Novel selective human melanocortin-3 receptor ligands: Use of the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold

Article abstract of DOI:10.1016/j.bmcl.2007.02.020

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-d-Phe-Arg-Tr

Full text of DOI:10.1016/j.bmcl.2007.02.020

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2492–2498
Novel selective human melanocortin-3 receptor ligands: Use of
the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold
Steven Ballet,a,* Alexander V. Mayorov,b Minying Cai,b Dagmara Tymecka,c
Kevin B. Chandler,b Erin S. Palmer,b Karolien Van Rompaey,a Aleksandra Misicka,c
a
b
´
Dirk Tourwe and Victor J. Hruby
aDepartment of Organic Chemistry, Vrije Universiteit Brussel, B-1050 Brussels, Belgium
bDepartment of Chemistry, University of Arizona, Tucson, AZ 85721, USA
cFaculty of Chemistry, Warsaw University, PL-02-093 Warsaw, Poland
Received 25 January 2007; revised 6 February 2007; accepted 7 February 2007
Available online 9 February 2007
Abstract—In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R
to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-
Phe-Arg-Trp-Lys]-NH2) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(20)-Arg-Trp-Lys]-NH2) by
replacing the His-D-Phe and His-D-Nal(20) fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetra-
hydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx = D-Phe/pCl-D-Phe/D-Nal(20)). Employment of the Aba mimetic yielded
novel selective high affinity hMC3R and hMC3R/hMC5R antagonists.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The a-melanocyte stimulating hormone (a-MSH, Ac-
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-
Val- NH2) plays a role in a wide range of biological
responses like feeding behavior, pain modulation, learn-
ing behavior, pigmentation, sexual function, energy
homeostasis, and thermoregulation.1 In particular, the
human melanocortin 4 subtype receptor is an attractive
drug target because of its role in regulation of feeding
behavior.2,3 Design of selective hMC4R antagonists is
considered to have great potential for the treatment of
anorexia.4,5 The hMC3R on the other hand has been
shown to play a role in the physiological process of ener-
gy partitioning and body weight.6 Controlled modula-
tion of these receptors could lead to promising results
in the field of feeding disorders.
conformational analysis of a variety of cyclic analogues
led to the discovery of a superpotent lactam analogue
MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2).9
This cyclic peptide analogue, first introduced by Al-Obe-
idi et al.9 was a very potent, but non-selective, agonist
for the human melanocortin receptor subtypes MC1R,
MC3R, MC4R, and MC5R. It also showed a very high
stability against all proteolytic enzymes and tissue
homogenates.9 Because of the lack in selectivity, finding
selective ligands for each of the four human melanocor-
tin receptors (hMC1R, hMC3R to hMC5R) was conse-
quently crucial for the determination of their individual
physiological roles. Development of selective melano-
cortin ligands can help to ascribe specific biological
functions for the corresponding receptor subtypes.
The principal pharmacophore groups of a-MSH were
found to be the side chains of the central tetrapeptide
His6-Phe7-Arg8-Trp9.7,8 Molecular modeling as well as
Several parameters of MT-II (e.g., ring size, introduction
of hydrophobic groups) were modified to design potent
and more selective peptide analogues.2,10,11 Recently,
Grieco et al. reported the influence of replacing the
His6 by Pro in MT-II, which resulted in retention of ago-
nist potency for most melanocortin receptors.11 Upon
substitution of His6 in SHU9119, a potent hMC3R/
hMC4R antagonist (Ac-Nle-c[Asp-His-D-Nal(20)-Arg-
Trp-Lys]-NH2),12 by conformationally restricted amino
acids, selective antagonists for the hMC3R and hMC4R
Keywords: Human melanocortin receptors; 4-Amino-1,2,4,5-tetrahy-
dro-2-benzazepin-3-ones; Cyclic lactam analogues; Conformational
restrictions; hMC3R/hMC5R antagonists.
*
Corresponding author. Tel.: +32 26293298; fax: +32 26293
304; e-mail: sballet@vub.ac.be
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.020

S. Ballet et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2492–2498
2493
were discovered.13 In particular, several conformational-
ly constricted amino acids such as Tic, Oic, Aic, etc.,
were introduced and subsequently provided information
about well-defined conformational spaces.13 Upon sub-
stitution of His6 by Tic a potent hMC3R (IC50 = 6.7 nM)
and hMC4R (IC50 = 3.7 nM) antagonist was obtained.
This work along with that of others14–16 provided clear
evidence of the importance of position 6 for potency
and melanocortin receptor selectivity in analogues of a-
MSH.
compound served as a precursor for the Aba-D-Xxx
dipeptomimetics 5a–c. Reductive amination of this
phthaloyl-protected o-formyl-Phe 2 with the corre-
sponding amino acid benzyl esters resulted in the sec-
ondary amines 3a–c, which were subsequently ring
closed with the activated carboxylic acids using DCC.
Removal of the benzyl-protecting moiety, followed by
phthaloyl-deprotection and final Fmoc-protection gave
Fmoc-Aba blocks 5a–c. These were building blocks in
the synthesis of the new MT-II and SHU9119 analogues
using Fmoc solid-phase peptide synthesis.36
The
4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one
scaffold (Fig. 1) has proven to be an excellent tool for
the design of novel peptide mimetics.17–22 Its qualities
can be described as two folded: (1) the scaffold can be
considered as a conformationally restricted Phe ana-
logue where the aromatic side chain is anchored to the
a-amine of the next residue by means of a methylene
bridge (dotted line);17 (2) on the other hand, one can
see the Aba template as a ‘privileged template’.23
Molecular modeling proved to be very useful in the de-
sign of these hybrid peptides of MT-II and SHU9119.
This study employed Macromodel 9.1, with the OPLS
2005 force field and a MCMM/LMCS (Monte Carlo
Multiple Minima/Low Frequency Mode) conformation-
al search method.27 We overlapped the NMR structure
of the cyclic lactam a-MSH analogue MT-II28 with the
global minima of Aba-2 (Ac-Nle-c-[Asp-Aba-D-Phe-
Arg-Trp-Lys]-NH2), Aba-3 (Ac-Nle-c-[Asp-Aba-pCl-D-
Phe-Arg-Trp-Lys]-NH2) and Aba-4 (Ac-Nle-c-[Asp-
Aba-D-Nal(20)-Arg-Trp-Lys]-NH2), and concluded that
the backbone overlap was very good (RMSD = 1.53,
In scaffold 1 only the g(+) (v1 = +60ꢁ) and trans
(v1 = 180ꢁ) staggered conformations are allowed for
the Ca–Cb bond. By applying this restraint bioactive
conformations can be fixed.17,18 Different substitution
patterns in 1 can induce the specific binding to certain
subtypes of receptors or enzymes, for example ACE
inhibitors,19 opioid receptors,20 B2 bradykinin recep-
tors,21 and farnesyl transferase inhibitors.22
˚
1.01, and 0.99 A, respectively; non-hydrogen backbone
pharmacophore atoms only) (see Fig. 2). It is evident
that the backbone conformations of all three Aba pep-
tides are remarkably similar. Moreover, the Aba-bearing
peptidomimetics preserve the amphiphilic character for
the message sequence (Aba-Xxx-Phe-Arg-Trp), a prop-
erty occurring in most of the high affinity ligands for
hMC1-, hMC3-, hMC4-, and hMC5R, needed for favor-
able interactions with the melanocortin receptors.28 The
hydrophobic part, bearing the side chains of Aba, D-
Xxx and Trp, is on one face, and the hydrophilic coun-
terpart, consisting of the side chain of Arg, is oriented
away from the aromatic moieties.
Thus, replacement of the His6-D-Phe7 dipeptide in
MT-II and His6-D-Nal(20)7 in SHU9119 by Aba-D-
Phe/Aba-pCl-D-Phe and Aba-D-Nal(20), respectively,
was expected to provide new structure–activity relation-
ships in the search for selective and potent ligands for
the hMC1–hMC5R.
All three a-amino protected dipeptomimetics Fmoc-
Aba-D-Phe-OH 5a, Fmoc-Aba-pCl-D-Phe-OH 5b, and
Fmoc-Aba-D-Nal(20)-OH 5c were synthesized using a
previously reported methodology based on an intramo-
lecular benzazepinone ring formation (see Scheme 1).24
The depicted synthetic pathway starts from (S)-ortho-
cyano-phenylalanine 1, obtained through an asymmetric
phase transfer catalysis reaction.25 Using the phthaloy-
lating agent MSB (methyl 2-[(succinimidooxy)carbon-
yl]benzoate), it was possible to efficiently protect
amino acid 1.26 The conversion of the nitrile to the alde-
hyde 2 was realized in an acetic acid/water/pyridine mix-
ture with Raney nickel as catalyst in good yield. This
The calculated dihedral angles for the Aba-Xaa block
loosely fit the criteria for a type IV b-turn, as defined
by Scheraga and co-workers,29 although examination
of the Ca(i)–Ca(i + 3) distances reveals that they are
larger than the Ca Asp5-Arg8 distance in MT-II (Aba-
˚
˚
˚
2 = 6.54 A, Aba-3 = 7.41 A, Aba-4 = 7.39 A, and MT-
˚
˚
II = 5.28 A), and are outside the cut off value of 7 A usu-
ally used to define a b-turn structure.29 The distances be-
tween the CO group of Asp5 and the NH group of Arg8
˚
˚
˚
(Aba-2, 2.70 A; Aba-3, 3.71 A; Aba-4, 3.71 A; MT-II,
˚
3.58 A) are also too large to expect a stable hydrogen
bond between these groups. These observations are in
accordance with our earlier reports on structural com-
parison of the b-turn-inducing properties of Aba with
those of the so-called Freidinger lactams,30 as we found
that only its spirocyclic derivative is capable of inducing
a b-turn conformation in an Ac-spiro-Aba-Xxx-NHMe
model25 or upon introduction in a biologically active
peptide hormone like bradykinin.21 The introduction
of this Aba-moiety also clearly avoids the typical stack-
ing between the side chains of residues 6 (His) and 7 (D-
Phe), which can be seen for MT-II (red) in Figure 2.
Nevertheless, introduction of the Aba block into the
MT-II structure resulted in largely preserved pharmaco-
Figure 1. The 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba)
scaffold.

2494
S. Ballet et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2492–2498
Scheme 1. Synthetic pathway for the Aba-Xxx-dipeptomimetics 5a–c.
Figure 2. Overlap of Aba-2 (blue), Aba-3 (yellow), Aba-4 (orange), and
MT-II (red).40
Figure 3. Overlap between the pharmacophore groups of MT-II and
Aba-2.
phore topography (Fig. 3), which was expected to lead
to good molecular recognition and high binding affinity
to the hMCRs. Another aspect rising from these model-
ing studies was the fact that the g(+) conformation of
the side chain of D-Phe was retained, analogously with
MT-II.
to the melanotropin receptors up to a concentration of
10 lM. This observation suggests that the Aba mimetic
alone cannot induce structural features necessary for
binding, such as a b-turn, and needs to be used in conjunc-
tion with a global conformational constraint. The cyclic
lactam analogue Aba-2 was found to show a good binding
affinity for the hMC3R (IC50 = 50 nM) and a weak affin-
ity for hMC5R (IC50 = 2.9 lM). No cAMP stimulation
could be detected leading to the conclusion that these data
are consistent with Aba-2 being a selective hMC3R antag-
onist (200-fold selective against the hMC4R, and about
60-fold selective against the hMC5R).
The affinity for the Aba-containing linear and cyclic lac-
tam analogues of MT-II Aba-1–Aba-3, as well as the
cyclic SHU9119 analogue Aba-4 was evaluated by com-
petition binding experiments carried out using HEK293
cells stably expressing the human MC1, MC3, MC4,
and MC5 receptors. The binding affinities, expressed
as IC50 values, are represented in Table 1.37–39
Halogenation of the para-position of D-Phe7 with F or
Cl typically enhances agonist activity at the hMCRs,12,31
Introduction of the Aba-D-Phe dipeptidomimetic in the
linear sequence of MT-II (Aba-1) resulted in no binding

Table 1. Names/sequences, binding-, activity-, and MPE values of Aba-peptidomimetics versus MT-II35–37
Name
Sequence
hMC1R
hMC3R
hMC4R
hMC5R
IC50
(nM)
EC50 (nM) Max IC50
effect (%) (nM)
EC50
(nM)
Max effect IC50
(%)
EC50
(nM)
Max IC50
effect (%) (nM)
EC50 (nM) Max effect (%)
(nM)
Aba-1
Ac-Nle-Asp-
Aba-D-Phe-
Arg-Trp-Lys-
NH2
>10,000
>10,000
>1,000
>10,000
>10,000
900 100
>2,500
0
>10,000
>10,000
>10,000
>1,000
0
>10,000
>10,000
>10,000
0
>10,000
>10,000
0
Aba-2
Aba-3
Aba-4
Ac-Nle-c[Asp-
Aba-D-Phe-
Arg-Trp-Lys]-
NH2
0
50
29
43
6
3
5
0
>10,000
0
2,900 300 >10,000
0
Ac-Nle-c[Asp-
Aba-p-Cl-D-
Phe-Arg-Trp-
Lys]-NH2
60
60
100
55
0
2,000 200 3,200
33
0
123 13
180 20
>10,000
3.3 0.7
45
0
Ac-Nle-c[Asp-
Aba -D-Nal-
Arg-Trp-Lys]-
NH2
580 70
>10,000
1700 200
1.07 0.3
>10,000
87 10
MT-II Ac-Nle-c[Asp-
His-D-Phe-Arg-
Trp-Lys]-NH2
0.2 0.01 0.3 0.04
1.25 0.2 1.85 0.2 100
2.87 0.52 100
7.47 0.23
100
IC50, concentration of compound at 50% specific binding. Values are means of three experiments; standard deviation is given.37
EC50, effective concentration of compound that was able to generate 50% maximal intracellular cAMP accumulation. Compounds were tested at a range of concentrations from 10ꢀ10 to 10ꢀ5 M.38

2496
S. Ballet et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2492–2498
and some weak agonist activity was indeed observed at
all receptor subtypes for Aba-3, which bears a pCl-D-
Phe at position 7. In addition, this substitution resulted
in somewhat improved binding affinities to all four
receptor subtypes. The Aba-D-Nal(20) analogue Aba-4
displayed high affinity hMC3R and hMC5R antagonist
properties (IC50 = 43 and 87 nM, respectively), and a
weak binding affinity to the hMC4R (IC50 = 1.7 lM),
developed melanotropin peptides will be used to clarify
the exact biological functions of the physiologically
important melanocortin-3 receptor.
Acknowledgments
This work was supported by the Institute for the Promo-
tion of Innovation through Science and Technology in
Flanders (IWT Vlaanderen), and by grants from the
U.S. Public Health Service, National Institutes of
Health DK-17420 and DA-06284. The opinions ex-
pressed are those of the authors and not necessarily
those of the USPHS.
a
weak partial agonist activity for the hMC1R
(EC50 = 2.5 lM, 60% max cAMP).
The observed lack of agonist activity points to possible
steric interference from Aba, analogous to the effect
of replacing His6 residue in cyclic a-MSH with bulky
Nle, recently presented by Mayorov et al.32 Their report
also describes markedly similar biological profiles
of Nle6 peptides, as the hMC3/4R agonist VJH085
(cyclo(5b ! 10e)-[succinyl5-His6-D-Phe7-Arg8-Trp9-Lys10]-
NH2) was thereby converted into a hMC3/4R antagonist
(cyclo(5b ! 10e)-[succinyl5-Nle6-D-Phe7-Arg8-Trp9-Lys10]-
NH2), with a significant decrease in binding affinities
(IC50 = 84 and 930 nM, respectively). The Nle6,
D-Nal(20)7 analogue (cyclo(5b ! 10e)-[succinyl5-Nle6-
D-Nal7-Arg8-Trp9- Lys10]-NH2) was also reported to
References and notes
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display hMC3R/hMC5R antagonist properties (IC50
=
12 and 17 nM, respectively).32 Notably, the hMC3/5R
antagonism was also observed in cyclic Nle4, D-
Nal(20)6-c-MSH analogues and was hypothesized to be
linked to steric hindrance of Arg7 binding space with
Nle4.27 It seems plausible that the steric effects of Aba
are responsible for the hMC3R and hMC3/5R antago-
nist properties of the Aba peptides described in this
report. Alternatively, the structural deviations of the
Aba-Xaa blocks from the type II b-turn structures
found in potent melanocortin agonists28 may indicate
the significance of type II b-turns for melanocortin ago-
nist activity. The unique conformational features of Aba
may also account for the enhanced receptor selectivity
displayed by these peptides.
8. Hruby, V. J.; Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.;
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12. Hruby, V. J.; Lu, D.; Sharma, S. D.; de Lauro Castrucci,
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Vrinten, D. H.; Garner, K. M.; Schaaper, W. M. M.;
Molecular modeling experiments40 have suggested that
the unique conformational properties of Aba mimetics
can be used to design and obtain novel melanotropin
peptides with significantly enhanced receptor selectivity.
The peptide design was based on the MT-II/SHU9119
cyclic lactam template, where the His6-Xaa7 residues
were replaced with Aba-Xaa block. The Fmoc-dipepti-
domimetics 5a–c were prepared in facile manner using
a previously reported method, which was based on a
reductive amination/cyclization sequence. The cyclic lac-
tam a-MSH analogues Aba-2–Aba-4 were synthesized
by Na-Fmoc solid-phase methodology. Competition
binding experiments, combined with the adenylate cy-
clase assay, were used to evaluate the activities of these
peptides at the human melanotropin receptors to reveal
new highly selective high affinity hMC3R antagonist
(Aba-2) and an hMC3R/hMC5R antagonist (Aba-4).
These results, in conjunction with earlier SAR work
on cyclic a- and c-MSH analogues, suggest that the
unique conformational and sterical attributes of the
Aba mimetic may be responsible for the observed
antagonist activities, and high hMC3R receptor
selectivity against the hMC1R and hMC4R. The newly

S. Ballet et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2492–2498
2497
Meloen, R. H.; Gispen, W. H.; Liskamp, R. M.; Adana,
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ninhydrin test. Upon completion of cyclization the peptide
sequence was finalized by coupling the Fmoc-Nle residue,
removal of the N-terminal Fmoc group, and acetylation of
the N-terminus. The strategy of performing the peptide
cyclization prior to removal of the Na-Fmoc protecting
group of the Asp residue was chosen to minimize the
competing aspartimide formation, as recently suggested by
Flora et al.,33 and indeed was found to be superior to the
previously reported procedure.13 The peptides were iso-
lated and purified as described previously27 in 30–35%
overall yield and were >95% pure as determined by
analytical RP-HPLC. The structures of the pure peptides
were confirmed by high-resolution electrospray ionization
(ESI) mass-spectrometry.
´
17. Tourwe, D.; Verschueren, K.; Frycia, A.; Davis, P.;
Porreca, F.; Hruby, V. J.; Toth, G.; Jaspers, H.; Verhey-
den, P.; Van Binst, G. Biopolymers 1995, 38, 1.
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21. Ballet, S.; De Wachter, R.; Van Rompaey, K.; To¨mbo¨ly,
Cs.; Feytens, D.; To¨th, G.; Quartara, L.; Cucchi, P.;
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psc.827.
22. Le Diguarher, T.; Ortuno, J.-C.; Shanks, D.; Guilbaud,
37. Competition binding experiments were carried out using
whole HEK293 cells stably expressing human MC1, MC3,
MC4, and MC5 receptors. HEK293 cells transfected with
hMCRs14,34,35 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 prepared MEM buffer containing 100% mini-
mum essential medium with Earle’s salt (MEM, Gibco)
and 25 mM sodium bicarbonate. Next, the cells were
incubated for 40 min at 37 ꢁC with different concentra-
tions of unlabeled peptide and labeled [125I]-[Nle4,D-Phe7]-
a-MSH (Perkin-Elmer Life Science, 20,000 cpm/well,
33.06 pM) diluted in a 125 lL of freshly prepared binding
buffer containing 100% MEM, 25 mM Hepes (pH 7.4),
0.2% bovine serum albumin, 1 mM 1,10-phenanthroline,
0.5 mg/L leupeptin, 200 mg/L bacitracin. The assay medi-
um was subsequently removed, the cells were washed once
with basic medium, and then lysed by the addition of
100 lL of 0.1 M NaOH and 100 lL of 1% Triton X-100.
The lysed cells were transferred to 12 · 75 mm borosilicate
glass tubes, and the radioactivity was measured by a
Wallac 1470 WIZARD Gamma Counter.
´
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38. Adenylate Cyclase assay: HEK 293 cells transfected with
human melanocortin receptors14 were grown to conflu-
ence in MEM 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 lL MEM buffer (Gibco). An aliquot
(100 lL) 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 lL) of
melanotropin peptides of varying concentrations 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 lL 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 2300g.
A 50 lL aliquot of the supernatant was transferred to
another 96-well plate and placed with 50 lL [3H]cAMP
and 100 lL protein kinase A (PKA) buffer in an ice bath
for 2–3 h. The PKA-buffer consisted of Tris/EDTA-buffer
with 60 lg/mL PKA and 0.1% bovine serum albumin by
weight. The incubation mixture was filtered through
1.0 lm glass fiber filters in MultiScreenTM-FB 96-well
plates (Millipore, Billerica, MA). The total [3H]cAMP
was measured by a Wallac MicroBeta TriLux 1450 LSC
and Luminescence Counter (Perkin-Elmer Life Science,
Boston, MA). The cAMP accumulation data for each
peptide analogue were determined with the help of a
cAMP standard curve generated by the same method as
described above.
36. All peptides in this study were synthesized manually, as
described previously,27 by the Na-Fmoc solid-phase meth-
odology on Rink amide AM (w/Nle) resin using DIC and
Cl–HOBt as the coupling reagents. Upon coupling the Asp
residue, the orthogonal allylic protection for the side
chains of Asp and Lys was removed using the well-
established procedure,27,33 and the peptide cyclizations
were found to proceed in facile manner with 6 equiv DIC,
6 equiv Cl–HOBt in THF (72 h), as determined by Kaiser
39. IC50 and EC50 values represent the mean of two experi-
ments performed in triplicate. IC50 and EC50 estimates

2498
S. Ballet et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2492–2498
and their associated standard errors were determined by
(Schro¨dinger, LLC, New York, NY, 2005) installed on a
Linux Red Hat 9.0 system, and were performed as
previously described using the OPLS 2005 force field.27
The superpositions of peptide structures were performed
using the a-carbons of the core sequence Xaa-D-Phe/D-
Nal(20)-Arg-Trp.
fitting the data using a nonlinear least squares analysis,
with the help of GraphPad Prism 4 (GraphPad Software,
San Diego, CA).
40. Molecular modeling experiments employed Macromodel
9.1 equipped with the Maestro 7.5 graphical interface