Endomorphin-1 analogs containing α-methyl-β-Amino acids exhibit potent analgesic activity after peripheral administration

Article abstract of DOI:10.1039/c7ob01115f

This study describes the design and synthesis of endomorphin-1 analogs containing C-Terminal aromatic α-methyl-β-Amino acids and an N-Terminal native tyrosine or 2,6-dimethyl-Tyrosine. We show that, in comparison with the parent peptide, these analogs exhibit improved bioactivity and blood-brain barrier penetration after intravenous administration, and have a lower tendency to induce constipation and sedation than morphine.

Full text of DOI:10.1039/c7ob01115f

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Endomorphin-1 analogs containing α-methyl-β-amino acids
exhibit potent analgesic activity after peripheral administration
Received 00th January 20xx,
Accepted 00th January 20xx
Yuan Wang, Junxian Yang, Xin Liu, Long Zhao, Dongxu Yang, Jingjing Zhou, Dan Wang, Lingyun
Mou, Rui Wang*
DOI: 10.1039/x0xx00000x
www.rsc.org/
13-18
This study describes the design and synthesis of endomorphin-1 pharmacological profiles,
with β-amino acid substituted
analogs containing a C-terminal aromatic α-methyl-β-amino acids opioid peptides attracting increased attention due to their
and an N-terminal native tyrosine or 2,6-dimethyl-tyrosine. We improved metabolic stability, remarkable physicochemical
19-24
show that, in comparison with the parent peptide, these analogs properties and bioactivities.
As a subclass of β-amino acids,
-amino acids are important building blocks of natural
after intravenous administration, and have a lower tendency to products, which makes their incorporation into opioid peptides
2, 3
exhibit improved bioactivity and blood−brain barrier penetration
β
induce constipation and sedation than morphine.
a
practical strategy for bioactivity and bioavailability
25-28
improvement.
Previously, we have developed a series of
2
,3
Pain significantly affects life quality, requiring the development
of safe and efficient pain killers. Despite the ongoing search for
new treatments, opioids are still the golden standard for
alleviating moderate to severe pain. However, the chronic use
of opioids causes undesired side effects, including respiratory
depression, constipation, tolerance development, and
novel β -amino acids (2-methylene-3-aminopropanoic acids)
containing a methylene group at the C position, with SAR
studies demonstrating that their incorporation at the C-
terminus of endomorphin-1 (EM-1) increases the biological
activity of the corresponding peptides.
α
29
1
-3
addiction. Opioid drugs primarily exert their analgesic effect
by binding to μ- (MOP), δ- (DOP) and κ-opioid receptors (KOP).
Among them, MOP is the main pharmacological target for
alleviating pain, with a large number of available opioid
4,5
analgesics binding to it.
As alternative pain killers,
endogenous opioid peptides exhibit better side-effect profiles,
6
-
opening up new possibilities for analgesic drug development.
9
Endomorphins (EMs) are opioid peptides isolated from
bovine brain and human frontal cortex, which have attracted
10,11
the attention of peptide chemists/pharmacologists,
due to
their high MOP affinities and specificities as compared with
those for DOP and KOP. Furthermore, EMs are believed not to
exhibit some of the undesirable side effects of morphine.
However, similar to other peptides, EMs have poor metabolic
stability and are unable to cross the blood−brain barrier (BBB),
with peripheral EMs administration thus producing almost no
1
2,13
detectable analgesic effects.
Thousands of EM analogs
Figure 1. Unnatural amino acids and structure of EM-1 analogs.
have been synthesized to understand the corresponding
structure-activity relationships (SARs) and thus improve their
Herein, we describe the synthesis and pharmacological
evaluation of novel EM-1 analogs containing α-methyl-β-
phenylalanine (AMBP) or α-methyl-β-furylalanine (AMBF) by
investigating the influence of introducing these residues at C-
terminus and determining the binding affinity and functional
activity of the synthesized analogs. In vivo activities after
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DOI: 10.1039/C7OB01115F
Table 1. Analytical Data for EM-1 Analogs.
+
a
TOF MS [M+H]
calcd
retention time
o
b
no.
sequence
H-Tyr-Pro-Trp-(R,R)-AMBP-NH
mp ( C)
purity (%)
found
(min)
analog 1
analog 2
analog 3
analog 4
analog 5
analog 6
analog 7
analog 8
2
625.3094
625.3094
615.2886
615.2886
653.3407
653.3407
643.3199
643.3199
625.3192
17.1
133-135
129-133
138-140
131-133
149-152
152-155
143-147
151-154
98
97
96
99
99
99
98
98
H-Tyr-Pro-Trp-(S,S)-AMBP-NH
2
625.3045
615.2964
615.2834
653.3489
653.3501
643.3255
643.3284
17.0
17.9
18.0
17.5
17.5
17.4
17.3
H-Tyr-Pro-Trp-(R,R)-AMBF-NH
2
H-Tyr-Pro-Trp-(S,S)-AMBF-NH
2
H-Dmt-Pro-Trp-(R,R)-AMBP-NH
2
2
H-Dmt-Pro-Trp-(S,S)-AMBP-NH
2
H-Dmt-Pro-Trp-(R,R)-AMBF-NH
H-Dmt-Pro-Trp-(S,S)-AMBF-NH
2
a
b
t
R
with Delta-Park C18 column (4.6 mm × 250 mm, 5 μm), A:B = 10:90 to A:B = 90:10 for 30 min, A:B = 90:10 to A:B = 10:90 for 5 min. Purity determination based
on analytical RP-HPLC.
intracerebroventricular (i.c.v.) and intravenous (i.v.)
administration were evaluated by tail-flick and formalin test,
and BBB permeability was determined using a peripherally
restricted opioid antagonist. Furthermore, the effects of the
above analogs on locomotor and gastrointestinal functions
were evaluated (experimental details are provided in the
Supplementary Information).
DOP affinities, their MOP-over-DOP selectivity was still
acceptable. Interestingly, Dmt substitution was reported to
1
enhance affinity to all three opioid receptor subtypes,
14,33
resulting in decreased selectivity.
All synthesized analogs
exhibited very low KOP binding affinities.
The functional activity profiles of EM-1 analogs were
assessed by an accumulation of cyclic adenosine
monophosphate (cAMP) assay using MOP-expressing HEK293
cells. The tested analogs inhibited forskolin-stimulated cAMP
in a dose-dependent manner, as shown in Table 2, with cAMP
accumulation assay results being in good agreement with
those of the radioligand binding assay. Analogs 1−4 exhibited
potencies between 10.5 and 22.9 nM, whereas combination
The (S,S)/(R,R)-AMBP and (S,S)/(R,R)-AMBF (Fig. 1) were
obtained by addition of titanium enolates to tert-butane-
30,31
sulfinyl imine,
and subsequently incorporated into EM-1
to afford analogs 1-8. Other non-natural amino acids were
sourced from commercially suppliers. All compounds were
prepared by solution-phase synthesis, with 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide
hydroxybenzotriazole (HOBt) used for peptide coupling
experimental details are provided in the Supplementary
(EDC)
and
1
4
4
of Dmt and AMBP /AMBF substitution produced analogs
−8 with improved subnanomolar-range potencies. The
(
5
3
2
Information). Crude peptides were purified by semi-
preparative reversed-phase high-performance liquid
cAMP accumulation assay indicated that all investigated
species behaved as MOP agonists, with the highest potency
observed for analogs 7 and 8.
chromatography (RP-HPLC) and identified by electrospray
ionization time-of-flight mass spectrometry (ESI-TOF MS),
with their purities determined as >95%. Detailed properties
of the above analogs are provided in Table 1.
The metabolic stability of EM-1 and its analogs was
assessed in mouse brain homogenate, with the
corresponding half-lives listed in Table 2. While EM-1 showed
A radioligand binding assay was employed to examine the
binding affinities and receptor selectivities of the synthesized
peptides in whole-cell preparations from HEK293 cells, which
4
a short half-life of 16.9 min, substitution of Phe with
4
4
AMBP /AMBF resulted in improved stability, with analogs
−4 exhibiting half-lives ranging from 112 to 142 min.
3
3
1
stably expressed MOP, DOP or KOP. [ H]DAMGO, [ H]DPDPE
1
3
Moreover, subsequent Dmt substitution increased the
metabolic stability even further (analog 5-8), e.g. analog 5
exhibited a ~16-fold enhanced metabolic stability compared
with that of EM-1. Thus, insertion of unnatural amino acids
into EM-1 probably impedes protease recognition of peptide
and [ H]U69,593 were used as the radioligand for MOP, DOP
and KOP, respectively. The results are summarized in Table 2.
Analogs 1−4 exhibited significant MOP affinities, with the
corresponding K values ranging from 6.3 to 21.2 nM. Among
i
these four species, analogs 2 and 4 containing (S,S)-
3
4
4
4
cleavage sites.
AMBP /AMBF exhibited higher MOP binding affinities than
4
In vivo antinociceptive effects of analog 7 and 8 were
further evaluated in the mouse tail-flick test after i.c.v.
administration and expressed as a percentage of maximum
possible effect (%MPE). As shown in Figure 2a−2c, all analogs
exhibited dose- and time-dependent inhibition of heat-
induced analgesia and exhibited better antinociceptive
their (R,R)-stereoisomers did. Replacement of Phe with
4
4
AMBP /AMBF resulted in decreased DOP binding affinity and
thus greatly increased MOP selectivity. Subsequent
1
1
1
replacement of Tyr with 2, 6-dimethyl-Tyr (Dmt ) afforded
analogs 5−8 with subnanomolar MOP affinities. Specifically,
analog 8 exhibited the highest MOP affinity among all
activities than the parent peptide did. The ED value of EM-1
synthesized compounds, corresponding to a K value of 0.12
50
i
2
9
1
equalled 0.304 nmol, whereas analog 7 and 8 were
nM. Although analogs containing Dmt exhibited increased
2
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DOI: 10.1039/C7OB01115F
Table 2. Opioid Receptor Binding Affinities, Functional Activity and Half-Lives of EM-1 and its Analogs.
a
e
Binding Affinities
Functional Activity
Half-lives
(min)
No.
μ
b
δ
c
κ
d
K
i
(nM)
K
i
(nM)
K
i
(nM)
K
i
ratio (μ/δ)
EC50(nM)
E
max (%)
EM-1
2.60 ± 0.21
21.2 ± 3.71
7.7 ± 1.94
6080 ± 640
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
1/2338
14.4 ± 0.6
22.9 ± 1.14
18.8 ± 2.01
13.7 ± 1.42
10.5 ± 0.67
0.28 ± 0.039
0.23 ± 0.026
0.16 ± 0.021
0.12 ± 0.011
83.1 ± 4.1
68.9 ± 3.3
70.1 ± 5.4
72.7 ± 6.2
79.2 ± 5.2
88.2 ± 7.3
90.9 ± 4.2
90.6 ± 3.9
93.1 ± 2.7
16.9 ± 2
112 ± 9
142 ± 11
133 ± 12
127 ± 8
276 ± 21
254 ± 15
163 ± 7
202 ± 6
analog 1
analog 2
analog 3
analog 4
analog 5
analog 6
analog 7
analog 8
-
>10000
-
9.3 ± 2.58
>10000
-
6.3 ± 0.31
>10000
-
0.75 ± 0.03
0.31 ± 0.02
0.47 ± 0.02
0.12 ± 0.008
551 ± 38
273 ± 12
297 ± 14
165 ± 3.4
1/735
1/881
1/632
1/1375
a
b
Displacement was done using whole cell preparations from transfected HEK293 cells expressing μ-opioid receptor, δ-opioid receptor or κ-opioid receptor, respectively.
3
c
3
d
3
Displacement of [ H]DAMGO (K
d
= 0.6 nM, μ-selective). Displacement [ H]DPDPE (K
d
= 2.8 nM, δ-selective). Displacement [ H]U69,593 (K
d
= 2.9 nM, κ-selective). K
i
values were calculated according to the Cheng−Prusoff equation: K
i
= EC50/(1 + [ligand]/K
d
), where the shown K
d
values were taken from isotope saturation
e
experiments. Data are expressed as the mean ± SEM, each performed in triplicate. 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. EC50 and Emax values were calculated by using the
GraphPad Prism software. Data are expressed as the mean ± SEM, each performed in triplicate.
significantly more potent, showing ED50 values of 0.019
0.006−0.036) and 0.016 (0.005−0.032) nmol, respectively.
(i.c.v.) (D). Each value represents the mean ± SEM for 8−12 mice. The asterisk
indicates that the response is significantly different from control (p < 0.05).
(
Pretreatment of opioid antagonist naloxone (Nlx, 0.2 nmol,
i.p.) significantly reduced the activities of tested compounds,
confirming the central role of the opioid system in
determining the effect. In addition, pretreatment with a MOP
antagonist β-funaltrexamine (β-FNA, 0.2 nmol, i.c.v.)
significantly reduced the analgesic activity of EM-1 analogs,
indicating that they mainly targeted the MOP (Figure 2D).
Thus, these results were in good agreement with binding
affinity and functional activity assays.
The antinociceptive effect of EM-1 analogs was tested
upon peripheral administration by i.v. injection and was re-
examined using the mouse tail-flick test. As shown in Figure
3a−3c, the tested analogs exhibited potent antinociceptive
activities, with ED50 values for analogs 7 and 8 equalling 0.089
(0.048−0.13) and 0.016 (0.009−0.024) mg, respectively.
Conversely, EM-1 displayed no obvious analgesic effect under
the same conditions. Naloxone methiodide (Nlx-M), a
peripherally restricted opioid antagonist, was used to
determine the action site of the tested analogs. Pretreatment
with Nlx-M (0.2 nmol, i.c.v.) significantly reduced the
antinociceptive effect of the above two compounds, whereas
the i.v. injection (0.2 mg) of this antagonist did not attenuate
their activity (Figure 3d). These results indicate that analogs 7
and 8 can penetrate the highly selective BBB and elicit their
activity in the central nervous system (CNS). The promising
pharmacological properties of EMs are offset by their lack of
activity upon peripheral administration and short action
duration, which limits their potential clinical usage. This work,
relying on the use of AMBP/AMBF as a building block,
2,3
indicated that incorporation of β -amino acids may improve
the BBB penetration ability of peptides, in agreement with
35
our previous reports.
In addition, the multiple site
modification method used in this study may serve as a useful
2
,3
strategy for future EM optimization: the aromatic β -amino
acids substituted at the C-terminus afford the peptide with
1
BBB permeability, and the Dmt replacement at the N-
terminus provids increased binding affinity and functional
activity.
Figure 2. Tail-flick test versus time curves of the antinociceptive effect of analog 7 (A)
and analog 8 (B) after i.c.v. administration. Dose-response curves for the analgesic
effects were presented as AUC data over the period 0 to 30 min (C). The
antinociceptive effect induced by analogs after injection of naloxone (i.p.) and β-FNA
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Figure 4. Dose-related analgesic effects of i.v. administration of VaienwaloAgrti7cle(AO)nalinnde
analog 8 (B) in the formalin test. Dose-response curves for the analgesic effects
DOI: 10.1039/C7OB01115F
induced by i.v. administration the tested analogs at different doses in the
phases I (C) and phases II (D). Each value represents the mean ± SEM for 8-12
mice. The asterisk indicates that the response is significantly different from the
saline-treated group (p < 0.05).
Although previous reports showed that the introduction
of β-Phe/β-homo-Phe into EM-1 results in low MOP
20,21
affinity,
the current work indicates that additional methyl
group modification at the α-carbon of the β-amino acid
imparts nanomolar MOP activity and improved bioavailability
after peripheral administration. Besides, it is reported that
incorporation of α-hydroxy-β-phenylalanine into EMs
afforded peptides with prolonged stability and varied activity
27
toward opioid receptors. Thus, these findings suggest that
tuning the C site structure of β-amino acids may afford EM
α
analogs with improved pharmacological profiles.
Figure 3. Tail-flick test versus time curves of the antinociceptive effect of analog 7 (A)
and analog 8 (B) after i.v. administration. Dose-response curves for the analgesic
effects were presented as AUC data over the period 0 to 30 min (C). The
antinociceptive effect induced by analogs after injection of naloxone (i.p.) and
naloxone methiodide (i.p. and i.c.v.). Each value represents the mean ± SEM for 8−12
mice. The asterisk indicates that the response is significantly different from control (p
<
0.05).
The warm water tail flick assay was used to test the acute
pain-killing activity, with analog 7 and 8 subsequently
evaluated in the formalin test (persistent pain model). As
shown in Figure 4, analog 8 exhibited higher analgesic activity
upon intravenous administration than analog 7 did in both
first (0-5 min) and second phase (15-30 min) of formalin test,
with the respective ED50 values in phase I equalling 0.021
(0.004−0.042) and 0.11 (0.07−0.151) mg, and ED50 values in
phase II equalling 0.013 (0.002−0.029) 0.061 and (0.041−0.08)
mg, respectively.
Figure 5. The effect of i.v. administrations of analog 7, analog 8 and morphine on the
locomotor activity in the rotarod test, and are expressed as the endurance time (A
and B). The effect of i.v. administrations of analog 7, analog 8 and morphine on the
fecal number (C) and fecal dry weight (D). Data are the mean ± SEM of 8–12 mice.
The asterisk indicates that the response is significantly different from control group (p
<
0.05).
Short-term side effects of analogs 7 and 8 were examined
and compared with those of morphine, using rotarod test to
determine the locomotor behaviour of mice after i.v.
administration of these compounds. Equal antinociceptive
doses affording MPE >95% were used for morphine (0.2 mg)
and EM-1 analogs (0.6 mg). As shown in Figure 5, morphine
administration significantly impaired the motor function
throughout the course of the examination, in contrast,
analogs 7 and 8 only interfered with motor function up to 20
min and 10 min after administration, respectively. These
results indicated that the analogs exhibited a lower tendency
to induce sedation effect. Since opioid-induced constipation
(OIC) is a common side effect of classical opioids, the
4
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propensity of the synthesized analogs to induce OIC was
determined by assessing the fecal pellet output of mice,
showing that their i.v. administration reduced the pellet
number and dry weight in a dose-dependent manner.
Compared with morphine (0.2 mg), analogs 7 and 8 (0.6 mg)
exhibited a significantly lower influence on gastrointestinal
mobility.
(13)
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A. Janecka, L. Gentilucci, Future Med. Chem., 2014, 6,
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In summary, a number of EM-1 analogs containing
(
2,3
aromatic β -amino acids were synthesized and characterized
by several in vitro and in vivo assays, which showed that such
modification can improve the bioactivity and bioavailability of
these peptides. The prepared EM-1 analogs were effective in
alleviating pain after peripheral administration, with in vivo
assays employing naloxone methiodide showing that they
may elicit their antinociceptive effect in the CNS. Additionally,
the above analogs displayed a low propensity to induce
locomotor impairment and constipation. Thus, this study
demonstrates the potential application of aromatic β -
amino acids in EMs optimization, with their further
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Acknowledgments
We are grateful for the grants from the National Natural
Science Foundation of China (Nos. 21432003, 21402076,
7
(
Debevec, L. E. Maida, P. Geer, M. Cazares, J. Misler, L. Li, C. Dooley,
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1473095, 81502904), the Program for Changjiang Scholars
and Innovative Research Team in University (No. IRT_15R27),
and Fundamental Research Funds for the Central Universities
(Grant lzujbky-2015-K11, lzujbky-2015-275, lzujbky-2015-232,
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