrodisc 13 mm syringe fil-
ters with 0.45 ␮M PTFE membranes (Pall Corporation, East Hills,
NY), and the clear filtrates were lyophilized. The resulting crude lin-
ear peptides were cyclized by air oxidation as follows: the obtained
crude peptides (∼200–250 mg) were dissolved in glacial acetic acid
(0.35 mL), diluted with water (450 mL) and acetonitrile (130 mL) to
a peptide concentration of about 0.5 mM, and to the resulting solu-
tion aqueous ammonia was added to adjust the pH to about 7.0,
as determined with pH paper. The resulting peptide solution in a
0.01 M ammonium acetate buffer was stirred for 36–72 h, until the
cyclization was complete, as determined by HPLC and MS analysis.
The peptide solution was then acidified by addition of glacial acetic
acid (1 mL), acetonitrile was removed under reduced pressure, and
the residual solution was lyophilized. The obtained white powders
(200–250 mg) were re-dissolved in glacial acetic acid (2 mL), the
resulting solutions were diluted with water (8 mL) to a peptide con-
centration of about 20 mg/mL, and passed through a Sephadex G-15
column (520 mm × 30 mm) using 1 M aqueous acetic acid as the
eluent. Fractions containing the target peptides, as determined by
TLC, were combined and lyophilized. Final purification was accom-
plished by preparative RP-HPLC on a C18-bonded silica column
The structural uniqueness of the Agouti protein pharma-
cophores presents intriguing opportunities for exploration of novel
melanocortin templates beyond the tetrapeptide pharmacophore
His-Phe-Arg-Trp of the endogenous agonists ␣-, ␤- and ␥-MSH to
expedite development of highly potent and selective melanocortin
ligands.
2. Materials and methods
2.1. Materials
N␣-Fmoc-amino acids, peptide coupling reagents and Rink
amide AM resin were obtained from Novabiochem (San Diego,
CA), except N␣-Fmoc-Glu(OAll)-OH, which was purchased from
NeoMPS (San Diego, CA). The following side chain protecting groups
␧
were used: Trp(Nin-Boc), Arg(N -Pbf), Cys(S-Trt). ACS grade organic
solvents were purchased from VWR Scientific (West Chester, PA),
and other reagents were obtained from Sigma–Aldrich (St. Louis,
MO) and used as commercially available. The polypropylene reac-
tion vessels (syringes with frits) were purchased from Torviq
(Niles, MI). The purity of the peptides was checked by analytical
reverse-phase HPLC using a Vydac C18 218TP104 column (Western
Analytical Products, Murrieta, CA) monitored at 230 and 254 nm,
and was determined to be >97% for all peptides reported herein.
Additionally, the purity of the peptides was evaluated by thin-layer
chromatography (TLC), which was performed using three differ-
ent solvent systems. Analytical thin-layer chromatography (TLC)
was carried out on 0.25 mm glass-backed silica gel 60 F254 plates
(EM Science 5715, VWR Scientific). The TLC chromatograms were
visualized by UV light, and by dipping in potassium permanganate
solution followed by heating (hot plate).
˚
(Vydac 218TP152022, 250 mm × 22 mm, 15–20 ␮m, 300 A) using
a Shimadzu SCL-10A HPLC system. The peptides were eluted with
a stepwise gradient of 10% (0–5 min), 20–22% (7–17 min), 34–35%
(20–30 min), and 38–40% (32–45 min) acetonitrile in 0.1% aqueous
TFA solution with 10 mL/min flow rate. The purified peptides were
isolated in 20–25% overall yield.
2.2. Peptide synthesis
All peptides in this study were synthesized manually by the
N␣-Fmoc solid-phase methodology [64,65], using Bromophenol
Blue pH indicator to monitor the extent of coupling reactions
as described by Krchnak et al. [58] Rink amide AM resin (4-
(2ꢀ,4ꢀ-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, 0.5 g,
0.637 mmol/g) was placed into a 50 mL polypropylene syringe with
the frit on the bottom and swollen in DMF (20 mL) for 1 h. The
Fmoc protecting group on the Rink linker was removed by 25%
piperidine in DMF (1× 5 min and 1× 15 min). The resin was washed
with DMF (4× 15 mL), then washed with 0.02 M HOBt solution in
DMF, stained with 0.05 mM solution of Bromophenol Blue in 0.02 M
HOBt/DMF solution, and washed with 0.02 M HOBt/DMF solution
2.4. Solid-phase lactam cyclodimerizations (method A)
The orthogonal allyl ester protection for the side chain of Glu
was removed with 0.1 equiv. Pd(PPh3)4/20 equiv. PhSiH3 in DCM
(2× 30 min) prior to the peptide cyclization [88]. The deprotected
resin-bound peptide was washed with DCM (6× 5 mL), and DMF
(3× 5 mL). The peptide cyclodimerizations were found to pro-
ceed in facile manner under the standard cyclization conditions
described previously [64], with 6 equiv. DIC, 6 equiv. Cl-HOBt in
THF (72 h) [81], and were monitored by Kaiser ninhydrin test [49].

A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
1897
Upon completion of cyclization the resin was treated with 5% solu-
tion of sodium diethyldithiocarbamate trihydrate in DMF (20 min)
to remove any remaining traces of the Pd catalyst [64,65], then
washed with DMF (5× 15 mL), DCM (3× 15 mL), methanol (5×
15 mL), and diethyl ether (5× 15 mL), and dried under reduced pres-
sure (16 h). The cyclized peptides were cleaved off the solid support
with 95% (v/v) TFA, 2.5% water, and 2.5% triisopropylsilane (5 mL,
3 h), and the crude peptides were precipitated out by the addi-
tion of a chilled 3:1 mixture of diethyl ether and petroleum ether
(50 mL) to give white precipitates. The resulting peptide suspen-
sions were centrifuged for 10 min at 6500 rpm, and the liquid was
decanted. The crude peptides were washed with diethyl ether (4×
50 mL), and after the final centrifugation, the peptides were dried
under vacuum (2 h). The resulting white residues were dissolved
in 2 M acetic acid, and the insoluble impurities were removed by
passing the solutions through Gelman Laboratory Acrodisc 13 mm
syringe filters with 0.45 ␮M PTFE membranes (Pall Corporation,
East Hills, NY), and the clear filtrates were lyophilized. Purifica-
tion was accomplished by preparative RP-HPLC on a C18-bonded
silica column (Vydac 218TP152022, 250 mm × 22 mm, 15–20 ␮m,
by methods A and B were determined to be identical according to
HPLC, HR-MS and 1H NMR analyses.
2.6. Receptor binding assay
Competition binding experiments were carried out using whole
HEK293 cells stably expressing human MC1, MC3, MC4, and MC5
receptors as described before [64–66]. HEK293 cells transfected
with hMCRs [7,26,35] were seeded on 96-well Plates 48 h before
assay (50,000 cells/well). For the assay, the cell culture medium
was aspirated and the cells were washed once with a freshly pre-
pared MEM buffer containing 100% minimum essential medium
with Earle’s salt (MEM, GIBCO), and 25 mM sodium bicarbon-
ate. Next, the cells were incubated for 40 min at 37 ◦C with
different concentrations of unlabeled peptide and labeled [125I]-
[Nle4,D-Phe7]-␣-MSH (PerkinElmer Life Science, 20,000 cpm/well,
33.06 pM) diluted in a 125 ␮L of freshly prepared binding buffer
containing 100% MEM, 25 mM HEPES (pH 7.4), 0.2% bovine serum
albumin, 1 mM 1,10-phenanthrolone, 0.5 mg/L leupeptin, 200 mg/L
bacitracin. The assay medium was subsequently removed, the cells
were washed once with basic medium, and then lysed by the addi-
tion of 100 ␮L of 0.1 M NaOH and 100 ␮L of 1% Triton X-100. The
lysed cells were transferred to 12 mm × 75 mm borosilicate glass
tubes, and the radioactivity was measured by a Wallac 1470 WIZ-
ARD Gamma Counter.
˚
300 A) using a Shimadzu SCL-10A HPLC system. The peptides
were eluted with a stepwise gradient of 10% (0–5 min), 20–22%
(7–17 min), 34–35% (20–30 min), and 38–40% (32–45 min) acetoni-
trile in 0.1% aqueous TFA solution with 10 mL/min flow rate. The
purified peptides were isolated in 20–25% overall yield. The molec-
ular compositions of the purified peptides were confirmed by 1H
NMR in DMSO-d6, and the dimeric structures were assigned by high
resolution electrospray ionization (ESI) mass-spectrometry using
an IonSpec Fourier transform mass spectrometer with a HiRes ESI
source.
2.7. Adenylate cyclase assay
HEK 293 cells transfected with human melanocortin receptors
[7] were grown to confluence in MEM medium (GIBCO) containing
10% fetal bovine serum, 100 units/mL penicillin and streptomycin,
and 1 mM sodium pyruvate. The cells were seeded on 96-well plates
48 h before assay (50,000 cells/well). For the assay, the cell culture
medium was removed and the cells were rinsed with 100 ␮L of
MEM buffer (GIBCO). An aliquot (100 ␮L) of the Earle’s balanced salt
solution with 5 nM isobutylmethylxanthine (IBMX) was placed in
each well along for 1 min at 37 ◦C. Next, aliquots (25 ␮L) of melan-
otropin peptides of varying concentration were added, and the cells
were incubated for 3 min at 37 ◦C. The reaction was stopped by
aspirating the assay buffer and adding 60 ␮L ice-cold Tris/EDTA
buffer to each well, then placing the plates in a boiling water
bath for 7 min. The cell lysates were then centrifuged for 10 min
at 2300 × g. A 50 ␮L aliquot of the supernatant was transferred to
another 96-well plate and placed with 50 ␮L [3H] cAMP and 100 ␮L
protein kinase A (PKA) buffer in an ice bath for 2–3 h. The PKA
buffer consisted of Tris/EDTA buffer with 60 ␮g/mL PKA and 0.1%
bovine serum albumin by weight. The incubation mixture was fil-
tered through 1.0 ␮m glass fiber filters in MultiScreenTM-FB96-well
plates (Millipore, Billerica, MA). The total [3H] cAMP was measured
by a Wallac MicroBeta TriLux 1450 LSC and Luminescence Counter
(PerkinElmer Life Science, Boston, MA). The cAMP accumulation
data for each peptide analogue was determined with the help of a
cAMP standard curve generated by the same method as described
above. IC50 and EC50 values represent the mean of two experiments
performed in triplicate. IC50 and EC50 estimates and their associated
standard errors were determined by fitting the data using a non-
linear least squares analysis, as implemented in GraphPad Prism 4
(GraphPad Software, San Diego, CA). The maximal cAMP produced
at 10 ␮M concentration of each ligand was compared to the amount
of cAMP produced at 10 ␮M concentration of the standard agonist
MT-II, and is expressed in per cent (as % max effect) in Table 2. The
antagonist properties of the lead compounds were evaluated by
their ability to competitively displace the MT-II agonist in a dose-
dependent manner, at up to 10 ␮M. The pA2 values were obtained
using the Schild analysis method [84].
2.5. Targeted synthesis of cyclodimeric analogue 12 (method B)
The Fmoc-Arg(Pbf)-D-Phe-Glu(OAllyl)-Trp(Boc)-NH2 tetrapep-
tide sequence was constructed on highly acid-labile Sieber amide
resin (0.46 g, 0.64 mmol/g) [86]. The Glu side chain allylic ester pro-
tection was removed as previously described [64,88]. The cleavage
of the resulting partially protected Fmoc-Arg(Pbf)-D-Phe-Glu(OH)-
Trp(Boc)-NH2 carboxylic acid peptide off the solid support was
achieved by treating the resin with 2% TFA/1% triisopropylsi-
lane (TIPS) in dichloromethane (5× 2 min), while collecting the
TFA/DCM solution into a 300 mL round-bottom flask, containing
50 mL pyridine and 100 mL methanol. After the cleavage process
was complete, the resin was washed thrice with methanol and
the washes were added to the same pyridine/methanol mixture.
The resulting solution was concentrated under reduced pressure
and the syrupy residue was triturated with deionized water to
remove pyridinium-TFA salts. The resulting white precipitate was
centrifuged for 10 min at 6500 rpm, and the liquid was decanted.
The crude peptide was washed with water (4× 50 mL), and after
the final centrifugation, the precipitate was dried under vac-
uum (12 h) to yield 172 mg of crude partially protected peptide
(48%). In parallel, the same sequence was prepared on Rink amide
AM resin (∼0.3 mmol scale), and its N-terminal Fmoc protection
was removed with 25% piperidine/DMF. The crude partially pro-
tected tetrapeptide Fmoc-Arg(Pbf)-D-Phe-Glu(OH)-Trp(Boc)-NH2
was coupled onto the exposed N-terminal amino group with
DIC/HOAt over 4 h, followed by washing the resin with DMF
(4×) and DCM (4×), and capping of the remaining amino groups
with acetic anhydride/pyridine/DCM (1:2:20, v/v/v) to preclude
cyclodimerization of the residual monomeric peptide during the
cyclization of the dimer. The N-terminal Fmoc and Glu side chain
allyl ester protecting groups were removed and macrocyclization
was induced with DIC/Cl-HOBt in THF as described above. TFA
cleavage/deprotection and purification followed the procedures
described above for the method A. The peptide 12 samples prepared

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A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
Fig. 2. Stereo view of the superimposed global minimum of analogue 6 (blue) obtained by MCLM (Monte Carlo/Low Frequency Mode)/OPLS 2005 (GB/SA) simulation with
˚
the NMR structure of non-selective super-agonist MT-II (gold) (rmsd = 0.12 A, non-hydrogen backbone atoms of the Xaa-D-Phe-Yaa-Trp pharmacophores only). Hydrogens
are omitted for clarity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.8. Computational procedures
Arg and Xaa, respectively, where the Xaa was to be an amino acid
with functionality suitable for macrocyclization of the hybrid pep-
tide analogue, i.e., a “scaffolding” residue. Such hybridization would
maintain the order of amino acid functionality characteristic for
the agouti proteins (Arg-aromatic AA-aromatic AA), while preserv-
ing the secondary structure and the spacing between the aromatic
amino acids observed in MSH analogues. The global constraint was
applied via macrocyclization using a cyclic disulfide template sim-
ilar to the one reported previously by our laboratories to furnish
a highly hMC4R-selective allosteric antagonist series [103], and a
lactam cyclodimerization. This design approach was expected to
result in the series of hybrid MSH/ASIP analogues that would retain
some biological properties of both parent compounds, leading to
novel SAR trends and new potent and highly selective melanotropin
peptides.
Molecular modeling experiments employed MacroModel
version 9.1 equipped with Maestro 7.5 graphical interface
(Schrödinger, LLC, New York, NY, 2005) installed on a Linux Red
Hat 9.0 system, and were performed as previously described
[3,64,65]. Peptide structures were built into extended structures
with standard bond lengths and angles, and they were mini-
mized using the OPLS 2005 force field [51] and the Polak–Ribier
conjugate gradient (PRCG). Optimizations were converged to a
gradient RMSD less that 0.05 kJ/Å mol or continued until a limit of
50,000 iterations was reached. Aqueous solution conditions were
simulated using the continuum dielectric water solvent model
˚
(GB/SA) [87]. Extended cut-off distances were defined at 8 A for
˚
˚
Van der Waals, 20 A for electrostatics and 4 A for H-bonds.
Conformational profiles of the cyclic peptides were investigated
by the hybrid Monte Carlo/Low Frequency Mode (MCMM/LMCS)
[56] procedure as implemented in Macromodel using the energy
minimization parameters as described above. MCMM torsional
variations and Low Mode parameters were set up automatically
within Maestro graphical user interface. A total of 20,000 search
steps were performed and the conformations with energy dif-
ference of 50 kJ/mol from the global minimum were saved. The
superimpositions of peptide structures were performed using the
␣-carbons of the core sequences Arg-Phe-Xaa-Trp and His-D-Phe-
Arg-Trp.
3.2. Molecular modeling
The hybrid Monte Carlo/Low Frequency Mode (MCMM/LMCS)
[56] simulations employed the OPLS 2005 force field [51] and
GB/SA implicit aqueous solvation model [87], as implemented
in Macromodel v9.1 modeling software package, and predicted
the expected ␤-turn-like secondary structure centered over the
Arg-Phe residues in the cyclic disulfide series, similar to the
3D structures of the previously reported cyclic disulfide N␣-
guanidinylbutyl-Cys8 analogues of ␣-MSH, obtained by simulated
annealing-MD/OPLS-AA experiments [103]. Comparison of the
structures of the hybrid ASIP/MSH peptides and the cyclic ␣-MSH
analogue MT-II revealed that the binding space of Arg8 (MT-II) is
blocked in the new peptides by the disulfide bridge (Fig. 2). While
steric hindrance of Arg8 binding space may be beneficial for devel-
opment of highly selective melanocortin peptides, as demonstrated
by our earlier reports [64,65], we recognized that it could also
impair the receptor–ligand interactions and lead to a decline in
binding affinity and agonist potency.
3. Results
3.1. Design of the MSH/ASIP hybrid pharmacophore
The design of the MSH/ASIP hybrid pharmacophore capitalized
on the reported 3D NMR structures of ASIP [67] and the cyclic ␣-
MSH analogue MT-II [104]. Both structures feature a ␤-turn-like
motif within the pharmacophore region, which spans over the first
two residues in the His-D-Phe-Arg-Trp MSH pharmacophore, and
the over the last two residues of the Arg-Phe-Phe-Arg ASIP phar-
macophore. The chosen approach to the hybridization of these two
seemingly distinct tetrapeptide sequences involved replacement
of His6 and Arg8 residues within the MSH pharmacophore with
The computational experiments predicted the cyclodimeric
peptides to possess C2-symmetrical secondary structures, stabi-
lized by two ␤-turns, also centered at the Arg-Phe residues (Fig.
3). The presence of the ␤-turn feature in the 3D structures of the
designed peptides, requisite for the melanocortin receptor recog-

A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
1899
Fig. 3. Stereo view of the global minimum of analogue 12, obtained by MCMM/LMCS (Monte Carlo multiple minima-low frequency mode)-OPLS 2005 (GB/SA) simulations.
Hydrogens are omitted for clarity.
Scheme 2. Solid-phase synthesis of cyclodimeric ␣-MSH/ASIP hybrid peptides.

1900
A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
Table 1
Sequences and the physicochemical properties of the cyclic homodimeric ␣-MSH/AgRP analogues.
b
No.
Sequence
m/z (M+H+)
m/z (M+2H+)
HPLC retention time,
mina
TLC Rf
Calcd.
Obsd. (ESI)
Calcd.
Obsd. (ESI)
1
2
1
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Ac-c[Cys-Arg-Phe-Cys]-Trp-NH2
Ac-c[Cys-Arg-D-Phe-Cys]-Trp-NH2
Ac-c[Cys-Arg-D-Phe-Cys]-D-Trp-NH2
Ac-c[Cys-Arg-Phe-Cys]-D-Trp-NH2
c[S(CH2)2CO-Arg-Phe-Cys]-Trp-NH2
c[S(CH2)2CO-Arg-D-Phe-Cys]-Trp-NH2
c[S(CH2)2CO-Arg-D-Phe-Cys]-D-Trp-NH2
c[S(CH2)2CO-Arg-Phe-Cys]-D-Trp-NH2
c[Arg-D-Phe-Glu]-Trp-NH2
753.2965
753.2965
753.2965
753.2965
696.2750
696.2750
696.2750
696.2750
618.3152
618.3152
1235.6227
1235.6227
1235.6227
1235.6227
1335.6534
1335.6534
1235.6227
1335.6534
753.2934
753.2931
753.2939
753.2946
696.2716
696.2733
696.2723
696.2733
618.3161
618.3159
1235.6351
1235.6264
1235.6173
1235.6046
1335.6915
1335.6729
1235.6147
1335.6855
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
15.667
14.742
14.567
15.725
16.525
15.283
15.050
16.350
14.700
14.825
17.200
17.325
17.292
17.283
19.650
19.742
16.958
20.042
24.958
23.517
23.650
25.675
25.583
23.792
23.358
24.967
24.892
23.533
28.083
26.642
26.858
27.450
31.400
31.442
27.875
32.000
0.64
0.64
0.63
0.61
0.64
0.65
0.64
0.64
0.54
0.53
0.57
0.62
0.61
0.56
0.61
0.66
0.59
0.66
0.38
0.41
0.39
0.43
0.47
0.45
0.43
0.41
0.35
0.34
0.34
0.34
0.33
0.33
0.34
0.41
0.33
0.40
–
–
–
–
c[Arg-D-Phe-Glu]-D-Trp-NH2
(c[Arg-Phe-Glu]-Trp-NH2)2
(c[Arg-D-Phe-Glu]-Trp-NH2)2
618.3153
618.3153
618.3153
618.3153
668.3306
668.3306
618.3153
668.3306
618.3221
618.3180
618.3127
618.3181
668.3377
668.3323
618.3183
668.3360
(c[Arg-D-Phe-Glu]-D-Trp-NH2)2
(c[D-Arg-Phe-Glu]-Trp-NH2)2
(c[Arg-Nal(2ꢀ)-Glu]-Trp-NH2)2
(c[Arg-D-Nal(2ꢀ)-Glu]-Trp-NH2)2
(c[D-Phe-Arg-Glu]-Trp-NH2)2
(c[D-Nal(2ꢀ)-Arg-Glu]-Trp-NH2)2
a
˚
HPLC column: Vydac 218TP104, 250 mm × 4.6 mm, 10 ␮m, 300 A; HPLC solvent system 1: solvent A, 0.1% TFA in water; solvent B, 0.09% TFA in acetonitrile; gradient:
10–90% B in A over 40 min, flow rate 1.0 mL/min; HPLC solvent system 2: solvent A, 0.1% TFA in water; solvent B, 0.09% TFA in methanol; gradient: 10–90% B in A over 40 min,
flow rate 1.0 mL/min.
b
TLC system 1: n-butanol/acetic acid/water/pyridine (4:1:2:1); TLC system 2: n-butanol/acetic acid/water (4:1:1).
nition, binding and receptor activation [92,104], encouraged us to
synthesize several analogues using both monomeric cyclic disul-
fide and cyclodimeric lactam templates, and explore the dihedral
space of these new peptides via D-amino acid substitutions of the
Arg, Phe, and Trp residues.
the residual monomeric peptide during the cyclization of the
dimer. The N-terminal Fmoc and Glu side chain allyl ester pro-
tecting groups were removed and macrocyclization was induced
with DIC/Cl-HOBt. TFA cleavage/deprotection followed by HPLC
purification gave the cyclodimeric analogue 12, which was chro-
matographically and spectroscopically undistinguishable from
the peptide obtained through a direct on-resin cyclodimeriza-
tion thus unambiguously establishing its intrinsically anti-parallel
cyclodimeric structure (Fig. 4).
3.3. Chemistry
The peptides of the cyclic disulfide series were synthe-
sized by standard Fmoc/tBu chemistry on Rink amide AM resin.
TFA deprotection/cleavage off the solid support provided the
crude linear peptides, which were cyclized by air oxidation in
0.01 M ammonium acetate buffer at pH 7.5. The peptide lactam
cyclodimerizations were achieved by construction of the linear
tetrapeptide sequences on polystyrene-based Rink amide AM resin
at 0.5–0.6 mmol/g loading, consecutive removal of the N-terminal
Fmoc and Glu(OAll) protecting groups, and exposure of the depro-
tected resin-bound peptide to DIC/6-Cl-HOBt in THF over 72 h
(Scheme 2). This procedure resulted in a predictably low yield
(<5%) of the monomeric cyclic tetrapeptide products due to the
high strain within their 11-membered lactam ring [57], and led
predominantly to the corresponding 22-membered cyclodimers,
which were isolated in 25-30% yields after TFA cleavage off the
solid support and HPLC purification (Scheme 2, method A).
The cyclodimeric structure was assigned to these peptides
on the basis of HR-MS and 1H NMR data (Table 1, Fig. 4),
and unequivocally confirmed through the targeted synthesis of
analogue 12 (Scheme 2, method B). The partially protected Fmoc-
Arg(Pbf)-D-Phe-Glu(OH)-Trp(Boc)-NH2 tetrapeptide was obtained
by constructing the sequence on highly acid-labile Sieber amide
resin (9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin) [86], Pd0-
mediated deprotection of the Glu side chain [88], and cleavage
of the resulting carboxylic acid peptide off the solid support
with 2% TFA/1% TIPS in dichloromethane. In parallel, the same
sequence was prepared on Rink amide AM resin, its N␣-terminal
Fmoc protection was removed, and the crude partially pro-
tected tetrapeptide Fmoc-Arg(Pbf)-D-Phe-Glu(OH)-Trp(Boc)-NH2
was coupled onto the exposed N␣-terminal amino group with
DIC/HOAt over 4 h, followed by capping of the remaining amino
groups with acetic anhydride to preclude cyclodimerization of
3.4. Competition binding and cAMP assays
Biological evaluation of the ASIP/␣-MSH hybrid peptides was
performed using human melanocortin receptors (hMC1R, hMC3-
5R) expressed in whole HEK293 cells, as summarized in Table 2.
The monomeric cyclic disulfide analogues 1–4 showed no bind-
ing affinity or receptor activation at the hMC1R, hMC4-5R. At the
same time, these peptides exhibited weak binding affinity to the
hMC3R within the 0.42–5.00 ␮M range, and competitively displac-
ing only 35-59% of 125I-labeled NDP-␣-MSH, similar to the previous
precedents [10,103]. In addition, analogue 2 exhibited a weak par-
tial agonist activity at the hMC3R (EC50 = 2.4 ␮M, 28% maximal
cAMP). Replacement of the chiral N-acetyl-Cys linker arm in the
cyclic disulfide template with an achiral ␤-mercaptopropionic acid
(Mpa) linker arm (analogues 5-8) resulted in a complete loss of both
binding affinity and agonist activity at all four receptor subtypes.
The small quantities of the monomeric cyclic lactam analogues
9 and 10, isolated as by-products of direct on-resin cyclodimeriza-
tions leading to the peptides 12 and 13, were examined in binding
and cAMP assays, and were found to possess neither binding affin-
ity nor agonist activity at all four human melanocortin receptor
subtypes.
The all L-diastereomer 11 showed faint non-competitive bind-
ing affinity at the hMC5R (IC50 = 2.6 ␮M) and a weak partial agonist
activity at the hMC1R (EC50 = 2.5 ␮M, 25% maximal cAMP). The D-
Phe7 analogue 12 displayed an improved competitive binding at
the hMC1R and the hMC4R (IC50 = 1.9 ␮M and 1.4 ␮M, respectively),
while binding at the hMC3R and the hMC5R remained apparently
non-competitive (IC50 = 2.9 ␮M, 45 and 62% binding efficiency,
respectively). At the same time, the cAMP assay revealed a nanomo-

A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
1901
Fig. 4. HPLC chromatograms of the crude cyclodimeric peptide 12 synthesized by methods A and B. 1H NMR spectra of the purified cyclodimeric peptide 12 synthesized by
methods A and B.
lar range full agonist activity at the hMC1R (EC50 = 120 nM) and
partial agonist activity at the hMC4R (EC50 = 22 nM, 46% cAMP stim-
ulation), and weak partial agonist activities at the other receptor
subtypes (hMC3R: EC50 = 1.7 ␮M, 70% cAMP stimulation; hMC5R:
EC50 = 1.6 ␮M, 59% cAMP stimulation). A similar pharmacological
profile was observed for the D-Phe7, D-Trp9 analogue 13, which
exhibited weak micromolar range binding at four receptor sub-
types, weak partial agonist activity at the hMC3R (EC50 = 4.2 ␮M,
60% cAMP stimulation) and the hMC5R (EC50 = 2.4 ␮M, 72% cAMP
stimulation), and nanomolar range partial agonist activity at the
hMC1R (EC50 = 240 nM, 75% cAMP stimulation) and the hMC4R
(EC50 = 26 nM, 40% cAMP stimulation).
binding efficiency), the hMC3R (IC50 = 80 nM, 23% binding effi-
ciency) and the hMC5R (IC50 = 450 nM, 45% of 125I-NDP-␣-MSH
displaced), and retained a weak partial agonist activity at the
hMC1R (EC50 = 2.7 ␮M, 42% cAMP stimulation). The D-Nal(2ꢀ)7 ana-
logue 18 showed diminished, albeit competitive, micromolar range
receptor binding, and slightly improved nanomolar range partial
agonist activity at the hMC1R (EC50 = 200 nM, 42% cAMP stimula-
tion).
4. Discussion
The outcome of the biological evaluation of analogues 1–18 was
analyzed from the perspective of the performance of the hybrid
MSH/ASIP pharmacophore in monomeric cyclic disulfide and lac-
tam templates, as well as the impact of its cyclodimerization on the
biological profiles of the resulting cyclodimeric peptides. The weak
non-competitive binding affinities and micromolar range partial
agonist activities determined for the monomeric cyclic disulfide
analogues 1-8 suggest that the designed ASIP/MSH hybrid pharma-
cophore in this template may not be sufficient to induce a potent
binding affinity and agonist activity at the human melanocortin
receptors-1, -4, and -5, while possessing a potential for develop-
ment of non-competitive (possibly, allosteric) molecular probes for
the hMC3R. Also, the observed strong influence of the linker arm
structure on the peptide binding affinity is completely congruent
with our previous reports [52,65,66].
Furthermore, the monomeric cyclic lactam analogues 9 and 10
exhibited no binding affinity or agonist activity at the hMC1,3-5R,
and one possible explanation for this result was found by exam-
ination of 1H NMR spectra of these peptides. The complex 1H
NMR spectra of the monomeric cyclic lactams (Fig. 5) indicates a
presence of three possible cis/trans ␻-rotamers, in dynamic equi-
librium with one another, as could be expected from a dihedrally
D-Arg6 substitution (analogue 14) resulted in slightly improved
non-competitive binding affinities at the receptor subtypes, com-
pared to the L-diastereomeric peptide 11. The weak hMC1R agonist
activity observed in analogue 11 also substantially increased in ana-
logue 14 to yield a potent (EC50 = 25 nM, 43% cAMP stimulation) and
highly selective (>400-fold) hMC1R partial agonist.
Phe7 → Nal(2ꢀ)7 substitutions (analogues 15 and 16) resulted in
improved competitive binding affinities, compared to the Phe7/D-
Phe7 peptide analogues 11 and 12, with the D-Nal(2ꢀ)7 peptide 16
showing IC50 values in the high nanomolar range of 360–800 nM.
Expectedly, the hMC1R partial agonist activity was retained in these
two peptides (analogue 15: EC50 = 470 nM, 34% cAMP stimulation;
analogue 16: EC50 = 25 nM, 55% cAMP stimulation), although selec-
tivity was lost, with partial agonist activity detected at the hMC3R
and the hMC5R.
Finally, the effects of cyclodimerization were examined on the
MSH pharmacophore D-Phe-Arg-Trp through inserting of a Glu
linker arm between Arg8 and Trp9 and subjecting the result-
ing D-Phe7-Arg8-Glu9-Trp10 sequence to cyclodimerization. The
obtained 22-membered cyclodimeric peptide 17 showed good par-
tial non-competitive binding at the hMC1R (IC50 = 120 nM, 22%

1902
A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
Fig. 5. The convoluted 1H NMR spectra of the monomeric cyclic lactam 9 indicates
presence of three possible cis/trans ␻-rotamers.
strained 11-membered lactam ring [57]. The lack of a well-defined
secondary structure in the monomeric cyclic lactam peptides
may impair receptor–ligand interaction, and, by the same token,
the less strained larger macrocycles may have more stable sec-
ondary structures, as evident from the 1H NMR spectra of the
cyclodimeric peptides (such as analogue 12, Fig. 4), which would
lead to augmented receptor binding affinity and agonist potency.
This hypothesis was put to the test in screening of the biolog-
ical properties of the cyclodimeric peptides 11–18. Indeed, the
cyclodimeric analogues exhibited a marked improvement in their
binding affinities as well as displayed nanomolar range full and
partial agonist activities at the hMC1R and the hMC4R. Thus, the
designed ASIP/MSH hybrid pharmacophore in the cyclodimeric
template clearly favors the hMC1R and the hMC4R subtypes, which
may be due to the tight secondary structure of the cyclodimeric
template (Fig. 3), unencumbered by steric or hydrophobic influ-
ence of the neighboring amino acids, which has been previously
shown to favor the hMC3R and the hMC5R, and produce agonists
and antagonists selective for these receptor subtypes [3,7,64,65].
D-substitution in the position 6 (analogue 14), normally asso-
ciated with complete loss of all binding affinity and potency in
MSH-derived templates [37], in the ASIP/MSH hybrid template led
to somewhat improved non-competitive binding affinities at the
receptor subtypes, compared to the L-diastereomeric peptide 11.
This finding suggests that the secondary structure requirements
for non-competitive (allosteric) agonists are not limited to a par-
ticular type of ␤-turn, as is the case for orthosteric agonists derived
from MSH peptides [104].
Phe7 → Nal(2ꢀ)7 substitutions in MSH peptides are generally
known to lead to enhanced binding affinity and conversion
of hMC3R/hMC4R agonists into antagonists, while retaining
hMC1R/hMC5R agonist activities [42]. Similar substitutions in
the ASIP/MSH hybrid pharmacophore-based cyclodimeric peptides
(analogues 15 and 16) indeed resulted in improved competitive
binding affinities, compared to the Phe7/D-Phe7 peptide analogues
11 and 12, with the D-Nal(2ꢀ)7 peptide 16 showing IC50 values in
the high nanomolar range of 360-800 nM, while retaining nanomo-
lar range hMC1R partial agonist activity. This SAR trend is in a good
agreement with our earlier reports, which linked the augmented
binding affinity with increased hydrophobicity of the Nal(2ꢀ)7/D-
Nal(2ꢀ)7 peptides [64,65].

A.V. Mayorov et al. / Peptides 31 (2010) 1894–1905
1903
Fig. 6. Schematic representations of putative receptor–ligand interactions of ␣-MSH-derived peptides, monomeric ASIP/MSH hybrid peptide 6, and a cyclodimeric peptide
12, showing one possible mode of receptor binding.
The MSH pharmacophore-bearing peptides 17 and 18 showed
reasonable (high nanomolar range) non-competitive binding affini-
ties at the hMC1,3,5R but unremarkable (micromolar range) partial
agonist activities at the hMC1R. This result indicates that the
cyclodimeric template described in this report was perhaps unable
to maintain the optimal topography for the MSH pharmacophore.
A more in-depth D-amino acid scan of the template will allow to
determine the full scope of its capabilities.
Acknowledgments
This research was supported by grants from the U.S. Public
Health Service, National Institutes of Health DK-17420 and DA-
06284. The opinions expressed are those of the authors and not
necessarily those of the USPHS.
Appendix A. Supplementary data
In summary, the design of a novel ASIP/MSH hybrid phar-
macophore has yielded peptides with hMC3R-selective non-
competitive binding affinities in the monomeric cyclic disulfide
series. The reported direct on-resin peptide cyclodimerization
yielded nanomolar range (25–120 nM) hMC1R-selective full and
partial agonists in the cyclodimeric lactam series, which represents
a 25-120-fold lower agonist potency than that of the super-
agonist MT-II but demonstrates an improvement over the previous
attempts at hybridization of MSH and agouti protein sequences
[46]. Among the possible reasons for the acquired agonist activ-
ity are the well-established factors, such as increased charge [64]
and increased hydrophobicity [64,65], or thermodynamic aspects,
such as the rotational entropy factor [2,78]. In addition, a possibility
that receptor recognition occurs at an overlap region of the dimeric
pharmacophore structures (Fig. 6) cannot be discounted.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.peptides.2010.06.026.
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