Pentapeptides displaying μ opioid receptor agonist and δ opioid receptor partial agonist/antagonist properties

Article abstract of DOI:10.1021/jm9007483

Chronic use of μ-opioid agonists has been shown to cause neurochemical adaptations resulting in tolerance and dependence. While the analgesic effects of these drugs are mediated by μ-opioid receptors (MOR), several studies have shown that antagonism or kn

Full text of DOI:10.1021/jm9007483

7724 J. Med. Chem. 2009, 52, 7724–7731
DOI: 10.1021/jm9007483
Pentapeptides Displaying μ Opioid Receptor Agonist and δ Opioid Receptor Partial
Agonist/Antagonist Properties†
Lauren C. Purington,‡ Irina D. Pogozheva,§ John R. Traynor,*,‡, and Henry I. Mosberg*,§
‡
§
Departments of Pharmacology and Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, and University of Michigan
Substance Abuse Research Center, Ann Arbor, Michigan
Received May 29, 2009
Chronic use of μ-opioid agonists has been shown to cause neurochemical adaptations resulting in
tolerance and dependence. While the analgesic effects of these drugs are mediated by μ-opioid receptors
(MOR), several studies have shown that antagonism or knockdown of δ-opioid receptors (DOR) can
lessen or prevent development of tolerance and dependence. On the basis of computational modeling of
putative active and inactive conformations of MOR and DOR, we have synthesized a series of
pentapeptides with the goal of developing a MOR agonist/DOR antagonist peptide with similar affinity
at both receptors as a tool to probe functional opioid receptor interaction(s). The eight resulting
naphthylalanine-substituted cyclic pentapeptides displayed variable mixed-efficacy profiles. The most
promising peptide (9; Tyr-c(S-CH2-S)[D-Cys-Phe-2-Nal-Cys]NH2) displayed a MOR agonist and
DOR partial agonist/antagonist profile and bound with equipotent affinity (Ki ∼ 0.5 nM) to both
receptors, but also showed κ opioid receptor (KOR) agonist activity.
Introduction
including respiratory depression and constipation as well as
tolerance. In particular, studies pointing to a role of the δ
opioid receptor (DOR) in modulating the development of
MOR tolerance have led to the hypothesis that both MOR
and DOR play major roles in the development of tolerance
after chronic morphine exposure. For example, work in DOR
knockoutrodentmodels2-4 or usingDORantagonists5-8 was
shown to prevent or lessen the severity of tolerance develop-
ment to chronic morphine exposure. More recent in vivo work
also points to a role of DOR in modulating morphine-induced
behavioral sensitization and conditioned place preference in
rodents.9-11 It has been hypothesized that the formation of
homo- or heterodimers of MOR and DOR leads to changes in
their pharmacological behaviors including alteration in toler-
ance or dependence development.6,12-14
The growing body of evidence implicating a role of DOR in
modulating MOR-induced tolerance suggests that opioid
ligands with similar affinities at MOR and DOR but display-
ing agonism at MOR and antagonism at DOR might be of
great clinical potential, especially for the treatment of chronic
pain conditions. Consequently, many groups have developed
compounds with MOR and DOR affinity, including pepti-
dic15-19 and nonpeptidic20-24 ligands displaying MOR agon-
ism and DOR antagonism. However, many of these com-
pounds, while displaying the desired efficacy profile, do not
have equivalent binding affinities to both MOR and DOR,
thus limiting their usefulness in probing MOR-DOR inter-
actions.
Mu-opioid receptor (MORa) agonists such as morphine are
commonly used in the treatment of moderate to severe pain.
However, use of such drugs is associated with side effects
includingthedevelopmentof tolerance, limiting the usefulness
of these compounds. It has been hypothesized that opioid
compounds displaying MOR agonism paired with a selective
δ- or κ-opioid receptor effect could lessen the severity of
limiting side effects surrounding current MOR agonist use,1
†This work is submitted in celebration of the 100th anniversary of the
Division of Medicinal Chemistry.
*To whom correspondence should be addressed. For J.R.T.: phone,
(734) 647-7479; fax, (734) 763-4450; E-mail: jtraynor@umich.edu; ad-
dress, University of Michigan, 1150 W. Medical Center Drive, Ann
Arbor MI 48109-5632.For H.I.M.: phone, (734) 764-8117; fax, (734)
763-5595; E-mail: him@umich.edu; address, University of Michigan,
428 Church Street, Ann Arbor MI 48109-1065.
a Abbreviations: Symbols and abbreviations are in accordance with
recommendations of the IUPAC-IUB Joint Commission on Biochemi-
cal Nomenclature: Nomenclature and Symbolism for Amino Acids and
Peptides. Biochem. J. 1984, 219, 345-373. All other abbreviations are as
follows: 1-Nal, 3-(1-naphthyl)alanine; 2-Nal, 3-(2-naphthyl)alanine; AcOH,
acetic acid; C6-MOR, C6 rat glioma cells stably expressing the μ-opioid
receptor; C6-DOR, C6 rat glioma cells stably expressing the δ-opioid
receptor; cAMP, cyclic adenosine monophosphate; CHO-KOR, Chinese
hamster ovary cells stably expressing the κ-opioid receptor; DAMGO,
[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; DIEA, diisopropylethylamine;
DMEM, Dulbecco's Modified Eagle Medium; DMF, dimethylformamide;
DOR, δ opioid receptor; EXL, extracellular loop; Fmoc, 9-fluorenylmethyl-
oxycarbonyl; FSK, forskolin; GPCR,
G protein-coupled receptor;
[
35S]GTPγS, [35S]-guanosine-50-O-(3-thio)triphosphate; HBTU, o-benzo-
triazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate; HOBT,
1-hydroxybenzotriazole; KOR, κ opioid receptor; MOR, μ opioid receptor;
NTI, naltrindole; rmsd, root-mean-square deviation; RP-HPLC, reverse
phase-high performance liquid chromatography; SNC80, 4-[(R)-[(2S,5R)-
4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)-methyl]-N,N-diethyl-
benzamide; TFA, trifluoroacetic acid; TM, transmembrane helixes; U69,593,
(5R,7R,8β)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzene-
acetamide.
Our previous work led to the synthesis of peptide 1 (Tyr-
1 dis-
c(S-CH2-S)[D-Cys-Phe-Phe-Cys]NH2).25 Peptide
played a promising mixed-efficacy profile at MOR and
DOR, binding with high affinity for both MOR and DOR
while exhibiting full agonism at MOR and the κ opioid
receptor (KOR) but only partial agonism at DOR. We wished
r
pubs.acs.org/jmc
Published on Web 09/29/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7725
Table 1. Binding Affinities and Efficacies of Cyclic Opioid Pentapeptides 1-9 at MOR, DOR, and KORa
Ki (nM) ( SEM
efficacy (%) ( SEM
peptide
sequence
linker
MOR
DOR
KOR
MOR
DOR
KOR
1
2
3
4
5
6
7
8
9
Tyr-c[D-Cys-Phe-Phe-Cys]NH2
Tyr-c[D-Cys-1-Nal-Phe-Cys]NH2
Tyr-c[D-Cys-1-Nal-Phe-Cys]NH2
Tyr-c[D-Cys-2-Nal-Phe-Cys]NH2
Tyr-c[D-Cys-2-Nal-Phe-Cys]NH2
Tyr-c[D-Cys-Phe-1-Nal-Cys]NH2
Tyr-c[D-Cys-Phe-1-Nal-Cys]NH2
Tyr-c[D-Cys-Phe-2-Nal-Cys]NH2
Tyr-c[D-Cys-Phe-2-Nal-Cys]NH2
S-CH2-S
S-S
S-CH2-S
S-S
S-CH2-S
S-S
S-CH2-S
S-S
0.016 ( 0.01
0.08 ( 0.04
0.33 ( 0.04
0.72 ( 0.05
0.47 ( 0.2
2.5 ( 0.9
0.61 ( 0.08
1.2 ( 0.3
0.47 ( 0.2
1.8 ( 0.8
34 ( 4.5
34 ( 0.7
55 ( 0.3
15 ( 1.7
7.1 ( 2.3
5.2 ( 0.4
11 ( 6.4
0.48 ( 0.2
2.5 ( 1.5
21 ( 1.7
5.8 ( 2.9
145 ( 11
69 ( 0.6
11 ( 1.8
6.0 ( 1.5
5.9 ( 0.8
1.3 ( 0.4
88 ( 1.1
33 ( 1.6
91 ( 4.5
34 ( 1.5
34 ( 3.8
88 ( 3.6
100 ( 3.8
90 ( 1.4
99 ( 1.8
45 ( 2.3
0
93 ( 3.4
59 ( 3.1
77 ( 6.3
7.5 ( 2.1
35 ( 4.0
100 ( 7.4
87 ( 2.4
49 ( 3.6
89 ( 3.6
23 ( 3.4
0.7 ( 1.0
23 ( 1.6
15 ( 1.5
22 ( 2.3
2.0 ( 1.0
7.0 ( 2.3
S-CH2-S
a 3-(1-Naphthyl)alanine and 3-(2-naphthyl)alanine substitution abbreviated as 1-Nal and 2-Nal, respectively. Cyclization abbreviated as S-S for
disulfide linkage and S-CH2-S for methylene dithioether linkage. Experiments were performed in C6-MOR, C6-DOR, or CHO-KOR cells. Binding
affinities (Ki) were obtained by competitive displacement of radiolabeled [3H]diprenorphine. Efficacy of pentapeptides at the three opioid receptors was
determined using the [35S]GTPγS binding assay. Efficacy is presented as percent of the maximal level of [35S]GTPγS binding obtained with standard
agonists for MOR, DOR, and KOR (DAMGO, SNC80, or U69,593, respectively) at a 10 μM concentration.
to improve peptide 1 by decreasing efficacy at DOR while
increasing affinity for this receptor, retaining both efficacy
and affinity at MOR, and reducing affinity at KOR. To
pursue this aim, we examined the docking of 1 into computa-
tional models of MOR and DOR. On the basis of modeling of
putative active and inactive conformations of MOR and
DOR26-29 and docking of 1 to these models, we focused on
steric constraints surrounding the third and fourth Phe resi-
dues of 1. We hypothesized that replacement of these Phe
residues with bulkier side chains would decrease ligand affi-
nity to the DOR active state but not the DOR inactive state
and not affect binding to MOR, thus favoring the desired
MOR agonist/DOR antagonist profile. Consequently, we
designed eight analogues of peptide 1 containing naphthyl-
alanine in place of Phe3 or Phe4 to more fully explore the steric
limits of the receptor binding pocket at either of these posi-
tions. We have previously used naphthylalanine substitution
to add steric bulk in cyclic peptides,30 and this has been more
recently applied to linear peptides.31 In vitro, our cyclic
peptidesdisplayedvariableMORefficacies andhad decreased
DOR efficacy. One compound (peptide 9; Tyr-c(S-CH2-S)-
[D-Cys-Phe-2-Nal-Cys]NH2) displayed full agonism at MOR
(99% stimulation compared with the full MOR agonist
DAMGO) while acting as an antagonist at DOR in the
[35S]GTPγS assay, but with partial agonist activity in the
adenylyl cyclase inhibition assay, and bound with similar
subnanomolar affinity to MOR and DOR stably and inde-
pendently expressed in C6 rat glioma cells. Compound 9 also
bound with high affinity to KOR and was an agonist at that
receptor. Thus, incorporation of a substitution based on
rational design and intended to highlight putative steric
constraints resulted in a compound that had similar affinity
for MOR and DOR but decreased DOR efficacy without
compromising MOR agonism. This is an important step
forward in the development of novel ligands presenting
MOR agonist and DOR antagonist effects.
full agonism at MOR and KOR, but only partial agonism at
DOR (Table 1). To understand the molecular mechanism
underlying the mixed-efficacy profile of 1, computational
models of MOR and DOR were utilized. Peptide 1 was
virtually docked in models of active and inactive conforma-
tions of MOR and DOR26-29 (Figure 1) in a similar manner
as MOR and DOR-selective tetrapeptides, JOM6 and
JOM13, respectively, which were previously positioned in
MOR and DOR models based on their structure-activity
relationships and receptor mutagenesis data.26,27,33,34 These
active and inactive receptor models were designed based on
the published crystal structure of the β2-adrenergic recep-
tor35 (as described in the Experimental Section). Though
the new adrenoreceptor-based models of opioid recep-
tors differ from our previously published rhodopsin-based
models26,28,29,33 by some helix shifts and an outward move-
ment of extracellular loop (EXL) 2 (rmsd in the range
˚
2.3-2.6 A for all CR-atoms, excluding EXL2), the ligand
binding mode and receptor-ligand interactions with resi-
dues from transmembrane helixes (TMs) 3, 5, 6, and 7 of
either DOR or MOR remained essentially the same. Thus,
we found that the docking mode of peptide ligands is more
influenced by its interactions with helix residues than resi-
dues from EXL2, the modeling of which is expected to be less
accurate.
Examination of the position of pentapeptide 1 inside the
binding pocket of active and inactive MOR (Figure 1A,B)
allowed us to obtain a low-energy conformation of 1 that did
not have steric hindrances or other adverse interactions with
residues in the receptor binding pocket in either receptor
state. Peptide 1 docked in MOR showed favorable aromatic
interactions between its Phe3 side chain and the Trp318 side
chain in TM7, which were more pronounced in the active
conformation of MOR. These aromatic interactions may
explain the preferential binding of 1 to MOR, as compared to
DOR or KOR, which have Leu300 or Tyr312 at the corre-
sponding position. The same conformation of 1, when fitted
into the active DOR model (Figure 1C), demonstrated some
steric overlap between Phe4 of the ligand and Trp284 from the
TM6 of DOR, which was not observed in the inactive DOR
model (Figure 1D). The docking of peptide 1 to both active
and inactive conformations of MOR and its better compat-
ibility with the inactive conformation of DOR is consistent
with the MOR agonist/DOR partial agonist profile of 1.
On the basis of the above, we hypothesized that incorpora-
tion of a bulkier naphthylalanine side chain in either the third
or fourth position of pentapeptide 1 would affect its binding
Results
Rationale for the Design of Pentapeptides Displaying MOR
Agonism and DOR Antagonism. Previous work by our group
led to the synthesis of the high-affinity, MOR-selective cyc-
lic pentapeptide 1 (Tyr-c(S-CH2-S)[D-Cys-Phe-Phe-Cys]-
NH2),25 which has picomolar affinity for MOR (Ki = 0.016
nM) and nanomolar affinities for DOR (Ki = 1.8 nM) and
KOR (Ki = 2.5 nM). When evaluated for efficacy at the three
opioid receptors using the [35S]GTPγS assay,32 1 displayed

7726 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Purington et al.
Figure 1. Modeling of peptide 1 in the binding pocket of putative active and inactive conformations of mouse MOR and human DOR. Peptide
1 docked in the putative active (A) and inactive (B) conformation of MOR shows no noticeable unfavorable interactions between ligand side
chains and residues from the receptor binding pocket. Peptide 1 docked in the active conformation of DOR (C) shows a steric overlap of the
peptide Phe4 side chain with the side chain of receptor Trp284 from TM6 (arrow), while peptide 1 in the inactive conformation of DOR (D) does
not show a similar steric hindrance.
three opioid receptors was generally noted as compared to
the highly potent pentapeptide 1. At MOR, the most sig-
nificant loss of affinity occurred with peptide 6 (p<0.001),
while at DOR four peptides had g10-fold decreased affinity
(2, 3, 4 p < 0.001, and 5 p < 0.05). At KOR, peptides 4 and 5
had >10-fold decreased binding affinity when compared to
1 (p < 0.001) (Table 1). Of the eight peptides synthesized,
only peptide 9 (Tyr-c(S-CH2-S)[D-Cys-Phe-2-Nal-Cys]-
NH2) showed similar low nanomolar binding affinity to
MOR, DOR, and KOR (Ki = 0.47, 0.48, and 1.3 nM,
respectively).
Peptides 2-9 were also analyzed for efficacy at MOR,
DOR, and KOR as determined by maximal stimulation of
[35S]GTPγS binding32 as a percentage of 10 μM standard
compounds DAMGO, SNC80, and U69,593 (Table 1). Pep-
Figure 2. Naphthylalanine-containing analogues of peptide 1.
and efficacy properties differentially at MOR and DOR
tides 3, 6, and 8 showed equivalent agonism at MOR as
peptide 1 (∼ 90% maximal stimulation), while peptides 2, 4,
and 5 had decreased efficacy at MOR (31-34% stimulation;
p<0.001). In contrast, peptides 7 and 9 showed greater
stimulation of [35S]GTPγS binding at MOR than 1 (p <
0.05), displaying the same maximal stimulation. It is note-
worthy that all peptides displaying decreased efficacy at
MOR (2, 4, and 5) were substituted at position 3 with
naphthylalanine. In contrast, analogues with naphthyl-
alanine substitution at position 4 (6-9) showed equal
or greater efficacy at MOR, indicating substitution at posi-
tion 3 by residues with bulky side chains is deleterious for
MOR agonism. One exception was peptide 3, which has
and could result in potent MOR agonist/DOR antagonist
ligands. To test this hypothesis, we replaced Phe3 and Phe4 of
1 with the bulkier 3-(1-naphthyl)alanine or 3-(2-naphthyl)-
alanine (Figure 2) to provide eight new cyclic pentapeptides
(2-9; Tables 1, 2). Peptides were cyclized via a disulfide bond
(S-S) or methylene dithioether (S-CH2-S) linkage to allow
for altered size and flexibility of the cycle.
Analysis of Synthesized Pentapeptides 2-9 for Binding
Affinity and Efficacy at Opioid Receptors. Most naphthyl-
alanine peptides demonstrated relatively high binding affi-
nities to MOR, DOR, and KOR as measured by competitive
displacement of the radiolabeled nonselective opioid antago-
nist [3H]diprenorphine. However, some loss in affinity to all

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7727
Figure 3. Pentapeptide 9 docked in the binding pocket of putative active and inactive conformations of mouse MOR and human DOR.
The 2-naphthylalanine4 side chain of peptide 9 shows minimal hindrance with receptor residue Lys303 in the MOR active conformation (A) but
an increased steric overlap with Trp284 side chain (arrow) in the DOR active conformation (C). These hindrances are absent in the
inactive conformations of both MOR (B) and DOR (D).
naphthylalanine substitution at position 3 and displayed
high efficacy at MOR. Peptide 3 was cyclized via a methylene
dithioether, possibly allowing greater flexibility of the pep-
tide ring structure and leading to increased stimulation
over the disulfide-cyclized counterpart 2. At DOR, peptides
2, 4, 8, and 9 behaved essentially as antagonists, providing
very little or no [35S]GTPγS stimulation. Compounds 3, 5, 6,
and 7 displayed partial agonism with maximal stimulation
varying from 15 to 23%. The eight peptides displayed varying
efficacy profiles at KOR, with most compounds behaving as
partial to full agonists (maximal stimulation 35-100%).
Docking of Peptide 9 to Modeled MOR and DOR Active
and Inactive Conformations. The binding and efficacy studies
described above identify peptide 9 as a candidate ligand
displaying the desired MOR agonism and DOR antagonism
profile. To better understand the mechanism of the de-
creased efficacy of 9 at DOR, we docked this peptide in the
modeled active and inactive conformations of MOR and
DOR similar to the docking of peptide 1 (Figure 3).
The lowest energy conformation of peptide 9 exhibited no
hindrance in the binding pocket of either active or inactive
conformations of MOR (Figure 3A,B) nor in the inactive
DOR conformation (Figure 3D). However, when compared
to peptide 1 (Figure 2C), 9 shows greater overlap between its
2-naphthylalanine4 and Trp284 in the active conformation of
DOR (Figure3C). Thisreduced compatibilityof9 relativeto1
for the active state of DOR supports the decreased agonism at
DOR. Modeling results also explain the high agonist efficacy
of 9 at MOR, because 9 fits both receptor states well and may
promote a conformational shift toward the active conforma-
tion of MOR where favorable aromatic interactions between
Phe3 and Trp318 of MOR are more prominent.
Characterization of the Functional Properties of Peptide 9
at Opioid Receptors. In the [35S]GTPγS binding assay, pep-
tide 9 behaved as a full agonist at MOR with EC50 of 1.2 (
0.05 nM (Figure 4A). On the other hand, at 10 μM concen-
tration 9 produced only 7% of SNC80-induced stimulation
of DOR-mediated [35S]GTPγS binding (Table 1).
The properties of 9 were further evaluated by measuring its
ability to inhibit SNC80-stimulated binding of [35S]GTPγS
to G-proteins. Peptide 9 produced a 3.1-fold rightward shift
in the dose-response curve of SNC80 in C6-DOR cells
(Figure 4B); the EC50 for SNC80 was shifted from 75 ( 3.8
to 188 ( 31 nM in the presence of 100 nM 9 (p = 0.02).
However, this shift and the calculated Ke value (48 ( 9.5 nM)
for 9 was not consistent with its high binding affinity to DOR
(Ki = 2.1 nM), indicating 9 may have some partial agonist
efficacy at DOR which cannot be fully observed using the
high efficacy-requiring [35S]GTPγS binding assay. To more
fully assess the extent of this partial agonism, we measured
the ability of 9 to inhibit cAMP accumulation as a measure of
adenylyl cyclase activity.36 Because of downstream signaling
amplification, it is easier to visualize partial agonism using
this system. Peptide 9 was shown to be more potent (EC50:
36 ( 4.8 nM) than SNC80 (EC50: 166 ( 43 nM; p = 0.01) and
behaved as a partial agonist, able to produce 55% inhibition
of that seen with SNC80 (Figure 4C). The DOR-selective
antagonist naltrindole (NTI) was without effect in this assay.
Discussion
The current studies were aimed toward the development of
a potent compound with mixed MOR agonist/DOR antago-
nist properties, a profile that would be valuable to probe
interactions of MOR and DOR and that has considerable

7728 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Purington et al.
to more fully explore the steric limits of the receptor binding
pocket at either of these positions. All peptides were evaluated
for their potential to interact at both MOR and DOR via
receptor binding and in vitro functional studies. The newly
synthesized peptides demonstrated MOR agonism with vari-
able efficacies and had greatly decreased DOR efficacy in the
[35S]GTPγS binding assay. One compound, peptide 9 (Tyr-
c(S-CH2-S)[D-Cys-Phe-2-Nal-Cys]NH2), bound with simi-
lar subnanomolar affinity toMOR and DORstably expressed
in rat glioma cells and was characterized as an agonist at
MOR and an antagonist or partial agonist at DOR depending
on the assay used. This latter difference highlights the im-
portance of the choice of assay in efficacy determination.37
The development of pentapeptide 9 represents a significant
step forward in the development of a mixed-efficacy MOR
agonist/DOR antagonist ligand. Previously reported mixed-
efficacy ligands did not show the same equipotent affinity
for both MOR and DOR15-17,19-24 or the same full MOR
agonist properties.18,21,22 The results also represent a valida-
tion of our receptor models and a novel demonstration of the
use of differences in modeled active and inactive states to
design ligands with prescribed properties. In this example,
steric differences in the binding site of the active and inactive
DOR models were exploited by incorporating bulkier
naphthylalanine in place of phenylalanine in residues 3 and
4 of lead peptide 1 to generate ligands with the desired MOR
agonist/DOR antagonist profile.
Although peptide 9 displays the desired MOR/DOR
mixed-efficacy profile, it also acts as a full agonist at KOR
in the [35S]GTPγS binding assay with EC50 of 12 ( 0.1 nM
although it is 10-fold selective in potency for MOR over KOR
(Figure 4A). This residual KOR activity is not surprising, as
compound 1 was initially developed in a series aiming to
improve KOR binding and efficacy.25 Using computational
models of KOR developed as described above for putative
active and inactive conformation models of MOR and DOR,
we are designing analogues that are intended to exhibit
reduced KOR affinity while retaining the desired MOR
agonist/DOR antagonist profile. Such compounds will allow
us to characterize MOR and DOR interactions without the
potential complication of concomitant KOR activation or
antagonism.
Figure 4. Pharmacological analysis of peptide 9. (A) Activity of
peptide 9 in the [35S]GTPγS binding assay at MOR, DOR, and
KOR. Results are plotted as percent stimulation compared to a
10 μM concentration of standard compounds (MOR standard
agonist DAMGO, DOR standard agonist SNC80, and KOR
standard agonist U69,593). Peptide 9 has 10-fold higher potency
at MOR (EC50: 1.2 ( 0.05 nM) than KOR (EC50: 12 ( 0.1 nM) and
10 μM 9 produces only 6.9 ( 2.3% of SNC80-induced stimulation at
DOR. (B) DOR antagonism of peptide 9 in the [35S]GTPγS binding
assay. Peptide 9 (100 nM) produces a 3.1-fold rightward shift in the
SNC80 dose-response curve, indicating DOR antagonism. Calcu-
lated Ke = 48 ( 9.5 nM. Results are plotted as percentage of
the maximum level of SNC80-stimulated [35S]GTPγS binding.
(C) Partial agonism of peptide 9 at DOR as illustrated by inhibition
of adenylyl cyclase. Results are shown as percent inhibition of 5 μM
forskolin-stimulated adenylyl cyclase production in C6-DOR cells.
Standard DOR agonist SNC80 (1 μM) gave 67 ( 4.4% inhibition of
adenylyl cyclase production with a calculated maximal effect of 76 (
8.8%, while 1 μM peptide 9 produced 37 ( 4.2% inhibition with a
calculated maximum of 43 ( 9.0% inhibition. Peptide 9 is more
potent (EC50 = 36 ( 4.8 nM) than SNC80 (EC50 = 166 ( 43 nM) in
this assay. The DOR antagonist naltrindole (NTI) did not inhibit
cAMP accumulation.
Experimental Section
Materials. All Fmoc-protected amino acids were obtained
from Advanced ChemTech (Louisville, KY) or Sigma-Aldrich
(St. Louis, MO). All other reagents for peptide synthesis and
characterization were from Sigma-Aldrich (St. Louis, MO)
unless otherwise indicated. Fetal bovine serum and all cell
culture media and additives were purchased from Gibco Life
Sciences (Grand Island, NY). [D-Ala2,N-Me-Phe4,Gly5-ol]-en-
kephalin (DAMGO) and all other biochemicals were obtained
from Sigma-Aldrich and were of analytical grade. 4-[(R)-
[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)-
methyl]-N,N-diethylbenzamide (SNC80) was obtained from the
Narcotic Drug and Opioid Peptide Basic Research Center at
the University of Michigan (Ann Arbor, MI). [35S]-guanosine-
50-O-(3-thio)triphosphate ([35S]-GTPγS; 1250 Ci (46.2TBq)/
mmol) and [3H]-diprenorphine were purchased from Perkin-
Elmer (Boston, MA). Cyclic AMP accumulation was deter-
mined by radioimmunoassay (GE Healthcare, Piscataway, NJ).
Structural Modeling. The homology modeling of the inactive
conformation of human DOR (residues 45-338, UniProt
accession code P41143) and mouse MOR (residues 64-354,
UniProt accession code P42866) was performed as previously
clinical promise. The eight newly synthesized cyclic pentapep-
tides represent modifications of our previously reported penta-
peptide 1 (Tyr-c(S-CH2-S)[D-Cys-Phe-Phe-Cys]NH2),25
which was characterized by high affinity binding to all opioid
receptors and a mixed efficacy profile. On the basis of our
computational modeling of active and inactive conforma-
tions of MOR and DOR and docking of 1 to these models
(Figure 1), we focused on receptor-ligand interactions sur-
rounding Phe3 and Phe4 residues of 1. We designed several
derivative peptides containing naphthylalanine substitution

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7729
Table 2. Analytical Data for Peptides 2-9
described26,27,38 using the more recent structure of the β2-adre-
nergic receptor fused with T4 lysozyme (PDB code 2rh1).35
Distance geometry calculations with DIANA39 were used to
provide helix shift and loop modeling. To model the active
receptor conformation, we included several structural con-
straints between TM 3, 5, and 6 that have been shown to be
compatible with active states of different GPCRs.27 Introduc-
tion of these constraints allowed us to reproduce the significant
movement of TM6 that has been suggested based on numerous
experimental studies of different GPCRs, as well as on the
comparison of the rhodopsin (1f88)40 and opsin (3dqb)41 crystal
structures.
MWa
HPLC (min; Rt)b
system A system B
35.23 31.97
peptide
calcd (MW)
obsd (MW þ 1)
2
3
4
5
6
7
8
9
728.2
742.3
728.2
742.3
728.2
742.3
728.2
742.3
729.237
743.260
729.241
743.260
729.238
743.255
729.248
743.265
35.13
35.71
35.81
35.88
35.79
35.85
35.92
31.60
32.14
32.18
33.22
32.71
32.84
33.09
3D structures of cyclic pentapeptides were generated by
QUANTA (Accelrys Inc.) using residue substitution of pre-
viously modeled pentapeptides25 followed by molecular me-
chanics computations using the CHARMm force field. Several
conformations of disulfide or methylene dithioether bridges and
different rotamers of residues in the third and fourth positions of
pentapeptides were tested during ligand docking. Several low
energy conformations (within 2 kcal/mol) were manually posi-
tioned inside the receptor binding cavity similarly to tetrapep-
tides JOM6 and JOM13, whose docking in DOR and MOR has
been previously justified using conformational and mutagenesis
analysis.26,27,33,34 During ligand docking, we selected those
low energy conformations that better satisfied the following
criteria: (1) provided interactions between Tyr1 and Phe3 and
functionally important receptor residues (Asp147 from TM3,
His297 from TM6, and Trp318 from TM7 of MOR27 or correspond-
ing Asp128, His278, and Leu300 in DOR), (2) had minimal steric
overlap, and (3) formed more hydrogen-bonds between receptor
and ligand polar groups. The docking pose of each ligand was
subsequently refined using the solid docking module of QUAN-
TA. The opioid receptor models are available upon request.
Solid Phase Peptide Synthesis. All peptides were synthesized
by solid phase methods42 on an ABI model 431A solid phase
peptide synthesizer (Applied Biosystems, Foster City, CA) as
previously published.25 Rink resin (Advanced ChemTech) was
used as the solid support for C-terminal carboxamide peptides.
Peptide elongation on the peptide-resin involved treating resin
with piperidine (Sigma-Aldrich) to cleave the Fmoc-protecting
group, diisopropylethylamine (DIEA) activation, followed by
coupling of the next amino acid with o-benzotriazol-1-yl-N,N,
N0,N0-tetramethyl uronium hexafluorophosphate (HBTU) and
1-hydroxybenzotriazole (HOBt, Applied Biosystems). These
steps were repeated until the entire peptide was assembled.
a Observed molecular weights determined by high resolution mass
spectrometry (HRMS). b Retention time assessed by analytical high-
performance liquid chromatography (HPLC) using two solvent systems;
(A) 0.1% trifluoroacetic acid (TFA) in water (w/v)/0.1% TFA in
acetonitrile with a gradient of 0-70% organic component in 70 min
and (B) 0.1% TFA in water/0.1% TFA in methanol with a gradient of
20-70% organic component in 50 min, monitored at 230 nm, samples in
H2O with 0.1% TFA (elution column heated at 35 °C). All peptides had
>97% purity determined by HPLC.
solvent removed in vacuo. The residue was dissolved in water,
filtered, and subjected to semipreparative RP-HPLC to obtain
the methylene dithioether cyclized peptide.
Characterization of Peptides. All final product peptides were
>97% pure as assessed by analytical RP-HPLC on a Vydac
218TP C-18 column (The Nest Group, Southboro, MA) using
two solvent systems: (A) 0.1% trifluoroacetic acid (TFA, w/v)
in water/0.1% TFA (w/v) in acetonitrile with a gradient of
0-70% organic component in 70 min and (B) 0.1% TFA
(w/v) in water/0.1% TFA (w/v) in methanol with a gradient of
20-70% organic component in 50 min, monitored at 230 nm.
All peptides displayed the appropriate molecular weights as
determined by high resolution mass spectroscopy (HRMS;
Table 2) (Protein Structure facility, University of Michigan,
Ann Arbor, MI).
Cell Lines and Membrane Preparations. C6-rat glioma cells
stably transfected with a rat μ (C6-MOR) or rat δ (C6-DOR)
opioid receptor43 and Chinese hamster ovary (CHO) cells stably
expressing a human κ (CHO-KOR) opioid receptor44 were used.
Cells were grown to confluence at 37 °C in 5% CO2 in either
Dulbecco’s Modified Eagle’s Medium (DMEM, C6 cells), or
DMEM-F12 medium (CHO-κ) containing 10% fetal bovine
serum. To prepare membranes for biochemical assays as
described previously,45 confluent cells were washed twice with
ice-cold phosphate-buffered saline (0.9% NaCl, 0.61 mM
Na2HPO4, and 0.38 mM KH2PO4, pH 7.4), detached from the
plates by incubation in harvesting buffer (20 mM HEPES, pH
7.4, 150 mM NaCl, and 0.68 mM EDTA) at room temperature,
and pelleted by centrifugation at 200g for 3 min. The cell pellet
was suspended in ice-cold 50 mM Tris-HCl buffer, pH 7.4, and
homogenized with a Tissue Tearor (Biospec Products, Inc.,
Bartlesville, OK) for 20 s at setting 4. The homogenate was
centrifuged at 20000g for 20 min at 4 °C and the pellet rehomo-
genized in 50 mM Tris-HCl with a Tissue Tearor for 10 s at
setting 2, followed by recentrifugation. The final pellet was
resuspended in 50 mM Tris-HCl, to 0.5-1.0 mg/mL protein
and frozen in aliquots at -80 °C. Protein concentration was
determined using the BCA protein assay46 (Thermo Fisher
Scientific, Rockford, IL) using bovine serum albumin as the
standard.
A
solution of trifluoroacetic acid/H2O/tri-isopropylsilane
(9:0.5:0.5, v/v/v) was used to cleave the linear peptide from the
resin and simultaneously remove the side chain-protecting
groups. The peptide solution was filtered from the resin and
then subjected to semipreparative reverse phase high-perfor-
mance liquid chromatography (RP-HPLC) to afford the linear
disulfhydryl-containing peptide.
General Method for Disulfide Cyclization of Peptides. To
obtain disulfide-cyclized peptides, linear disulfhydryl-contain-
ing peptide was dissolved in a 1% (v/v) acetic acid (AcOH) in
H2O solution (saturated with N2) at 5 °C (1 mg linear peptide/
mL of aqueous AcOH solution). The pH of the peptide solution
was raised to 8.5 using NH4OH, followed by the addition of 4
equiv of K3Fe(CN)6. The reaction mixture was stirred for 2 min
and quenched by adjusting the pH to 3.5 with AcOH. The
mixture was then subjected to semipreparative RP-HPLC to
afford the disulfide-cyclized peptide.
General Method for Methylene Dithioether Cyclization of
Peptides. To form methylene dithioether-containing cyclic pep-
tides, linear disulfhydryl peptide was added to dimethylforma-
mide (DMF) and maintained at 5 °C under a N2 atmosphere
(0.1 mg linear peptide/mL DMF). Approximately 10 equiv of
potassium t-butoxide was added to the peptide solution, fol-
lowed by the addition of 10 equiv of dibromomethane. The
reaction was quenched with 5 mL AcOH after 2 h and the
Radioligand Binding Assays. Opioid ligand-binding assays
were based on the competitive displacement of [3H]dipre-
norphine by the test compound from membrane preparations
containing opioid receptors as described above.25 The assay
mixture, containing membrane suspension (10-20 μg protein/
tube) in 50 mM Tris-HCl buffer (pH 7.4), [3H]diprenorphine
(0.2 nM), and increasing concentrations of test peptide, was
incubated at 25 °C for 1 h to allow binding to reach equilibrium.
Subsequently, the samples were filtered rapidly through GF/C

7730 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Purington et al.
filters (Whatman, Middlesex, UK) using a Brandel harvester
and washed three times with ice-cold 50 mM Tris-HCl buffer.
The radioactivity retained on dried filters was determined by
liquid scintillation counting after saturation with EcoLume
liquid scintillation cocktail (MP Biomedicals, Solon, OH) in
a Wallac 1450 MicroBeta (PerkinElmer, Waltham, MA).
Nonspecific binding was determined using 10 μM naloxone.
Ki values were determined from nonlinear regression analysis to
fit a logistic equation to the competition data using GraphPad
Prism 5.01 software (GraphPad Software, La Jolla, CA). The
results presented are the mean from at least three separate
assays, each performed in duplicate.
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pharmacophore: hydrophobic substituents at the C-terminus en-
dow delta antagonists to manifest mu agonism or mu antagonism.
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potent analgesic effect, no physical dependence, and less tolerance
than morphine in rats. J. Med. Chem. 1999, 42, 3520–3526.
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[35S]GTPγS Binding Assay. Agonist stimulation of [35S]-
GTPγS binding was measured as described previously.32
Briefly, membranes (20-30 μg of protein/tube) were incubated
in GTPγS binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM
NaCl, and 5 mM MgCl2) containing 0.1 nM [35S]GTPγS,
100 μM GDP, and varying concentrations (0.001-10000 nM)
or a maximum concentration (10 μM) of opioid peptides,
compared with standards DAMGO, SNC80, or U69,593
(10 μM) in a total volume of 500 μL for 1 h at 25 °C. The
reaction was terminated by rapidly filtering through GF/C
filters and washing four times with 2 mL of ice-cold GTPγS
binding buffer. Retained radioactivity was measured as
described in radioligand binding assay methods. Experiments
were performed at least three times in duplicate. EC50 values
were determined by nonlinear regression analysis using Graph-
Pad Prism 5.01 software as described above. To determine
antagonism of 9, [35S]GTPγS binding was determined for
SNC80 in the presence or absence of 100 nM 9. The IC50 value
in the presence of 100 nM 9 was divided by the IC50 value for
SNC80 alone, and this ratio (DR) was employed to calculate the
Ke value using the equation Ke = [antagonist]/(DR-1).
Whole Cell Acute Inhibition of Adenylyl Cyclase. Inhibition of
adenylyl cyclase by opioid standards or test peptides was
measured in C6-DOR cells grown to confluence in 24-well
plates.36 Cells were washed in serum-free DMEM at least
30 min prior to the start of the assay and incubated with vehicle
or various concentrations (0.1-1000 nM) of SNC80, naltrin-
dole, or peptide 9 in serum-free media containing 5 μM forskolin
(FSK) and 1 mM 3-isobutyl-1-methylxanthine for 10 min at
37 °C. The assay was quenched by replacing media with 1 mL
ice-cold 3% perchloric acid and 30 min incubation at 4 °C.
A 400 μL aliquot of sample was neutralized with 2.5 M KHCO3
and centrifuged 1 min at 11000g. Cyclic AMP (cAMP) was
measured from the supernatant using a radioimmunoassay kit
from GE Healthcare (Piscataway, NJ) according to the manu-
facturer’s instructions. Inhibition of cAMP accumulation by 9
or standard opioid ligands was calculated as a percent of FSK-
stimulated cAMP accumulation in vehicle-treated cells. EC50
values were calculated for each compound using GraphPad
Prism 5.01 software. Experiments were performed in duplicate
and repeated a minimum of three times.
Statistical Analysis. Data were analyzed using Student’s
two-tailed t test or a one-way analysis of variance followed by
Bonferroni’s posthoc test using GraphPad Prism version 5.01 for
Windows (GraphPad Software, San Diego, CA www.graphpad.
com). p values less than 0.05 were considered to be significant.
Acknowledgment. This work was funded by NIH grants
DA04087 (J.R.T.) and DA03910 (H.I.M.). L.C.P. was sup-
ported by DA007281 and DA007267. We thank Hui-Fang
Song for technical assistance with the [3H]diprenorphine
binding assays and Jessica Anand for assistance with the
HRMS analyses.
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