Synthesis and electrophysiological characterization of cyclic morphiceptin analogues

Article abstract of DOI:10.1016/j.bcp.2004.02.011

A challenge in opioid peptide chemistry and pharmacology is the possibility to develop novel peptides with peripheral selectivity. An enzymatically stable opioid peptide could involve an antidiarrheal effect. For this reason, we constrained the highly selective and potent tetrapeptide morphiceptin with a 6-atom bridge, resulting in a cyclic amide and an ester analogue, 2 and 3, respectively. Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying K⁺ (GIRK1/GIRK2) channel, either the wild-type μ-, κ-, δ- or a mutated μ-opioid receptor (hMORS329A) was functionally co-expressed with GIRK1/GIRK2 channels and a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes. The two-microelectrode voltage clamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. Both cyclic analogues were equally potent via the wild-type μ-opioid receptor hMORwt (EC₅₀ value 976.5±41.7 for 2 and 1017.7±60.7 for 3), while the EC₅₀ value for Tyr-Pro-Phe-D-Pro-NH₂ measured 59.3±4.8 nM. These three agonists displayed a four to five times decreased potency via hMORS329A as compared to the wild type. Interestingly, no effect on κ- and δ-opioid receptors was observed. The intramolecular bridge created by cyclization of morphiceptin prevents dipeptidyl peptidase IV from interacting with these analogues. We conclude that constraining morphiceptin with a 6-atom bridge resulted in enzymatically stable peptidomimetics that are exclusively active on μ-opioid receptors. These analogues provide an interesting template in the promising approach for the design of potential antidiarrheal agents.

Full text of DOI:10.1016/j.bcp.2004.02.011

Biochemical Pharmacology 67 (2004) 1887–1895
Synthesis and electrophysiological characterization
of cyclic morphiceptin analogues
Joost Pila, Pieter Van der Vekenb, Gunther Balb, Koen Augustynsb,
Achiel Haemersb, Jan Tytgata,*
aLaboratory of Toxicology, Faculty of Pharmaceutical Sciences, University of Leuven, Van Evenstraat 4, 3000 Leuven, Belgium
bDepartment of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, 2610 Antwerpen, Belgium
Received 6 November 2003; accepted 3 February 2004
Abstract
A challenge in opioid peptide chemistry and pharmacology is the possibility to develop novel peptides with peripheral selectivity. An
enzymatically stable opioid peptide could involve an antidiarrheal effect. For this reason, we constrained the highly selective and potent
tetrapeptide morphiceptin with a 6-atom bridge, resulting in a cyclic amide and an ester analogue, 2 and 3, respectively.
Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying Kþ
(GIRK1/GIRK2) channel, either the wild-type m-, k-, d- or a mutated m-opioid receptor (hMORS329A) was functionally co-expressed
with GIRK1/GIRK2 channels and a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes. The two-microelectrode voltage
clamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. Both cyclic analogues were equally
potent via the wild-type m-opioid receptor hMORwt (EC50 value 976:5 Æ 41:7 for 2 and 1017:7 Æ 60:7 for 3), while the EC50 value for
Tyr-Pro-Phe-D-Pro-NH2 measured 59:3 Æ 4:8 nM. These three agonists displayed a four to five times decreased potency via
hMORS329A as compared to the wild type. Interestingly, no effect on k- and d-opioid receptors was observed. The intramolecular
bridge created by cyclization of morphiceptin prevents dipeptidyl peptidase IV from interacting with these analogues.
We conclude that constraining morphiceptin with a 6-atom bridge resulted in enzymatically stable peptidomimetics that are exclusively
active on m-opioid receptors. These analogues provide an interesting template in the promising approach for the design of potential
antidiarrheal agents.
# 2004 Elsevier Inc. All rights reserved.
Keywords: Cloned opioid receptors; Xenopus oocytes; Morphiceptin analogues; Voltage clamp; RGS; GIRK
1. Introduction
sage domain (enkephalins, endorphins, dynorphins) and the
other containing the Tyr-Pro-Phe/Trp sequence. This latter
group comprises the highly selective endogenous m-opioid
receptor ligands endomorphin-1 (Tyr-Pro-Trp-Phe-NH2)
and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) that produce
potent and prolonged analgesia in test animals [2].
Morphiceptin (Tyr-Pro-Phe-Pro-NH2), a synthetic tetra-
peptide amide with a similar sequence to the endomorphins,
is a potent and selective agonist at the m-opiate receptor. This
peptide was originally synthesized as an analogue posses-
sing the N-terminal tetrapeptide fragment of b-casomor-
phin. Morphiceptin is about 50–100 times more active than
b-casomorphin in receptor binding assays and in the guinea
pig ileum test. Its potency in the mouse vas deferens assay
and its affinity for d-receptors are much lower [3].
Opioid receptors are activated by two main kinds of
molecules. The first group comprises the opiates, the non
peptide molecules like morphinans, benzomorphinans, phe-
nylpiperidines, and oripavines [1], the second group consists
of the opioid peptides. Two types of endogenous opioid
peptides exist, one containing Tyr-Gly-Gly-Phe as the mes-
Abbreviations: Boc, t-butoxycarbonyl; BOP, benzotriazole-1-yloxy-
tris(dimethylamino)phosphoniumhexafluorophosphate; Fmoc, fluoren-9-
ylmethylsuccinimidyl carbonate; 32 GIRK1/GIRK2, G-protein-coupled
inwardly rectifying potassium channel; hDOR, human wild-type d-opioid
receptor; hKOR, human wild-type k-opioid receptor; hMOR, human wild-
type m-opioid receptor (hMOR); RGS4, regulator of G-protein signaling;
TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-
borate
* Corresponding author. Tel.: þ32-16-32-34-03; fax: þ32-16-32-34-05.
Besides the central role of opiates in analgesia, mor-
phiceptin analogues have also been proposed as peripheral
E-mail address: jan.tytgat@pharm.kuleuven.ac.be (J. Tytgat).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.02.011

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J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
agents. Firstly, morphiceptin analogues showed antidiar-
rheal activity. Intracerebroventricular or subcutaneous
administration of Tyr-Pro-NMePhe-D-Pro-NH2, a potent
morphiceptin derivative, inhibited diarrhea and decreased
the gastrointestinal transit [4]. This observation provides
an interesting approach to the development of peripheral
opioid receptor agonists as antidiarrheal agents. These
drugs would lack the central effects of analgesia and
sedation, and would not cause constipation. On the other
hand, morphiceptin and other b-casomorphins inhibited
cell proliferation of human prostate cell lines, by a
mechanism partly involving opioid receptors [5,6]. Very
recently, Janecka et al. [7] presented a series of new
morphiceptin analogues, modified at the pharmacophoric
position 3 and their binding to m-opioid receptors in
experimental mammary adenocarcinoma. These therapeu-
tical possibilities encourage further studies on this group of
opioids, focusing on peripheral selectivity.
Since the discovery that morphiceptin is one of the most
selective agonists for the m-receptor, several structure–
activity studies of this tetrapeptide have been carried out
[8,9]. They modified morphiceptin by substitutions at the
second, third, and fourth amino acid residues. Substitution
of the fourth amino acid, L-Proline with D-Proline was the
most effective. This analogue (Tyr-Pro-Phe-D-Pro-NH2,
1), relative to morphiceptin, is about 15 times more active
on m-receptor binding (Fig. 1).
carried out with the Newton–Raphson method until the
maximum derivative was less than 0.001 kcal molÀ1 using
Sybyl 6.5 (MIPS3-IRIX6.2). Conformational energies
were estimated as the sum of nonbonded Van der Waals
interactions, Coulombic interaction, intrinsic torsional
potentials, and energies of deformation of bond lengths
and bond angles. After energy minimization, 10 confor-
mers with minimum energy were selected. The geometry
optimization for the selected conformers was performed
with the semi-empirical AM1 and PM3 method [11]
employing MOPAC 6 software [12]. The minimum energy
conformation showed great similarity with the conforma-
tion described by Yamazaki, adopting a cis Tyr-Pro amide
bond, a trans conformation for the side chains of Tyr and
Phe, and characteristic distances between the aromatic
rings.
These data allowed us to design a 6-atom bridge between
the terminal amide group and the Pro2-4 position, in order
to provide a rigid cyclic peptide. A global minimum energy
structure was deduced for Tyr-Pro-Phe-D-Pro-NH2 in
which Pro2 was replaced by (S)-4-hydroxyproline, where
the 4-hydroxy group is linked through a 5-C chain to the
terminal amide. For the binding of the bridging unit with
this hydroxygroup, we used an amide and an ester bond, as
shown in structures 2 and 3 (Fig. 1).
To investigate the effect of this new cyclization on the
potency of the tetrapeptide we performed an electrophy-
siological characterization of these products. Taking
advantage of the functional coupling of the opioid receptor
with the heteromultimeric G-protein-coupled inwardly
rectifying potassium channel (GIRK1/GIRK2), either the
human wild-type m-opioid receptor (hMOR), the human
wild-type k-opioid receptor (hKOR), the human wild-type
d-opioid receptor (hDOR), or the mutated receptor
(hMORS329A) was functionally co-expressed with
GIRK1/GIRK2 channels together with a regulator of G-
protein signaling (RGS4) in Xenopus laevis oocytes. The
two-microelectrode voltage clamp technique was used to
measure the opioid receptor activated GIRK1/GIRK2
channel responses, and from that the EC50 values were
obtained. Co-expression of RGS4, the brain-expressed
isoform of RGS proteins [13,14], reconstitutes the native
gating kinetics by accelerating GIRK1/GIRK2 channel
deactivation [15]. The role of Serine 329 in the m-opioid
receptor on the potency of DAMGO, morphine, fentanyl,
and b-hydroxyfentanyl was previously described [16]. In
brief, this mutation demonstrated the dual role of Serine
329 in the potency of various agonists. The potency of
DAMGO, a peptide agonist, decreased, whereas the
potency of nonpeptide agonists, like morphine and fentanyl
increased when this residue was mutated to alanine.
This experimentally created model [17] provides a
defined population of functionally active opioid receptor
(hMORwt, hKOR, hDOR, or hMORS329A) and an excel-
lent tool for measuring the efficacy and potency of a ligand
for a certain receptor [16,18].
In this study, we present two new cyclic analogues of
morphiceptin, 2 and 3, respectively (Fig. 1). These com-
pounds were designed using NMR data and molecular
modeling. Yamazaki et al. defined some main topochem-
ical requirements for the m-opioid receptor [10]. A prob-
able bioactive conformation for a potent m-agonist Tyr-Pro-
NMePhe-D-Pro-NH2 was proposed. Looking for bridging
units to constrain D4-morphiceptin 1 (Tyr-Pro-Phe-D-Pro-
NH2), we used their data to determine a cyclic bioactive
conformer. Systemic conformational energy search was
O
O
H
N
H2N
NH2
N
N
O
O
1
HO
O
O
H2N
N
X
O
O
O
N
HN
H
N
HO
2: X = NH
3: X = O
Fig. 1. Chemical structures of peptides 1, 2, and 3.

J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
1889
2. Materials and methods
que. A Rink amide MBHA resin and coupling reagents
HOBt/BOP were used on a Rainin PS 3 automated solid-
phase peptide synthesizer. Crucial synthesis steps were
monitored by means of the Kaiser test. The resin was
cleaved by treatment with 95% TFA in water for 2 h. The
peptide was precipitated as TFA-salt with cold diethyl
ether and dried in vacuo.
2.1. Compounds: peptide synthesis
2.1.1. Tyr-Pro-Phe-D-Pro-NH2 1
This linear morphiceptin analogue was synthesized
according to the Fmoc/t-Bu chemistry solid-phase techni-
HO
O
1. phtalimide,
Ph P/DEAD
3
O
NH NH .H O
N
2
2
2
N
Z
O
O
O
N
Z
4
O
5
H N
O
2
1. Ph P/DEAD
3
2. H
2
HN
N
Boc
O
7
H
H
N
Z
2
Boc-NH-(CH ) -COOH
2 4
O
O
TBTU
N
6
O
Z
8
O
X
N
Boc
O
H
OH
X
N
Boc
1. Fmoc-ONSu
H
1. Resin-D-Pro-L-Phe
2. Z-Tyr(Bn)-OH/TBTU
3. TFA
N
O
OH
O
O
N
H
O
9 : X = NH
10 : X = O
11: X = NH
12: X = O
O
O
ZHN
N
X
NH2
O
O
O
O
ZHN
N
COOH
HN
X
N
O
O
O
O
HOBt, TBTU
N
H
HN
N
O
14 : X = NH
15 : X = O
16: X = NH
17: X = O
O
O
H2N
N
X
O
O
O
N
H
HN
H2
N
HO
2: X = NH
3: X = O
Fig. 2. Synthesis of cyclic peptides 2 and 3.

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J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
2.1.2. Cyclic morphiceptin analogues 2 and 3 (Fig. 2)
GIRK2 [13]. The polylinker in each of these vectors is
flanked by Xenopus globin 50 and 30 untranslated regions,
resulting in an enhanced protein expression after injection
of in vitro transcribed cRNA [20]. For in vitro transcription,
plasmids were first linearized either with EcoRI (for pSP/
GIRK1), or with SalI (for pBScMXT/GIRK2). Next the
cRNAs were synthesized from the linearized plasmids
using the large-scale SP6 mMessage mMachine (for
pSP/GIRK1) or T3 mMessage mMachine (for pBScMXT/
GIRK2) transcription kit (Ambion).
The hMOR, hKOR, hDOR, and rat RGS4 were sub-
cloned into pGEMHE as described [17]. For in vitro
transcription, each clone was linearized with NheI. Next
the capped cRNA’s were synthesized from the linearized
plasmids using the large-scale T7 mMessage mMachine
transcription kit (Ambion).
2.1.2.1. (1S,4S,10R,19S)-21-(L-Tyr)-4-benzyl-2,5,11,17-
tetraoxo-3,6,12,18,21-pentaaza-tricyclo[17,2,1,06,10]-
docosane (2). N-Z-(2S,4R)-4-hydroxyproline benzyl ester
was treated with phtalimide in a Mitsunobu reaction
affording its N-phthalimido (S)-4-amino-derivative. The
phthalimido group was cleaved with hydrazine to obtain
the free amine, N-Z-(2S,4S)-4-aminoproline benzyl ester.
The amino group was coupled to Boc-protected 5-
aminopentanoic acid with TBTU. After removal of
both Z- and benzylprotections with hydrogenolysis, the
proline moiety was Fmoc-protected with fluoren-9-
ylmethylsuccinimidyl carbonate. This protected 4-(5-
aminopentanoyl)proline was a suitable building block to
be used in a solid-phase synthesis procedure and was
incorporated in a linear pseudopeptide with Fmoc-D-
Pro, Fmoc-L-Phe, and N-Z-L-tyrosine-O-benzyl ether.
2.3. Construction of mutant human m-opioid receptor
To prevent diketopiperazine formation
a 2-chloro-
tritylpolystyrene resin was used as polymeric support
with TBTU as coupling reagent. After Boc- and resin
cleavage by treatment with a 95% TFA solution in
water, a linear pseudopeptide was obtained. Cyclization
was achieved by coupling the C-terminal carboxylic acid
to the amino terminus of the bridging unit, in highly
diluted solution, with 3 equivalents TBTU, 3 equivalents
HOBt, and 9 equivalents diisopropyletylamine as base,
yielding the Z-and benzyl protected target compound,
(1S,4S,10R,19S)-21-[Z(O)-benzyl)L-Tyr]-4-benzyl-2,5,11,
17-tetraoxo-3,6,12,18,21-pentaaza-tricyclo[17,2,1,06,10]-
docosane. Removal of the protecting groups by hydro-
genolysis afforded compound 1.
Ser329 in hMOR was mutated to Ala329 using the
Quickchange site-directed mutagenesis kit (Stratagene).
The primers were designed in such way that a silent MluI
restriction site was introduced simultaneously: 50-GCT-
CTAGGTTACACAAACGCGTGCCTCAACCCAGTCC-
30 and 50-GGACTGGGTTGAGGCACGCGTTTGTGT-
(codon and complementary codon are underligned, palyn-
dromic sequence is in bold). Cycling parameters were set
according to the manufacturer’s guidelines. The cDNAs
from eight single colonies were digested with MluI to
identify possible mutants. All eight clones contained the
MluI restriction site, which was introduced by the mutant
primers. A 313 basepair fragment containing the desired
mutation was isolated by a double restriction digest with
NsiI and BglII. The mutant cDNA was then loaded on an
agarose gel, the fragment of interest was cut out, gene-
cleaned (QIAQUICK, QIAGEN) and ligated with T4 DNA
ligase (Promega) into the corresponding sites of the WT
hMOR/pGEMHE. The same mutant fragment was sub-
cloned into pGEM7Zf(þ) (Promega) for DNA sequencing
(Eurogentec). For in vitro transcription, the mutant
hMORS329A/pGEMHE was linearized with NheI. Next
the capped cRNA’s were synthesized from the linearized
plasmids using the large-scale T7 mMessage mMachine
transcription kit (Ambion).
2.1.2.2. (1S,4S,10R,19S)-21-(L-Tyr)-4-benzyl-2,5,11,17-
tetraoxo-18-oxa-3,6,12,21-tetraaza-tricyclo[17,2,1,06,10]-
docosane (8.1b) (3). For the synthesis of cyclic peptide 3 in
which the bridging moiety was introduced by means of an
ester bond, Boc-protected 5-aminopentanoic acid, was
linked to the starting N-Z-(2S,4R)-4-hydroxyproline
benzyl ester under Mitsunobu conditions. The incor-
poration of N-Z-(2S,4S)-4-[4(N-Boc-aminobutyl)carbo-
nyloxy]-proline benzyl ester in a linear pseudopeptide,
the cyclization and the final deprotection of (1S,4S,
10R,19S)-21-[Z(O)-benzyl)L-Tyr]-4-benzyl-2,5,11,17-te-
traoxo-18-oxa-3,6,12,21-tetraaza-tricyclo[17,2,1,06,10]-
docosane was performed as described for amide analogue 2.
2.4. Experimental model
2.2. Subcloning and in vitro transcription of cDNA
clones encoding GIRK1/2 channels; human m-, k-,
and d-opioid receptors (hMOR, hKOR, and hDOR);
and RGS4
Xenopus laevis oocytes were prepared for injection as
described [21]. Oocytes were co-injected with 0.5 ng
50 nlÀ1 GIRK1, 0.5 ng 50 nlÀ1 GIRK2 and 10 ng 50 nlÀ1
RGS4 cRNA, with the addition of 10 ng 50 nlÀ1 of either
hMOR or hMORS329A cRNA. Injected oocytes were
maintained in ND-96 solution (composition in mM: KCl
2, NaCl 96, MgCl2 1, CaCl2 1.8, HEPES 5, pH 7.5)
supplemented with 50 mg mlÀ1 gentamicin sulphate and
incubated at 16 8C.
Plasmids containing the entire coding sequence for the
mouse GIRK1 and the mouse GIRK2 channel were sub-
cloned into the vector pSP35T and pBScMXT, respec-
tively, and designated as pSP/GIRK1 [19] and pBScMXT/

J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
1891
2.5. Electrophysiological recordings
and 0% was taken as the control current level. Current
percentages were used for the calculation of the EC50-
value, using the Hill equation:
Whole-cell currents from oocytes were recorded one day
after injection using the two-microelectrode voltage clamp
technique (Geneclamp 500, Axon Instruments). Resis-
tances of voltage and current electrodes were kept as
low as possible (approx. 200 kO) and were filled with
3 M KCl. To eliminate the effect of voltage drop across
the bath-grounding electrode, the bath potential was
actively controlled. All experiments were performed at
room temperature (19–23 8C). At the start and the end of
each experiment, oocytes were superfused with low-potas-
sium (ND-96) solution (composition in mM: KCl 2, NaCl
96, MgCl2 1, CaCl2 1.8, HEPES 5, and pH 7.5). During
application of increasing concentrations of ligands,
oocytes were superfused with high-potassium (HK) solu-
tion (composition in mM: KCl 96, NaCl 2, MgCl2 1, CaCl2
1.8, HEPES 5, and pH 7.5). In HK solution, the Kþ
equilibrium potential is close to 0 mV and enables Kþ
inward currents to flow through inwardly rectifying Kþ
channels at negative holding potentials. A gravity con-
trolled fast perfusion system (Warner Instruments) was
used to ensure rapid solution exchanges. Application of
opioid ligands did not evoke an increase of the conductance
in uninjected oocytes (n ¼ 30). In each experiment,
oocytes were clamped at a holding potential of À70 mV
for approximately 10 min and superfused with ND-96
solution. Next, the superfusion was switched from ND-
96 to HK solution, after which increasing concentrations of
an opioid receptor agonist were applied. Each concentra-
tion was applied for as long as needed to achieve a steady
state GIRK1/GIRK2 current activation. Each ligand con-
centration was washed out by superfusing with HK solu-
tion. During this washout period, the channels return to the
control current level as a result of deactivation process that
is dramatically accelerated in the presence of RGS4, as
described [15]. At the end of each experiment, the oocyte
was superfused with HK solution containing 300 mM
BaCl2, causing block of the net GIRK1/GIRK2-gated
inward current. Finally, the superfusion was switched
back to ND-96 solution to confirm complete reversibility.
To avoid that the receptor expression level affects the
EC50-values of the investigated agonists in the study, the
expression system was standardized as previously
described [17].
Imax
I ¼
nH
1 þ ðEC50=AÞ
where I represents the current percentage, Imax the max-
imal current percentage, EC50 the concentration of the
agonist that evokes the half-maximal response, A the
concentration of agonist, and nH the Hill coefficient.
Averaged data are indicated as mean Æ S:E:M: and were
calculated using n experiments, where n indicates the
number of oocytes tested. Statistical analysis of differ-
ences between groups was carried out with Student’s t
test and a probability of 0.05 was taken as the level of
statistical significance.
Substrate activity for DPPIV was determined as
described [22].
3. Results
Each receptor was individually co-expressed with
GIRK1/GIRK2 channels and RGS4, mimicking the native
neuronal G-protein-mediated pathway of Kþ channel acti-
vation [14]. We used the two-microelectrode voltage clamp
technique to measure the opioid receptor-activated GIRK1/
GIRK2 channel response as the in increase of the inward
Kþ current at À70 mV, evoked by the application of in-
creasing concentrations of opioid ligands. In our study, we
examined the potency of 1 and the cyclic peptides 2 and 3
(Fig. 1) on hMORwt, the mutant receptor hMORS329A,
hKOR, and hDOR.
Fig. 3 shows representative current traces of agonist-
gated currents evoked from oocytes expressing hMORwt
by 1 (Fig. 3A), 2 (Fig. 3B) and 3 (Fig. 3C). The agonists
could not activate hKOR nor hDOR up to a concentration
of 1 mM. Fig. 4 illustrates this finding with a representative
current trace of an oocyte expressing hKOR upon applica-
tion of 1 mM 2.
Table 1 summarizes EC50 values calculated for 1, 2, and
3 via hMORwt and hMORS329A. Averaged data are
indicated as the mean Æ S:E:M. and were calculated using
five to seven experiments.
Concentration-response relationships (Fig. 5A–C) are
shown for 1, 2, and 3, respectively.
2.6. Data analysis
Table 1
EC50 values calculated for GIRK1/GIRK2 channel activation via hMORwt
or hMORS329A, co-expressed with RGS4 in Xenopus laevis oocytes
The pCLAMP program was used for data acquisition
and data files (Axon Instruments) were imported in Micro-
soft Excel. The percentage activated current was calculated
using the equation
Receptor
EC50 values
hMORwt
hMORS329A
percentage activation
ꢀ
1
2
3
59.3 Æ 4.8 nM
976.5 Æ 41.7 nM
1017.7 Æ 60.7 nM
292.8 Æ 26.9 nM
4.68 Æ 0.41 mM
4.31 Æ 0.54 mM
ꢁ
activated current amplitude
control current amplitude
¼
 100 À 100

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J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
Fig. 3. Representative current traces evoked from Xenopus laevis oocytes co-expressing GIRK1/GIRK2 channels and RGS4 with hMORwt. Agonist-gated
currents were evoked at À70 mV by application of increasing concentrations of 1 (A), 2 (B), or 3 (C). ND-96 solution contains (in mM): KCl 2, NaCl 96,
MgCl2 1, CaCl2 1.8, and HEPES 5. HK solution is composed of (in mM): KCl 96, NaCl 2, MgCl2 1, CaCl2 1.8, and HEPES 5.
The linear morphiceptin analogue 1 (EC50 value:
59:3 Æ 4:8 nM) was about 16 times more potent than the
cyclic analogues via the wild-type m-opioid receptor.
Both cyclic analogues were as potent via the hMORwt
(EC50 value 976:5 Æ 41:7 nM for 2 and 1017:7 Æ 60:7 nM
for 3).
On the mutant receptor, the EC50 values for the three
agonists were four to five times higher as compared to the
wild-type receptor (EC50 values 292:8 Æ 26:9 nM for 1,
4:68 Æ 0:41 mM for 2, and 4:31 Æ 0:54 mM for 3). Statis-
tical analysis revealed that there was no difference in
potency between 2 and 3. 1 displayed also on the mutant
hMORS329A a 16-fold higher potency than the cyclic
peptides.
The cyclic proline analogues were found to be stable
against hydrolysis by DPPIV. Under identical conditions
the corresponding linear analogues showed half-life of
about 30 min. Degradation was monitored by HPLC and
a disappearance curve was constructed from the integrated
peak areas versus time. To verify the specificity of the

J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
1893
4. Discussion
In this study, we present the synthesis and pharmaco-
logical profile of two new cyclic morphiceptin analogues.
In search for biologically active and stable peptides, we
designed a 6-atom bridge between the terminal amide and
the Pro2-4 position: a tetramethylene group linked to the
Pro residue through an amide or ester bond. Focusing on
peripheral selectivity could afford interesting therapeutic
products, which would not be associated with opiate side
effects linked to central opioid activity (sedation, analge-
sia). Such analogues need certain structural features favor-
ing a local gastrointestinal activity: these products should
be stable against breakdown by peptidases in the gastro-
intestinal tract, while their lipophilicity should be limited,
in order to prevent absorption from the intestine and
potential side effects.
Fig. 4. Representative current trace evoked from Xenopus laevis oocytes
co-expressing GIRK1/GIRK2 channels and RGS4 with hKOR. Currents
were evoked at À70 mV by application of 1 mM of 2.
reaction, HPLC analysis was also performed after inacti-
vation of the enzyme by a selective irreversible inhibitor
(diphenyl Pro-Prophosphonate). No peptide degradation
was observed.
The p-hydroxyphenethylamine (tyramine) moiety is a
structural feature shared by all fused ring opiates and the
N-terminal tyrosine of opioid peptides. The presence of
this tyramine moiety is widely accepted as a requirement
1
for high m-affinity. H NMR spectroscopic measurements
and computer simulations were carried out on 10 stereo-
isomeric analogues related to morphiceptin to obtain a
topochemical model that explains the bioactivity of mor-
phiceptin [10]. The relative spatial arrangements of the
pharmacophoric groups, the amine and phenolic groups of
the Tyr1 residue and the aromatic group of the Phe3 residue
were defined by a set of eight bond conformations. The
torsional angles around these eight bonds required for
morphiceptin bioactivity were determined. The most inter-
esting characteristic of the model is a requirement of a
cis amide bond linking residues Tyr1 and Pro2, although
energetically less favored.
Recently, 1H NMR spectral analysis provided evidence
for the cis Tyr-Pro amide bond for morphiceptin and
endomorphin-2, using pseudoproline-containing analo-
gues [23]. Furthermore, it has been suggested that the
fourth residue of endomorphin has less stringent stereo-
chemical or conformational requirements. Molecular
dynamics simulations and NMR data indeed show that
this position is conformationally flexible and independent
of the preceding three amino acids [24], the structure of
residue 4 does not affect recognition at the receptor site
[10].
In the search for bridging units to constrain the peptide,
we used the model by Yamazaki et al. [10] to determine a
possible constrained bioactive conformer. The minimum
energy conformation of our analogues showed great simi-
larity with the conformation described in this model.
On the wild-type receptor, there was no difference in
EC50 value for both cyclic analogues: 976 Æ 41:7 nM and
1017:7 Æ 60:7 nM for 2 and 3, respectively. As compared
to the EC50 value of the linear tetrapeptide Tyr-Pro-Phe-D-
Pro-NH2 (59:3 Æ 4:8 nM), the cyclic derivatives are
approximately 16 times less potent. This may be due to
Fig. 5. Concentration–response curves for GIRK1/GIRK2 channel activa-
tion by increasing concentrations of 1 (A), 2 (B), and 3 (C). Agonist-gated
currents were evoked from Xenopus laevis oocytes co-expressing GIRK1/
GIRK2 and RGS4 with hMORwt (^) or hMORS329A (&). The agonist-
gated increase of GIRK current at each concentration was normalized to a
maximal response of 100%. Each point represents the average current
activation evoked from five to eight oocytes (mean Æ S:E:M:).

1894
J. Pil et al. / Biochemical Pharmacology 67 (2004) 1887–1895
an unoptimized conformation. The potency of the linear
peptide 1 towards the wild-type receptor is similar to the
potency of fentanyl (EC50 value 64:9 Æ 7:8 nM) and only
slightly lower as compared to [D-Ala2,NMePhe4,Gly5-ol]-
enkephalin (DAMGO) [18]. The greater flexibility of
DAMGO that may allow additional binding modes could
contribute for this difference [24].
receptors. This selectivity together with the resistance to
enzymatic hydrolysis provides an interesting pharmaceu-
tical template in the promising approach for the design of
potential antidiarrheal agents.
Acknowledgments
The potency of all agonists was decreased four to five
times by mutating the wild-type hMOR to hMORS329A.
This residue is a part of the seventh transmembrane seg-
ment of the receptor and lies deep within the binding cavity
of the receptor. Recently, our group showed that mutation
of this residue to alanine caused dual effects depending on
the nature of the ligand [16]. We noticed a similar decrease
in potency with DAMGO as we can observe here with 1,
the cyclic amide and the ester derivative, suggesting simi-
lar hydrophilic contacts deep within the binding cavity of
the seventh transmembrane helix.
GIRK1 cDNA was a gift from Kazutaka Ikeda (The
Institute of Physical and Chemical Research, RIKEN,
Wako, Japan). GIRK2 and RGS4 were kindly donated
by Henry Lester (California Institute of Technology, Pasa-
dena, USA). The hMOR cDNA was a gift from Lei Yu
(University of Cincinnati, Cincinnati, USA). The hKOR
cDNA was kindly provided by Lee-Yuan Liu-Chen (Tem-
ple University, Philadelphia, USA). The hDOR cDNA was
a gift from Henry Yamamura (University of Arizona,
Tucson, USA).
Interestingly, the cyclic derivates and the linear tetra-
peptide are selective for the m-opioid receptor. None of
these agonists could activate GIRK1/GIRK2 channels
coupled to hDOR or hKOR. Endomorphins have the high-
est specificity for m receptors than any of the endogenous
substances heretofore described [25]. This selectivity fea-
ture is of major importance in the quest for new therapeutic
agents.
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