A new class of highly potent and selective endomorphin-1 analogues containing α-methylene-β-aminopropanoic acids (map)

Article abstract of DOI:10.1021/jm300664y

A new class of endomorphin-1 (EM-1) analogues were synthesized by introduction of novel unnatural α-methylene-β-amino acids (Map) at position 3 or/and position 4. Their binding and functional activity, metabolic stability, and antinociceptive activity were determined and compared. Most of these analogues showed high affinities for the μ-opioid receptor and an increased stability in mouse brain homogenates compared with EM-1. Examination of cAMP accumulation and ERK1/2 phosphorylation in HEK293 cells confirmed the agonist properties of these analogues. Among these new analogues, H-Tyr-Pro-Trp-(2-furyl)Map-NH₂ (analogue 12) exhibited the highest binding potency (K_i^μ = 0.221 nM) and efficacy (EC ₅₀ = 0.0334 nM, E_max = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these analogues with the opioid receptors. We found that, compared to EM-1, the incorporation of our synthesized Map at position 4 would bring the analogue to a closer binding mode with the μ-opioid receptor.

Full text of DOI:10.1021/jm300664y

Article
pubs.acs.org/jmc
A New Class of Highly Potent and Selective Endomorphin-1
Analogues Containing α-Methylene-β-aminopropanoic Acids (Map)
Yuan Wang,§ Yanhong Xing,§ Xin Liu,§ Hong Ji, Ming Kai, Zongyao Chen, Jing Yu, Depeng Zhao,
Hui Ren, and Rui Wang*
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Institute of Biochemistry
and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou, 730000, P. R. China
S
* Supporting Information
ABSTRACT: A new class of endomorphin-1 (EM-1)
analogues were synthesized by introduction of novel unnatural
α-methylene-β-amino acids (Map) at position 3 or/and
position 4. Their binding and functional activity, metabolic
stability, and antinociceptive activity were determined and
compared. Most of these analogues showed high affinities for
the μ-opioid receptor and an increased stability in mouse brain
homogenates compared with EM-1. Examination of cAMP
accumulation and ERK1/2 phosphorylation in HEK293 cells
confirmed the agonist properties of these analogues. Among
these new analogues, H-Tyr-Pro-Trp-(2-furyl)Map-NH2 (ana-
logue 12) exhibited the highest binding potency (Kiμ = 0.221
nM) and efficacy (EC50 = 0.0334 nM, Emax = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in
comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these
analogues with the opioid receptors. We found that, compared to EM-1, the incorporation of our synthesized Map at position 4
would bring the analogue to a closer binding mode with the μ-opioid receptor.
system (CNS).10 Therefore, it is essential to enhance their
bioavailability in order to facilitate therapeutic use.
For the past several years, hundreds of EM based compounds
have been designed and synthesized to explore the importance
INTRODUCTION
The opioid system is one of the most studied pain relieving
systems, and it consists of three subtype receptors: the μ opioid
■
receptor (MOR), the δ opioid receptor (DOR), and the κ
of specific residues and to gain insight into the structural
opioid receptor (KOR).1 Opioid peptides serve as endogenous
requirements for bioavailability.9,11 According to the “message−
neurotransmitters and exert their pharmacological functions
address” concept, EMs can be divided into two components:
through these receptors.2 Opioid peptides have been studied
extensively since their discovery, and many efforts have been
the biologically important N-terminal Tyr-Pro-Trp/Phe frag-
ment (message sequence) and the remaining C-terminal
dedicated to the determination of their intrinsic nature.3 In
fragment (address sequence).12,13 In the message sequence,
1997, the MOR endogenous tetrapeptides, endomorphin-1
Tyr1 is widely considered to be the most conserved residue and
(EM-1, H-Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (EM-2,
essential to the opioid activity of EMs, the aromatic groups at
H-Tyr-Pro-Phe-Phe-NH2), were isolated from the bovine brain
positions 1 and 3 of endomorphins are required for MOR
recognition,14 and Pro2 is thought to be a spacer residue that
and the human cortex. These peptides exhibited the highest
affinity for the MOR and a high MOR over DOR selectivity.4,5
Furthermore, EMs are thought to inhibit pain without some of
the undesirable side effects of morphine. Particularly, the
rewarding effect of EMs can be separated from analgesia,6 and
they are less prone to induce respiratory depression and
cardiovascular effects at effective antinociceptive doses.7 For
these reasons, EMs have attracted much attention from the
peptide chemist/pharmacologist teams and have been consid-
ered as useful pharmacological tools with a tremendous
potential in pain alleviation.8,9 However, EMs still suffer from
serious limitations including lack of oral activity, short duration
of action, poor metabolic stability, and relative inability to cross
the blood−brain barrier (BBB) and access the central nervous
joins the two pharmacophore residues Tyr1 and Trp3/Phe3. On
the other hand, the address sequence (Phe4-NH2) is considered
to influence mainly binding affinity and selectivity.15,16 The
main synthetic strategies in the chemical modification of EMs
involve the insertion of unnatural amino acids, alteration of the
peptide backbone, modification of the pharmacophore groups,
and introduction of side chain/backbone conformational
constraints.17−20
β-Amino acids are an interesting class of physiologically
active compounds for medicinal chemists.21−23 Endomorphins
Received: May 11, 2012
Published: June 22, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of Unnatural β-Amino Acids Map
a
Reagents and conditions: (a) catalyst 20% mol, K2CO3 (1.5 M, 1.2 equiv), toluene, 0 °C, 24 h, 83−92%; (b) NaOMe (2.2 equiv), THF, −15 °C, 30
min; (c) (HCHO)n (5 equiv), −15 °C, 8 h, 89−93%; (d) LiOH (aq, 1.5 equiv), THF, rt, 12 h, 95−98%; (e) HCl/EA (v/v = 1:4), rt., 2 h, 95−99%.
containing β-amino acids are generally more stable toward
proteolysis, and in some cases replacement of the α-amino acids
with the β-amino acids results in improved activity.24−29
However, the β-amino acids improve the activity of EMs only
when they were placed at position 2 as a stereochemical spacer.
When they were incorporated at other positions, a great
decrease of affinity was observed.26,27 Considering the
structural features of a β-amino acid, its flexible backbone
structure possibly hindered the EMs from choosing an ideal
space orientation.
Table 1. Maps Used in This Study
In this paper, we introduced a series of novel unnatural β-
amino acids (2-methylene-3-aminopropanoic acids (Map)) into
EM-1. Unlike normal β-amino acids, Map is characterized by a
methylene at the Cα position. This CC bond generates a
conformational constraint on the backbone structure and
provides more rigidity to the molecule. A new methodology
that had been developed by our group was employed to obtain
this kind of unnatural β-amino acids.30 The strategy was
fulfilled by asymmetric addition of N-acylpyrrole phosphonates
to N-tert-butyloxycarbonyl (N-Boc) imines generated in situ
catalyzed by a bifunctional thiourea catalyst. Then the
corresponding Mannich adducts were further transformed to
aza-Morita−Baylis−Hillman (MBH) products by Horner−
Wadsworth-Emmons (HWE) olefination with good to
excellent enantioselectivities. Subsequently, the desired Map
could be afforded by saponification of the corresponding
methyl ester (Scheme 1). Importantly, compared with
conventional aza-MBH reaction, which often provides N-Tos
products in which the tosyl group was not easily deprotected,
the present method could produce the required Map protected
by the N-Boc group which could be removed under mild acidic
conditions in a normal approach of polypeptide synthesis.
Furthermore, the side chains of these unnatural amino acids
could be replaced with many other aromatic or heteroaromatic
groups to provide more Map derivatives with different
pharmacophore elements (Table 1).
The Map were employed to substitute the residue at position
3 or/and position 4 in the hope of improving the bioactivity
and enzyme resistance of EM-1. The opioid receptor affinity
and in vitro opioid activities of these analogues were
determined by radioligand binding experiments with
[3H]DAMGO in HEK293 cells stably expressing MOR and
GPI/MVD bioassays,31,32 respectively. Then the agonist/
antagonist profiles of these analogues were assessed in cyclic
AMP accumulation and ERK1/2 phosphorylation assays.
Furthermore, the degradation rate of these analogues was
examined in the presence of mouse brain homogenate, and
their in vivo antinociceptive activities were characterized by a
tail-flick test after intracerebroventricular (icv) administrations
in mice. Finally, molecular modeling approaches were
employed to gain more insight into the structure−activity
relationship between the analogues and MOR.
RESULTS
■
Synthesis. Endomorphins and their analogues were
obtained by solution-phase methods with segment-coupling
peptide synthesis strategy. Chiral 2-methylene-3-aminopropa-
noic acids (Map) were prepared by using the method we have
published (see the Experimental Section and Supporting
Information for experimental details).30 Synthesis of all the
analogues was conducted by performing an active ester
reaction, and EDC and HOBt were used as coupling agents.
Peptides were purified using semipreparative RP-HPLC and
characterized by RP-HPLC, TLC, ESI-TOF MS, [α]D20, and
mp. Purities were determined to be 95−99% and characterized
by analytical RP-HPLC. Table 1 outlined the Map used in this
study. The detailed analytical properties of the synthetic
analogues are provided in Table S1 in the Supporting
Information.
Radioligand Binding and Selectivity. The affinity and
selectivity of EMs and the analogues were evaluated by
radioligand binding assay in whole cell preparations from
HEK293 cells expressing the MOR or DOR. [3H]DAMGO and
[3H]DPDPE were used as MOR and DOR specific radio-
ligands, respectively. EM-1 and EM-2 were also characterized
for comparison, and the affinity values were in agreement with
those published previously.4,28,33 The opioid receptor binding
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Table 2. Opioid Receptor Binding Affinities and in Vitro Pharmacological Activity of EMs and Analogues
d
IC50 (nM)
selectivity
ac
,
bc
,
e
peptide
sequence
Kiμ (nM)
Kiδ (nM)
Kiδ/ Kiμ
GPI
MVD
MVD/GPI
1
Tyr-Pro-Trp-Phe-NH2
2.60 0.21
3.20 0.13
6080 640
2338
2006
576
14.1 1.7
9.33 1.12
20.9 2.37
6.81 0.80
38.1 1.2
31.5 1.5
15.3 3.2
7.66 0.51
16.6 3.7
84.2 2.0
14.3 1.7
2.92 0.31
3.94 0.60
33.9 6.2
18.2 3.8
30.4 2.6
21.6 3.4
>10000
2.2
2.3
2
Tyr-Pro-Phe-Phe-NH2
6420 330
3
Tyr-Pro-(Ph)Map-Phe-NH2
103
2
59290 5680
56010 5180
10980 1680
17040 2050
10810 1340
5820 450
4
Tyr-Pro-Trp-(Ph)Map-NH2
0.535 0.076
15.7 0.4
104692
699
7.53 1.22
166 34
130 14
36.7 7.0
69.4 7.4
365 14
299 14
432 10
15.8 0.9
10.2 1.2
84.4 6.8
1.1
4.4
4.1
2.4
9.1
22
5
Tyr-Pro-(Ph)Map-(Ph)Map-NH2
Tyr-Pro-Trp-(4-FPh)Map-NH2
Tyr-Pro-Trp-(4-ClPh)Map-NH2
Tyr-Pro-Trp-(3-ClPh)Map-NH2
Tyr-Pro-Trp-(2-ClPh)Map-NH2
Tyr-Pro-Trp-(4-MeOPh)Map-NH2
Tyr-Pro-Trp-(piperonyl)Map-NH2
Tyr-Pro-Trp-(2-furyl)Map-NH2
Tyr-Pro-Trp-(3-furyl)Map-NH2
Tyr-Pro-Trp-(1-naphthyl)Map-NH2
Tyr-Pro-Trp-(2-naphthyl)Map-NH2
6
13.7 0.9
1244
1518
1668
2724
2112
2418
226290
185876
3264
3097
7
7.12 1.05
3.49 0.25
5.48 0.38
4.83 0.91
7.73 1.02
0.221 0.014
0.274 0.066
26.0 3.5
8
9
14930 1620
10200 1430
18690 1330
50010 2880
50930 6710
84680 10490
84850 9650
10
11
12
13
14
15
3.6
30
5.4
2.6
2.5
7.7
27.4 0.8
141
6
a
b
c
Displacement of [3H]DAMGO (Kd = 0.6 nM, μ-selective). Displacement [3H]DPDPE (Kd = 2.8 nM, δ-selective). Displacement was done using
whole cell preparations from transfected HEK293 cells expressing μ-opioid receptor and δ-opioid receptor, respectively. Ki values were calculated
according to the Cheng−Prusoff equation: Ki = EC50/(1 + [ligand]/Kd), where the shown Kd values were taken from isotope saturation experiments.
d
e
Data are expressed as the mean SEM, n ≥ 3, each performed in triplicate. Values represent the average of 10−15 measurements. Potency ratio.
a
Table 3. Functional Activity of EMs and Analogues
peptide
sequence
EC50 (nM)
Emax (%)
0
DAMGO
3.04 0.32
98.14
83.13
82.75
70.78
97.94
60.26
81.45
6
4
4
2
3
6
6
1
Tyr-Pro-Trp-Phe-NH2
14.40 0.62
11.80 0.23
36.50 2.45
0.16 0.09
2
Tyr-Pro-Phe-Phe-NH2
3
Tyr-Pro-(Ph)Map-Phe-NH2
4
Tyr-Pro-Trp-(Ph)Map-NH2
5
Tyr-Pro-(Ph)Map-(Ph)Map-NH2
Tyr-Pro-Trp-(4-FPh)Map-NH2
Tyr-Pro-Trp-(4-ClPh)Map-NH2
Tyr-Pro-Trp-(3-ClPh)Map-NH2
Tyr-Pro-Trp-(2-ClPh)Map-NH2
Tyr-Pro-Trp-(4-MeO)Map-NH2
Tyr-Pro-Trp-(Piperonyl)Map-NH2
Tyr-Pro-Trp-(2-Furyl)Map-NH2
Tyr-Pro-Trp-(3-Furyl)Map-NH2
Tyr-Pro-Trp-(1-Naphthyl)Map-NH2
Tyr-Pro-Trp-(2-Naphthyl)Map-NH2
45.09 4.01
7.35 1.02
6
7
12.00 0.98
0.72 0.08
85.57 11
8
92.53
82.57
85.56
83.90
97.14
98.73
71.01
67.46
4
4
3
5
5
5
4
5
9
0.84 0.03
10
11
12
13
14
15
10.91 0.83
10.70 1.09
0.0334 0.0012
0.0342 0.0018
72.30 6.00
70.34 4.67
a
Effects of peptides on forskolin stimulated cyclic AMP accumulation by μ-opioid receptor. HEK293 cells expressing MOR were stimulated with
increasing concentrations of the indicated peptides as described in the Experimental Section. EC50 and Emax values were calculated by using the
GraphPad Prism software. Data were means SEM, n ≥ 3, each performed in triplicate.
properties of the analogues were summarized in Table 2. The
analogue in which Trp3 was replaced by (Ph)Map (3) exhibited
about 39.6-fold lower μ-affinity and 9.8-fold lower δ-affinity
than EM-1. However, substitution of Phe4 with (Ph)Map gave
noteworthy to mention that 8 displayed the highest δ-affinity in
all the synthesized analogues. Analogues 12 and 13 bearing a
furyl ring were the two most potent peptides. Their μ-affinity
increased about 10-fold (Kiμ of 0.221 and 0.274 nM,
respectively), and their δ-affinity decreased about 8-fold over
EM-1, consequently giving a remarkable increase in μ-
selectivity (Kiδ/Kiμ of 226 290 and 185 876, respectively),
which made analogues 12 and 13 also the most μ-selective
analogues. Introduction of (1-naphthyl)Map (14) and (2-
naphthyl)Map (15) to the sequence decreased their binding μ-
affinities by 10- to 11-fold, and these analogues displayed
similar μ-selectivity compared to the parent peptide.
analogue 4 with about 4.9-fold increase in μ-affinity (Kiμ
=
0.535 nM) and 9.2-fold decrease in δ-affinity (Kiδ = 56,010
nM), thus resulting in a remarkable increase in μ-selectivity.
The corresponding substitution, performed at both positions 3
and 4, led to analogue 5 which exhibited about 6-fold lower μ-
affinity compared with the parent. Thus, subsequent
modifications were performed mainly on the aromatic group
of position 4. Substitution of the Phe4 with (4-FPh)Map
afforded analogue 6 with reduced potency in comparison with
parent peptide. Analogues 7−11 displayed relatively high μ-
affinity, with binding affinities ranging from 3.49 to 7.73 nM,
and their selectivities were similar to that of EM-1. It was
In Vitro Pharmacological Activity. Pharmacological
activities were evaluated in vitro using isolated guinea pig
ileum (GPI) for MOR and mouse vas deferens (MVD) for
DOR. The former tissues contained predominantly μ-opioid
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receptor but also κ-opioid receptor, whereas the latter included
predominantly δ-opioid receptor but contained μ- and κ-opioid
receptors as well. The potencies of these analogues to inhibit
electrically evoked neurotransmitter release and the resulting
muscle contractions in GPI and MVD preparations were
summarized in Table 2. In the GPI assay, the potency of
analogues 7−9 and 11 were found to be similar to that of EM-
1. In agreement with the radioligand binding affinity assays,
analogues 12 and 13 were the two most potent analogues,
which exhibited inhibition constants of 2.92 and 3.94 nM,
respectively. Analogues 3, 5, and 15 with radioligand binding
affinities lower than EM-1 showed similar GPI potency as EM-
1. Although analogue 8 exhibited slightly decreased affinity
toward MOR in radioligand binding assays, its potency was
observed to be nearly 2-fold higher in the GPI assays. In MVD
assays, all analogues were relatively weak DOR agonists except
4, 12, and 13. The three analogues showed relative potent in
MVD potency, though they exhibited low Kiδ values. In order
to make clear this discrepancy, the MVD activity of EMs and all
the analogues was examined using a specific DOR antagonist,
naltrindole. The results showed that the MVD activity of
analogues was partly inhibited (23−65%) by naltrindole as well
as that of EMs, suggesting the MVD activity of these analogues
was mainly produced by the coexisting μ-opioid receptor in the
MVD tissues. Such discrepancy between the MVD potency and
DOR binding affinity was also observed in previous
studies.34−36
Inhibition of Forskolin-Stimulated cAMP Increase. It
was widely known that inhibition of cAMP production was one
of the physiological consequences of agonist binding to the
MOR.37,38 The potencies of the new EM-1 analogues were
evaluated via functional [3H]cAMP competitive assays using
HEK293 cells expressing MOR.27,37,38 The results of the
analogue stimulated [3H]cAMP functional assays are summar-
ized in Table 3. All of the analogues showed inhibition of
forskolin (10 μM) stimulated cAMP production in a
concentration-dependent manner. Potency (EC50) and efficacy
(Emax) values were compared with those of EMs and DAMGO.
Analogues 4 and 6−13 exhibited higher potencies than EMs,
but the replacements proceeded at Trp3, resulting in analogues
3 and 5 displaying lower potencies than the native peptides.
Analogues 12 (EC50 = 0.0334 nM) and 13 (EC50 = 0.0342 nM)
showed about 431 and 421 times higher potency than EM-1
(EC50 = 14.4 nM). Endomorphins exhibited comparable
potency but lower efficacy (Emax of 83.13% and 82.75%,
respectively) compared with DAMGO, confirming that the
EMs were partial agonist and DAMGO was a full agonist.39,40
Analogues 4, 12, and 13 exhibited the highest efficacies (Emax of
97.94%, 97.14%, 98.73%, respectively), acting as full agonists.
Analogues 5−11, 14, and 15 displayed slightly lower efficacy
than DAMGO, which indicated that those analogues remained
partial agonist properties.
Figure 1. Analogues stimulated pERK1/2 phosphorylation in HEK293
cells expressing human μ-opioid receptor. HEK293 cells stably
expressing μ-opioid receptor were treated with analogues 4, 12, 13,
and native EM-1 for the indicated concentrations (mole). Whole cell
lysates were prepared and analyzed for pERK and ERK content by
Western blot. Results are representative of at least three independent
experiments.
concentration-dependent manner. Compared with EM-1, all
of the tested analogues were able to activate ERK1/2 by
stimulating MOR at 10 nM, but the magnitudes of the
phosphorylation for analogues 4, 12, and 13 were reduced
much more slowly than the native EM-1. A statistical difference
was observed at 1 pM: the magnitudes of the phosphorylation
for analogues 4, 12, and 13 were much stronger than that of
EM-1, which indicated that the three analogues exhibited
higher potencies for ERK1/2 phosphorylation.
Antinociception. Antinociceptive potencies of EM-1 and
its analogues were studied in the mice tail-flick test.
Antinociception was expressed as a percentage of maximum
possible effect (% MPE), and analogues 4, 8, 12, and 13 were
selected for determination of ED50 values based on their
potential binding affinities and functional activities (Table 4).
Table 4. In Vivo Antinociceptive Activities of EM-1 and Its
Analogues Given icv To Produce Tail-Flick Inhibition in the
Mouse
a
peptide
ED50 (nmol/kg)
1
15.2 (13.1−19.3)
2.33 (1.74−3.03)
9.28 (6.66−12.5)
1.42 (1.11−1.88)
1.55 (1.09−2.06)
4
8
12
13
a
The ED50 values were estimated at the time of peak activity and are
given as the mean value with its 95% confidence limits.
Effect of the Analogues on ERK1/2 Activation by the
μ-Opioid Receptor. It had been reported that ERK1/2
phosphorylation could result from MOR activation.41,42 To
examine whether stimulation of the MORs by the analogues
had a downstream effect, alteration of ERK1/2 phosphorylation
was evaluated in HEK293 cells expressing MOR. Analogues 4,
12, and 13 were selected considering their high bioactivity.
Serum-starved HEK293 cells stably expressing the MOR were
treated with analogues 4, 12, and 13 for the times indicated in
Figure 1. EM-1 and analogues 4, 12, and 13 obviously
stimulated ERK1/2 phosphorylation in 10 min in a
The ED50 value for EM-1 after icv administration was 15.2
nmol/kg, which was in agreement with a previous report.43
Analogues 4, 8, 12, and 13 displayed a better analgesic effect
and produced dose- and time-dependent inhibition at doses
ranging from 0.67 to 20 nmol/kg compared with the parent
peptide (Figure 2A−E). Analogue 12 displayed the highest
analgesic effect, about 10.9-fold more potent than EM-1, and
analogues 4, 8, and 13 also showed a more potent analgesic
effect with ED50 values of 1.6- to 9.5-fold higher, respectively.
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Figure 2. Time course of the antinociceptive effect in icv EM-1 (A) and analogues 4 (B), 8 (C), 12 (D), and 13 (E) in the mouse tail-flick test.
Groups of mice were administered an icv injection of saline or different doses of EM-1 and its analogues. The doses used are shown in the figure.
The tail-flick responses were measured at 5, 10, 15, 20, 30, 40, 50, and 60 min after the injection. Each value represents the mean SEM for 10−15
mice. The icv injection saline versus each dose of the above drugs are significantly different (p < 0.05) (Student’s t test). The antinociceptive effects
induced by EM-1, analogues 4, 8, 12, and 13 at 20 nmol/kg were significantly antagonized by naloxone (2 mg/kg) (p < 0.05) (F). Naloxone was
administered ip 10 min before icv administration of drugs. The error bar indicates the SEM of the mean, and the asterisk indicates that the response
was significantly different from control.
Moreover, antinociceptive effects of analogues 4, 8, 12, and 13
were inhibited by naloxone (2 mg/kg, ip), suggesting that
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MOR was involved in producing analgesia (Figure 2F). All the
other analogues produced antinociceptive activities in dose-
related manner at a dose of 20 nmol/kg (Table S2).
Metabolic Stability. The metabolic stability of EM-1 and
its analogues was assessed in mouse brain homogenate. Table 5
extracellular surface. This analogue formed three hydrogen
bonds with Thr218, Glu229, and Lys233, respectively.
Analogue 4 was also stabilized by many stacking interactions
with the aromatic moieties of the receptor. The side chain of
Tyr1 pointed toward a hydrophobic pocket composed mainly of
the aromatic residues Tyr148, Phe152, Phe237, and Trp293.
The aromatic groups of Trp3 and (Ph)Map4 were involved in
stacking interactions with the side chains of Phe221 and
Trp318. Site-directed mutagenesis experiments had already
determined the importance of Asp147, Tyr148, Glu229, H223,
and Trp318, and our proposed model was in good agreement
with the available mutation studies.45−51 In addition, our
modeling complex shared a similar ligand binding site with the
recently resolved crystal structure of human MOR bound to
antagonist β-FNA:52 eight residues (Asp147, Tyr148, Met151,
Lys233, Trp293, Ile296, His297, and Val300) that had direct
interactions with β-FNA were also found to make close
contacts with the analogue (Supporting Information Table S3),
indicating that our modeling results were in accordance with
the crystal structure. In analogue 5, Tyr1 was inserted inside the
aromatic clusters of Tyr148, Phe152, Phe237, and Trp293 and
formed a salt bridge with Asp147. However, this analogue lost
its contact with Phe221, and its interaction with Trp318 was
also weakened. In addition, analogue 5 formed only one
hydrogen bond, between (Ph)Map4 and Asp216, with the
receptor. Analogue 12 bound in the same aromatic pocket as
described for analogue 4 and formed the same hydrophilic
interactions with Asp 147, Thr218, and Glu229. Furthermore,
the oxygen on the furyl ring of (2-furyl)Map4 formed two
hydrogen bonds with the side chains of His223 and Lys233,
respectively.
Table 5. Half-Lives of EM-1 and Its Potent Analogues in
a
Mouse-Brain Membrane Homogenate
b
c
peptide
100 × k (min−1
)
half-life
1
4.10 0.14
1.11 0.05
0.77 0.18
0.81 0.19
0.78 0.22
16.9 1.2
62.4 3.1
89.9 9.3
85.9 9.2
88.3 8.2
4
8
12
13
a
Values are arithmetic mean of at least three individual experiments
SEM. The protein content of the brain homogenate was 2.1 mg/mL.
b
c
Velocity constants. Half-lives were calculated on the basis of pseudo-
first-order kinetics of the disappearance of the peptides.
summarized the half-lives determined for EM-1 and some of its
potent analogues in twice-washed 15% mouse brain membrane
homogenate. Within the brain homogenate, EM-1 disappeared
rapidly with a half-life of 16.9 min. The replacement of Phe4 in
EM-1 with Map resulted in an enhancement of the stability,
giving analogues 4, 8, 12, and 13 the half-life range from 62.2 to
89.9 min. Furthermore, analogue 8 exhibited a significant
enhancement of metabolic stability of about 5.3-fold compared
with EM-1.
Molecular Modeling. To further investigate the interaction
modes between the analogues and receptor, analogues 4, 5, and
12 were subjected to molecular docking and molecular
dynamics (MD) simulations in view of their biological activities
and structural diversity. The three analogues were flexibly
docked into a model of human MOR, and then the resultant
docking models were further optimized with molecular
dynamics. The binding modes of these analogues established
after MD simulations were shown in Figure 3. The obtained
binding orientation of analogue 4 was similar to that of EM-1
that was reported previously by our group:44 the Tyr1 lay at the
bottom of the binding site, and its protonated nitrogen moiety
interacted with the carboxyl group of Asp147 to form a salt
bridge, and the rest of the analogue was orientated toward the
DISCUSSION
■
For many peptide hormones and neurotransmitters, peptides
could be thought of as being composed of a “message” region
and an “address” region. The message sequence was the part of
the peptide structure that was necessary for agonist activity and
signal transduction. The address region was the part of the
structure that was primarily important for recognizing and
binding to the receptor. Previous structure−activity relationship
(SAR) studies demonstrated that the endomorphin sequence
could also be divided into a N-terminal tripeptide message
Figure 3. Binding modes of the three peptides obtained after MD simulations optimization: analogue 4 (A), analogue 5 (B), analogue 12 (C).
Predicted peptide binding poses are shown in line mode with their carbon atoms in magenta. Residues making close interactions with the peptides
are labeled with their carbon atoms in green. Oxygen, nitrogen, and hydrogen atoms are colored as red, blue, and white, respectively. Hydrogen
bonds are represented as black dashed lines.
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domain (Tyr1-Pro2-Phe3/Trp3) and a C-terminal address
domain (Phe4-NH2). C-Terminal residues played an important
role in the biological activity of MOR-specific ligands, and
many investigators proposed that the C-terminal sequence
could help in choosing or stabilizing a favorable conformation
among multiple conformations, and it was indispensable for the
high binding activity to MOR. In this study, a series of
unnatural β-amino acids, 2-methylene-3-aminopropanoic acids
(Map), were synthesized and incorporated into EM-1 mainly at
the address domain. Compared with classic β-amino acid with
flexible backbone, our unnatural amino acid was constrained by
introduction of a double bond at the Cα. The introduction of
Map into the different positions in the EM-1 resulted in
analogues with varied in vitro and in vivo activities.
13 displayed the highest receptor functional potencies. As we
know, stimulation of the MOR by the agonist had a
downstream effect to alter the phosphorylation of EKR1/2. It
was reported that the phosphorylation of ERK1/2 might
contribute to the pain regulation.60 The tested analogues
revealed an increase in ERK1/2 phosphorylation by stimulating
MOR in 10 min in a concentration-dependent manner. Rapid
ERK1/2 phosphorylation was compared among all the tested
peptides. All tested analogues showed significant increase in
ERK1/2 phosphorylation at 1 pM, which was parallel to
inhibition of cAMP accumulation and binding affinity assays.
The cAMP accumulation and ERK1/2 phosphorylation results
suggested that the C-terminal group of EMs had a profound
influence on the activation state of MOR.
In our study, the binding affinity and in vitro bioassays
indicated that the incorporation of (Ph)Map at position 3 or at
positions 3 and 4 simultaneously was detrimental to structural
replacement. This is in agreement with previous studies in
which substitution of Phe3/Trp3 with corresponding β-amino
acid resulted in a great decrease of binding affinity.26,27
Surprisingly, the replacement taken at position 4 led to
analogue 4 with remarkable increase in binding affinity and
bioactivity, and this analogue also exhibited improvement in μ-
selectivity with respect to EM-1. Compared with the β-Phe4
replacement,26 (ph)Map4 primarily constrained the space
orientation of the C-terminal amide. The NH2 group was
regarded as an important pharmocophore for determining the
activity of EMs.53,54 Consistent with previous reports our data
further emphasized that the space orientation of C-terminal
amide also exerted great influence in the regulation of the
opioid pharmacological property. In other words, extending the
C-terminal backbone structure with appropriate structural
constraint would improve the bioactivity of EMs. Analogues
6−11, which contained fluorine, chlorine, methoxyl, or
piperonyl group at different positions on the aromatic ring of
Map4, exhibited decreased MOR affinity and selectivity
compared with analogue 4. A similar tendency was observed
when incorporation of a subunit with different lipophilicity or
electronic character at the third aromatic ring of opioid peptide
was shown to be detrimental to the activity of MOR.35,36,55,56
This data suggested that these kinds of modifications might
interfere with the interaction between the aromatic ring of
Map4 and the residues on the receptor. In addition, the binding
affinities of analogues 7−9 could be found in the order of 8 > 9
> 7, indicating that the meta-position modification of the
phenyl ring might be more favored. Four analogues with
heteroaromatic ring at position 4 (analogues 12−15) were also
synthesized and evaluated. It was in agreement with previous
study that analogues containing a naphthyl ring (14 and 15)
showed reduced activity.57−59 Interestingly, 12 and 13 were
found to be the most active and selective analogues with
incorporation of a furyl ring. These results confirmed that the
size and atomic composition of the third aromatic ring could be
a very important feature to determine the activity of EM-1.
Furthermore, our results also indicated that, compared with
phenyl ring, the furyl ring could reinforce the interaction
between the peptide and the receptor.
The antinociceptive activities of peptides were assessed by
the tail-flick test after icv injection. Most of the analogues
express approximate or even higher analgesic potency and
extension of acting time. That was probably related to their
high efficacy to the MOR. In addition, it was possible that the
enhanced stability in brain homogenate also contributed to the
increase of in vivo activities, since this factor might lead to a
longer duration of the peptide in the brain. Furthermore, it had
been reported that a variety of enzymes were involved in
peptide degradation. Carboxypeptidase Y and proteinase A
could reveal deamidase activity, hydrolyzing the endomorphins
into peptide acids, and then cleave off the C-terminal Phe.10
Insertion of Map into position 4 probably impeded these
proteases from recognizing the cleavage site.
Computational studies were performed to understand the
interaction characteristics of these analogues. The analysis
through the results of molecular docking and molecular
dynamics simulations of the ligand−receptor systems suggested
a good agreement between our study and the site-directed
mutagenesis data. We found that the interaction between the
C-terminal and the receptor could be essential for the binding
affinities of these analogues. For analogues 4 and 12, close
interactions with the receptor was observed and both of their
C-terminal residues formed hydrogen bonds with Thr218 and
Glu229. It was noteworthy that the incorporation of a furyl ring
into the C-terminal residue (analogue 12) further stabilized the
peptide conformation. The oxygen on the furyl ring could act as
a hydrogen acceptor and therefore form two hydrogen bonds
with His223 and Lys233, respectively. In a binding pose of
analogue 4, His223 was not involved in any hydrogen bond
with the analogue and Lys233 was used to contact with Trp3 as
well. These results indicated that introducing a pharmacophore
feature of a hydrogen bond acceptor into the C-terminal
residue might enhance the interaction between the peptide and
the receptor. For analogue 5, the C-terminal residue lost its
contact with the residues mentioned above on the receptor but
formed a hydrogen bond with Asp216. The aromatic
interaction between analogue 5 and the receptor was also
weak. As a result, this analogue did not have sufficient
interactions with the receptor. Taking the binding affinities into
consideration, we presumed that the binding pocket formed by
Thr218, His223, Glu229, and Lys233 might be responsible for
the binding of the C-terminal sequence of endomorphin
peptide. This polar moiety probably anchored the address
domain of the peptide at the right position inside the receptor
so that the peptide could have close interactions with the
receptor.
The functional activities of the analogues were determined by
measuring the inhibition of forskolin-stimulated cAMP
accumulation and phosphorylation of MAPK kinase in
HEK293 cells stably expressing the MOR. The [3H]cAMP
binding assay results proclaimed that most of the analogues
exhibited higher potencies than the parent. Analogues 12 and
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microplate counter. The scintillation cocktail was obtained from
Perkin-Elmer, Boston, MA.
CONCLUSIONS
■
In the present study, we developed a class of highly potent
MOR agonists by incorporating conformational constrained
unnatural β-amino acids (2-methylene-3-aminopropanoic acids
(Map)) into EM-1. From a combination of multiple in vitro
and in vivo assays, we found that Map would facilitate the
interaction between the analogues and the receptor when they
were placed in the C-terminal region of EM-1. Besides, the
bioactivity of these analogues varied when the side chain
element of Map changed. Analogues 12 (H-Tyr-Pro-Trp-(2-
Furyl)Map-NH2) and 13 (H-Tyr-Pro-Trp-(3-Furyl)Map-NH2)
behaved as the most active agonists. In binding affinity assays
they exhibited extraordinary affinities compared with the EM-1
and were even ∼431 times more potent in the inhibition of
forskolin-stimulated cAMP accumulation. These analogues also
displayed increased metabolic stability in mouse brain
homogenate. In vivo assays proved that these two analogues
showed enhanced antinociceptive activities. Overall, the present
study demonstrated that the modification used in this research
was successful for increasing the bioactivity of EM-1, and these
analogues might be of great importance in potential application
as drug candidates as analgesic for pain relief.
General Procedure for Synthesis of the Boc Protected
Unnatural Amino Acid Map (Boc-Map-OH). Boc-Map-OH was
derived from our group’s published product.30 HWE reagent (325.2
mg, 1.5 mmol) and catalyst (87.6 mg, 0.2 mmol, 20 mol %) were
dissolved in toluene (10 mL) at 0 °C. Then α-amidosulfones (1
mmol) were added followed by addition of an aqueous solution of
K2CO3 (1.5 M, 0.8 mL). After the reaction was finished, the
intermediate product was quickly isolated use column chromatography
(PE/EA = 1:1). Then it was dissolved in THF (6 mL) and added to a
precooled solution of MeONa (2.2 equiv) in MeOH (2 mL) slowly at
−15 °C. After the mixture was stirred for 30 min, paraformaldehyde (5
equiv) was added, and the mixture was stirred for another 8 h. The
reaction process was monitored by TLC. When the reaction was
completed, saturated aqueous NaCl was added and extraction was with
ethyl acetate. Drying was over Na2SO4. After evaporation of the
solvents, the residue was purified on a silica gel column (PE/EA =
7:1). Then the corresponding intermediate product was dissolved in
THF (5 mL) at 0 °C and added to an aqueous solution of LiOH (1M,
2.5 mL). The mixture was stirred at the room temperature for 12 h.
Upon completion, the mixture was extracted with DCM and dried
over Na2SO4. After concentration of the solvents, the residue was
purified on a silica gel column (PE/EA = 1:2) to give the
corresponding product.
Peptide Synthesis. Endomorphins and their analogues were
obtained by solution-phase methods with segment-coupling peptide
synthesis strategy. Synthesis of all the peptides that contain the Map
residue has been accomplished by performing an active ester reaction
with the EDC/HOBt coupling method. The first step in the synthesis
of all the analogues involved the preparation of the amide of the
unnatural amino acid Map, which was easily achieved by EDC/HOBt
coupling with ammonia. Then the N-terminal dipeptide (Boc-Tyr-Pro-
OH) and C-terminal dipeptide (Boc-Trp-Map-NH2, Boc-Map-Phe-
NH2, or Boc-Map-Map-NH2) were obtained by the same method.
After synthesis of fragments, the active ester method with EDC/HOBt
as the coupling agent was performed with N- and C-terminal
fragments coupling. All the intermediates were characterized by TLC,
1H NMR, and ESI-TOF MS. Deprotection of Boc was performed
EXPERIMENTAL SECTION
■
Materials and Methods. Mass spectra were measured with a
Maxis 4G ESI-TOF analyzer (Bruker, U.S.). Melting points were
determined on a micromelting point apparatus (AII-E, China). Optical
rotations were determined with AUTOPOL IV (Rudolph Research
Analytical). Purities were determined by ascending TLC performed on
precoated plates and by analytical RP-HPLC. The purity of the
analogues was confirmed by ascending TLC performed on precoated
plates (silica gel 60 F254, Yinlong, China) in the following solvent
systems (all v/v): (I) ethyl acetate−methanol−ammonia water
(30:10:1) and (II) acetone−glacial acetic acid−water (8:1:1). UV
light and I2 vapor were applied to visualize the TLC spots. Analytical
RP-HPLC was carried out with a Waters Delta 600 instrument
equipped with a Waters Deltapak C18 column (4.6 mm × 250 mm, 5
μm). Absorbance was monitored at λ = 220 nm. The solvents for
analytical RP-HPLC were as follows: A, 0.05% TFA in acetonitrile; B,
0.05% TFA in water. The column was eluted at a flow rate of 1 mL/
min with a linear gradient of A/B = 10:90 to A/B = 90:10 for 30 min
and a gradient of A/B = 90:10 to A/B = 10:90 for 5 min. The
retention time was reported as tR (min). The final purity of the
analogues was ≥95%. The detailed analytical properties of the
synthetic analogues are provided in Table S1 in the Supporting
Information.
Guinea pigs (300−350 g, National Institute of the Biological
Products, Gansu, People’s Republic of China) were used for guinea pig
ileum (GPI) assay. Male Kunming mice (30−35 g, Animal Center of
Medical College of Lanzhou University, Gansu, People’s Republic of
China) were used for mouse vas deferens (MVD) assay. Male
Kunming mice (18−22 g, Animal Center of Medical College of
Lanzhou University, Gansu, People’s Republic of China) were
employed for analgesia studies. They were housed in a temperature-
controlled environment (22 2 °C) under standard 12 h light/dark
conditions and received food and water ad libitum. All animals were
cared for, and experiments were carried out in accordance with the
principles and guidelines of the American Council on Animal Care. All
protocols were approved by the Ethics Committee of Lanzhou
Medical College.
using HCl/EA, and final products were purified by semipreparative
RP-HPLC.
Synthesis of Boc-Tyr-Pro-OH. A solution of Boc-Tyr-OH (10
mmol, 2.81 g), DCC (12 mmol, 2.472 g), and HOSu (12 mmol, 1.38
g) in distilled THF (50 mL) was stirred at 0 °C. H-Pro-OH (1.2
mmol, 1.38 g) was dissolved in saturated aqueous NaHCO3, adjusting
the pH to 9−10, and then the reaction mixture containing Boc-Tyr-
OSu and H-Pro-ONa was stirred at 0 °C for 30 min and at room
temperature overnight. When the reaction was completed, the
appropriate portion was extracted with THF. The extract was washed
successively with 5% citrate acid (10 mL), twice, and brine, dried over
Na2SO4, and evaporated at reduced pressure.The residue was purified
on a silica gel column (EA/MeOH = 10:1). Yield: 3.24 g (86%). Rf =
0.38 (PE/EA/HOAc = 10:20:1). [α]20 −18.3 (c 1.0, MeOH). Mp:
D
103−106 °C. ESI-TOF MS: m/z [M + H+] calcd, 379; found, 379.2.
General Procedure for the Synthesis of Boc-Trp-Map-NH2. A
solution of Boc-Trp-OH (10 mmol, 3.04 g), EDCI (16 mmol, 3.072
g), HOBt (14.5 mmol, 1.96 g), and DIEA (40 mmol, 6.50 mL) in
distilled DCM (50 mL) was stirred at 0 °C. Then H-Map-NH2 (1.2
mmol) dissolved in distilled DCM (10 mL) was added slowly into the
mixture, and the mixture was stirred at 0 °C for 30 min and at room
temperature overnight. When the reaction was completed, the
appropriate portion was extracted with DCM. The extract was washed
successively with 5% citrate acid (10 mL), saturated NaHCO3 (10
mL), twice, and brine, dried over Na2SO4, and evaporated at reduced
pressure. The residue was purified on a silica gel column (DCM/
MeOH = 15:1) with a yield of 75−90%.
[3H]DAMGO (50 Ci/mmol), [3H]DPDPE (43 Ci/mmol), and
[3H] cAMP (50 Ci/mmol) were purchased from Perkin-Elmer,
Boston, MA. Anti-phospho-ERK1/2 (Thr202/Tyr204) antibodies,
anti-ERK1/2 antibodies, and HRP-conjugated secondary antibody
were purchased from Cell Signaling Technology Inc. Protein kinase A,
forskolin, and IBMX were the products of Sigma-Aldrich (St. Louis,
MO, U.S.). The radioactivities were measured by a Perkin-Elmer 2460
General Procedure for the Synthesis of H-Tyr-Pro-Trp-Map-
NH2. A solution of N-terminal fragments Boc-Tyr-Pro-OH (1.0 mmol,
0.38 g), EDCI (1.6 mmol, 0.31 g), HOBt (1.45 mmol, 0.19 g), and
DIEA(4.0 mmol, 0.66 mL) in distilled DCM (10 mL) was stirred at 0
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°C. Deprotection of the Boc groups of C-terminal fragments (1.2
mmol) was performed with HCl/EA (1:4, v/v). Then the reaction
mixture containing Boc-Tyr-Pro-OBt and deprotected C-terminal
fragments H-Trp-Map-NH2 was stirred in distilled DCM at 0 °C for
30 min and at room temperature overnight. When the reaction was
completed, it was extracted with DCM. The extract was washed
successively with 5% citrate acid (10 mL), saturated NaHCO3 (10
mL), twice, and brine, dried over Na2SO4, and evaporated at reduced
pressure. The crude protected tetrapeptides residue was purified on a
silica gel column (EA/MeOH = 15:1) with a yield of 60−80%.
The Boc-group deprotection was performed by treatment with
HCl/EA (1:4, v/v) at room temperature for 2 h. After extraction with
DCM/MeOH = 10:1, the solvent was evaporated at reduced pressure.
The residue was purified by semipreparative RP-HPLC and
characterized by TLC, TOF-MS, [α]D20, mp, and RP-HPLC, which
revealed that all the peptides were the desired peptides with 95−99%
purity. Data are reported in Table S1 in the Supporting Information.
Establishment of HEK293 Cells Stably Expressing μ- or δ-
Opioid Receptor. HEK293 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
calf serum. The eukaryotic vector, pcDNA3.1-FLAG-μ-OR or
pcDNA3.1-Myc-δ-OR, was introduced into HEK293 cells by Lip-
ofectmine2000 according to the manufacturer’s instruction. The day
after transfection, G418 (800 μg/mL) was added to the medium for 2
weeks. Then the antibiotic-resistant clones derived from a single cell
were selected and further characterized by RT-PCR and Western
blotting to ensure the expression of human MOR or DOR. The
cellular function of MOR or DOR was confirmed by the μ-opioid
receptor antagonist naloxone and δ-opioid receptor antagonist
naltrexone. Briefly, HEK293-μ-OR or HEK293-δ-OR cells were
preincubated with or without 1 μM naloxone or 1 μM naltrexone in
DMEM medium for 30 min. Then the cells were stimulated using 1
μM EM-1 or 1 μM DPDPE and 10 μM forskolin to measure the
cAMP accumulation level.
min at 37 °C. An amount of 100 μL of serum-free medium containing
various concentrations of the appropriate ligand (5 × 10−11 M to 5 ×
10−5 M) using 50 μM forskolin was added to the cells, and the samples
were incubated at 37 °C for 30 min. They were removed from the
medium. Then 0.2 N HCl was added to the cells at room temperature
for 30 min. The clarified lysates were neutralized with 10 N NaOH.
Lysates were transferred to microcentrifuge tubes and micro-
centrifuged 2 min at top speed at room temperature. Then 50 μL of
neutralized lysates, 100 μL of PKA (60 μg/mL), and 50 μL of
[3H]cAMP were mixed briefly and incubated for ≥2 h at 4 °C. The
chilled charcoal suspension was added to adsorb free cAMP from
solutions. An amount of 200 μL of the supernatant was transferred to a
scintillation vial. Scintillation fluid was added, and radioactivity was
quantified in a scintillation counter (liquid scintillation counter,
PerkinElmer). Analysis of the data was performed using the GraphPad
Prism software (version 5.0, San Diego, CA).
Detection of MAPK Phosphorylation. ERK phosphorylation
was measured by immunoblotting as described.68−70 HEK293 cells
stably expressing the FLAG-tagged μ-opioid receptor were seeded in
12-well plates. At 16 h before the addition of the ligands, the culture
medium was removed and replaced by fresh serum-free medium. For
rapid ERK1/2 phosphorylation assay, the cells were treated with EM-
1, 4, 12, and 13 at different concentrations and incubated at 37 °C for
10 min. Cell monolayers were rinsed with ice-cold PBS, and whole
lysates were prepared by the addition of RIPA lysis buffer containing
10 μM PMSF and phosphatase inhibitor cochtails (P5726, Sigma-
Aldrich) for 5 min. Soluble proteins were separated by centrifugation
at 15 000 rpm for 10 min. Protein concentration was determined by
using BCA protein assay kit (Pierce, Thermo Scientific, U.S.). A total
amount of 20 μg of protein from each sample was prepared for 10%
SDS−polyacrylamide gel electrophoresis and electroblotted onto
polyvinylidene difluoride membranes. Membranes were probed with
primary antibody against phosphor-ERK1/2 or ERK1/2 (1:1000
dilution in blocking solution, Cell Signaling Technology Inc.).
Immunoreactive proteins were visualized using a horseradish
peroxidase sensitive ECL chemiluminescent Western blotting kit
(Pierce, Thermo Scientific, U.S.).
In Vitro Assays on Isolate Tissue Preparation. In vitro opioid
activities of peptides were tested in the guinea pig ileum (GPI) and
mouse vas deferens (MVD) bioassays as reported elsewhere. For the
GPI assay, the myenteric plexus longitudinal muscle was obtained from
guinea pig (300−350 g, National Institute of the Biological Products,
Gansu, People’s Republic of China) as described by Rang.31 For the
MVD assay, the vas deferens of male Kunming strain mice (30−35 g,
Animal Center of Medical College of Lanzhou University, Gansu,
People’s Republic of China) was prepared as described by Hughes et
al.32 The GPI tissue and MVD tissues were mounted in a 10 mL bath
containing aerated 95% O2, 5% CO2 with Krebs−Henseleit solution at
37 and 36 °C, respectively. Both tissues were used for field stimulation
with bipolar rectangular pulses of supramaximal voltage. Dose−
response curves were constructed, and IC50 values (concentration
causing a 50% decrease in electrically induced twitches) were
calculated graphically. Moreover, in both assays, three to four washings
were done at intervals of 15 min between each dose. The values were
the arithmetic mean of 10−15 measurements. In order to measure
whether δ-opioid-receptor-mediated antagonism occurred in the
MVD, naltrindole, a selective δ-receptor antagonist, was added to
the tissue preparation after 5 min of incubation, the test analogue was
added at the IC50 dose value, and the percentage recovery (reversal
rate) of electrically evoked contraction was then calculated.35
Antinociception Test. Antinociceptive responses were deter-
mined using the warm water tail-flick test. For the analgesia studies,
male Kunming mice weighing 18−22 g were employed. They were
obtained from the Animal Center of Medical College of Lanzhou
University. Various doses of drugs were injected icv according to the
procedure adopted by Haley and McCormick, and the warm water tail-
flick responses were measured at different times after the injection.
Nociception was evoked by immersing the mouse tail in hot water (50
0.2 °C) and measuring the latency to withdrawal. Before treatment,
each mouse was tested, the latency to tail-flick [control latency (CL)]
Cell Culture. HEK293 cells stably expressing either the FLAG-
tagged μ-opioid receptor or the Myc-tagged δ-opioid receptor were
grown in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal calf serum, 100 units/mL penicillin, and 0.1
mg/mL streptomycin at 37 °C in a humidified atmosphere containing
5% CO2 as described.61
Radioligand Binding Assay. In the experiments designed to
define peptide specificity for μ- and δ-opioid receptors, whole cells
expressing either MOR or DOR ((2.5−3.5) × 106 cells/tube) were
incubated with 1.7 nM [3H]DAMGO or 1.0 nM [3H]DPDPE and
10−10−10−4 M unlabeled ligands for each experiment. Nonspecific
binding was measured in the presence of 10 μM naloxone or 10 μM
naltrexone. The reaction was performed at 25 °C for 60 min in freshly
prepared binding buffer (25 mM Hepes, 5 mM MgCl2, 1 mM CaCl2,
2.5 mM ethylenediaminetetraacetic acid [EDTA], and 0.4% bovine
serum albumin [BSA], pH 7.4).62,63 The reaction was stopped by rapid
vacuum filtration through GF/C filters (Whatman, Maidstone, U.K.)
using a cell harvester. The filters were washed twice with 6 mL of ice-
cold buffer and then dried for 1 h at 80 °C. The radioactivity was
measured by liquid scintillation counting (liquid scintillation counter,
PerkinElmer). The affinity constants (Ki) were calculated according to
Cheng and Prusoff with GraphPad Prism 5.0 software (GraphPad
μ
Software Inc., San Diego, CA). The dissociation constant (Kd = 0.6
δ
nM, Kd = 2.8 nM) and the number of binding sites (Bmax) were
calculated by Scatchard analysis using at least seven concentrations of
[3H]DAMGO or [3H]DPDPE in the range 0.085−8.5 or 0.10−5.05
nM. Nonspecific binding was assessed in the presence of 10 μM
naloxone and 10 μM naltrexone.
Measurements of cAMP Accumulation. Measurements of
adenylyl cyclase activity were performed as described in the
literature.64−67 Briefly, the day before the assay, HEK293 cells stably
expressing the μ-opioid receptor were seeded at 80% confluency in 24-
well microtiter plates. Before the start of the assay, the culture medium
was aspirated and each well was washed twice with serum-free medium
warmed to 37 °C. Then 400 μL of prewarmed serum-free medium
containing 1 mM IBMX was added to each well and incubated for 10
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was recorded, and selection was the one at approximately 3−5 s. The
latency to tail-flick was defined as the test latency (TL). The
corresponding cutoff time TL was set at 10 s, and 0.9% saline was used
as control. The antinociceptive response was expressed as percentage
of maximal possible effect (% MPE), calculated by the following
equation: % MPE = 100 × (TL − CL)/(10 − CL). The ED50 values
and their 95% confidence limits were determined by using the graded
dose−response procedure.
2). The average structure of the last 1 ns trajectory was considered as
the typical structure of the MD simulations.
The molecular dockings were performed with AUTODOCK4.78
This program suite uses an automated docking approach that allows
ligand flexibility, and it employs the Lamarckian genetic algorithm to
treat the ligand−receptor interaction. The initial conformations of the
three analogues were built based on the NMR structure of
endomorphins that we solved previously,79 and the amide group of
Tyr1 was protonated. Partial charges, necessary for the docking
protocol, were assigned according to the Gasteiger−Marsili scheme.
The grid maps representing the opioid receptors in the docking
process were calculated with AutoGrid. The docking process was
performed in two steps.80,81 First, a box of 42 Å × 42 Å × 42 Å,
centered on one of the oxygen atoms of the Asp147, was used with a
grid resolution of 5.5 Å. The initial position of the ligand was random.
The population size was 100. The maximum number of generations
was 27 000, and the maximum number of energy evaluations was 2
500 000. The lowest docking energy conformations or the lowest
docking energy conformations included in the largest cluster were
considered to be the most stable orientations. In the second step, a box
of 40 Å × 30 Å × 50 Å centered on the best conformations obtained in
the first step was used with a resolution of 0.3 Å. The number of
energy evaluations was changed to 25 000 000, and the population size
was raised to 500. The resulting orientations were scored based on the
docking and binding energies and on the distance of Asp147 to the
protonated nitrogen of the ligand. The docked energies were
calculated using a modified scoring function.82 Experimental studies
suggested that the protonated nitrogen moiety interacts with the
carboxyl group of Asp147 to form a putative salt bridge and this
residue mutated to Ala/Asn or Glu, leading to diminished binding
affinities. This residue was believed to be the primary binding site.45 In
the next stage of the study, MD simulations of the all the docking
models were performed using the GROMACS program to get more
reasonable ligand−receptor binding modes, where the flexibility of the
receptor was considered. Each ligand−receptor complex was subjected
to a 5 ns MD simulation in the previously described membrane bilayer.
The topologies of the ligands were generated by PRODRG.83 The
time evolutions of the rmsd of Cα and total energy were analyzed to
evaluate the stability of each system. As shown in Supporting
Information Figures S1 and S2, all systems became stable after about 3
ns of MD simulations. Figures were drawn by the means of PyMOL.84
Metabolic Stability. Mouse brain homogenate was used for
enzymatic degradation studies. The 15% mouse brain homogenate was
prepared as described previously.71 Protein content of the suspension
was confirmed by BCA potein assay kit (Thermo, Rockford, IL, U.S.).
A final protein concentration of 2.1 mg/mL in 50 mM Tris buffer, pH
7.4, was used for all incubations. RP-HPLC analysis determined the
stability of peptides. Approximately 10 μL of peptide stock solution
was digested with 190 μL of rat brain homogenate at 37 °C in a final
volume of 200 μL for incubation. An amount of 20 μL of the aliquots
was withdrawn from the mixture at 0, 5, 10, 15, 30, 60, 120, 240 min,
and 90 μL of acetonitrile was added immediately for precipitated
proteins, placing the tube on ice for 5 min and addeding 90 μL of 0.5%
acetic acid at the required time to prevent further enzymatic
breakdown. The aliquots were centrifuged at 13000g for 15 min at 4
°C. The obtained supernatants were filtered with filters of 0.22 μm,
and 50 μL of the filtrate was analyzed by RP-HPLC on a Waters Delta
Pak C18 column (4.6 mm × 250 mm, Milford, MA), using the solvent
system of 0.05% TFA in acetonitrile (A) and 0.05% TFA in water (B)
with a linear gradient of A/B = 10:90 to A/B = 90:10 for 30 min and
A/B = 90:10 to A/B = 10:90 for 5 min. The column was eluted at a
flow rate of 0.8 mL/min. The degradation rate constants (k) were
determined by least-squares linear regression analysis of logarithmic
tetrapeptide peak area [(ln(At/A0)] vs time courses, with at least five
time points. The rate constants obtained were used to establish the
degradation half-lives (t1/2) as ln 2/k.
Molecular Modeling. Using the homology modeling method,
Mosberg had reported a kind of MOR model based on the bovine
rhodopsin structure,49 and we also constructed a homology MOR
using the same template.72 By comparison of the two models, it was
found that the model built by Mosberg et al. was more suitable for
docking peptide agonists because structural alterations were
incorporated into this model to obtain an active state receptor
structure. Recently, the crystal structure of human MOR was resolved
with antagonist β-FNA.52 The Mosberg model showed a good
agreement with the crystal structure. Both structures represented
exposed binding pockets that would facilitate the ligand binding
process. Moreover, the overall Cα root-mean-square deviation (rmsd)
between the two structures was 3.64 Å, indicating that the
computational model preserved the main structural architecture of
MOR. Considering that the crystal structure probably represented an
“inactive state” of MOR, the Mosberg “active” MOR model was
chosen as the working receptor model.49 The model was then refined
using 15 ns MD simulations in the phospholipid bilayer. The receptor
was inserted into a pre-equilibrated 70 Å × 70 Å DPPC bilayer slab,
and the protein membrane system was embedded in a SPC water box.
Counterions were added to the system in order to produce a neutral
charge on it. MD simulations were performed with the GROMACS
Data Analysis. The data are expressed as the mean
SEM.
Responses were analyzed with a one-way ANOVA followed by
Dunnett’s test for comparison of multiple groups with one saline
control group and by Student’s t test for comparisons between two
groups. p < 0.05 was used as the statistical significance level.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and characterization data for new
analogues. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-9318912567. E-mail: wangrui@lzu.edu.cn.
■
3.3.3 package employing NPT and periodic boundary conditions.73
A
modification of GROMOS87 force field was applied for protein and
the lipid.74 A twin cutoff of 9 Å was used for the short-range
interaction, and a cutoff of 12 Å was used for the Lennard-Jones
interaction. Particle Mesh Ewald algorithm was used for the calculation
of electrostatic contributions to energies and forces.75 Bond length was
constrained using the LINCS algorithm.76 The systems were coupled
to a temperature bath at 300 K, with a coupling constant of 0.1 ps.
Semiisotropic coupling with a time constant of 1 ps was applied to
keep the pressure at 1.0 bar.77 The system was first energy-minimized
using the steepest descent integrator for 5000 steps. Then a
progression of position restraint was performed for 300 ps. Finally, a
15 ns simulation was performed with a time step of 2 fs. The root-
mean-square deviation was calculated for the backbone atoms of
MOR. The receptor became stable after approximately 7.5 ns (Figure
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
The manuscript was written with contributions from all
authors. All authors have given approval to the final version
of the manuscript.
ACKNOWLEDGMENTS
We are grateful for the grants from the National Natural
Science Foundation of China (Grants 90813012 and
■
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20932003), the Key National S&T Program “Major New Drug
Development” of the Ministry of Science and Technology
(Grant 2012ZX09504001-003), and Fundamental Research
Funds for the Central Universities (Grant lzujbky-2011-86).
We acknowledge computing resources and time on the Gansu
Supercomputing Center, Cold and Arid Environment and
Engineering Research Institute of Chinese Academy of
Sciences.
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ABBREVIATIONS USED
■
Boc, tert-butyloxycarbonyl; DAMGO, H-Tyr-D-Ala-Gly-NMe-
Phe-Gly-ol; DCM, dichloromethane; DIEA, N,N′-diisopropyle-
thylamine; DMF, dimethylformamide; DMSO, dimethyl
sulfoxide; DOR, δ opioid receptor; DPDPE, Tyr-c(D-Pen-Gly-
Phe-D-Pen); ED50, median effective dose; EDC, N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide; EM-1, endomorphin-1;
EM-2, endomorphin-2; ESI-TOF MS, electrospray ionization
time-of-flight mass spectrometry; GPI, guinea pig ileum; HOBt,
1-hydroxybenzotriazole; IC50, 50% inhibitory concentration;
icv, intracerebroventricular (dosing); ip, intraperitoneally;
KOR, κ opioid receptor; MD molecular dynamics; MOR, μ
opioid receptor; MVD, mouse vas deferens; RP HPLC,
reversed-phase high performance liquid chromatography;
THF, tetrahydrofuran; TLC, thin-layer chromatography
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