MEL-N16: A Series of Novel Endomorphin Analogs with Good

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MEL-N16: a series of novel endomorphin analogs with good analgesic activity and a favorable side effect profile Xin Liu, Long Zhao, Yuan Wang, Jingjing Zhou, Dan Wang, Yixin Zhang, Xianghui Zhang, Zhaojuan Wang, Dongxu Yang, Lingyun Mou, and Rui Wang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00097 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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ACS Chemical Neuroscience

MEL-N16: a series of novel endomorphin analogs with good analgesic activity and a favorable side effect profile Xin Liu, Long Zhao, Yuan Wang, Jingjing Zhou, Dan Wang, Yixin Zhang, Xianghui Zhang, Zhaojuan Wang, Dongxu Yang, Lingyun Mou, Rui Wang*

Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Department of Pharmacology, Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, 730000, P. R. China

*Corresponding Author: Rui Wang, Ph.D., Dean & Chair Professor Address: Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, 199 Donggang West Road, Lanzhou, 730000, P.R.China. Tel: +86-931-8912567. Fax: +86-931-8912567. E-mail: [email protected] 1

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Abstract: Opioid peptides are neuromodulators that bind to opioid receptors and reduce pain sensitivity. Endomorphins are one of the most active endogenous opioid peptides, and they have good affinity and selectivity toward the µ opioid receptor. However, their clinical usage is hindered by their inability to cross the blood–brain barrier, and their poor in vivo activity after peripheral injection. In order to overcome these defects, we have designed and synthesized a series of novel endomorphin analogs with multiple site modifications. Radioligand binding, cAMP accumulation, and β-arrestin-2 recruitment assays were employed to determine the activity of synthesized endomorphin analogs toward opioid receptors. The blood–brain barrier permeability and antinociceptive effect of these analogs were determined in several rodent models of acute and persistent pain. In addition, the side effects of the analogs were examined. The radioligand binding assay and functional activity examination indicated that the MEL-N16 series of compounds were more active agonists against µ opioid receptor than were the parent peptides. Notably, the analogs displayed biased downstream signaling toward G-protein pathways over β-arrestin-2 recruitment. The analogs showed highly potent antinociceptive effects in the tested nociceptive models. In comparison with endomorphins, the synthesized analogs were better able to penetrate the blood–brain barrier and exerted their pain regulatory activity in the central nervous system after peripheral injection. These analogs also have lower tendency to cause side effects than morphine does at similar or equal antinociceptive doses. The MEL-N16 compounds have highly potent and efficacious analgesic effects in various pain models with a favorable side effect profile. Abbreviation ADME, absorption, distribution, metabolism, and excretion; AUC, area under the curve; BBB, blood-brain barrier; cAMP, cyclic adenosine monophosphate; CNS, central

nervous

system;

CPP,

H-Tyr-D-Ala-Gly-NMePhe-Gly-ol;

conditioned DCM,

place

preference;

dichloromethane;

DAMGO, D-NMeAla,

N-methyl-D-alanine; DIEA, N,N′-diisopropylethylamine; Dmt, 2,6-dimethyltyrosine; 2


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DOR, δ opioid receptor; EDC, 1-ethyl-3-(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; HEK293, human embryonic kidney;

HOBt,

intraperitoneally;

hydroxybenzotrazole; i.v.,

intravenous;

i.c.v., KOR,

intracerebroventricular; κ

opioid

receptor;

i.p., Map,

α-methylene-β-aminopropanoic acids; MOR, µ opioid receptor; MVD, mouse vas deferens; NOP, nociceptin receptor; nor-BNI, nor-binaltorphimine; RP-HPLC, reversed-phase high performance liquid chromatography; β-FNA, β-funaltrexamine; MPE, maximum possible effect; NLX, naloxone; NTI, naltrindole; TLC, thin layer chromatography. Keywords: antinociception, blood–brain barrier, endomorphin, side effect, stability, opioid peptide.

Introduction Opioids, as the main category of analgesics, are the most effective drugs for alleviating moderate to severe pain.1, 2 Currently, morphine is a commonly used as opioid analgesic drug.3 However, the potential for addiction and abuse of opioids has seriously hindered their clinical application.4 The classical opioid system consists of three receptor subtypes: the µ opioid receptor (MOR), the δ opioid receptor (DOR), and the κ opioid receptor (KOR). More recently, an additional opioid receptor was identified with a high degree of homology to the classical opioid receptors. This receptor is regarded as a new member of the opioid class of receptors and has been named the nociceptin receptor (NOP). The binding of opioids to these receptors decreases neurotransmitter release and blockade of the transmission of nerve impulses, resulting in analgesic effects.5, 6 Opioid peptides are a class of endogenous neurotransmitters, and have been extensively studied since their discovery. In 1997, two endogenous opioid receptor ligands, endomorphin-1 (EM-1, H-Tyr1-Pro2-Trp3-Phe4-NH2) and endomorphin-2 (EM-2, H-Tyr1-Pro2-Phe3-Phe4-NH2), were isolated from the human cortex and bovine brain.7 These ligands are involved in numerous physiological regulatory 3



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processes, and particularly those involved in pain regulation. Endomorphins (EMs) have a favorable therapeutic profile compared to those of other µ selective opioids, and reduce the pain response without inducing the side effects associated with morphine usage.8, 9 Therefore, they are considered to have the potential to be used as analgesic drugs to replace morphine. However, their poor enzyme stability, short duration of action, and difficulty in penetrating the blood–brain barrier (BBB) and accessing the central nervous system (CNS) has limited their clinical application. Therefore, the development of a drug with a strong analgesic effect and few side effects is of crucial importance.6, 10 The development of EM analogs with improved properties requires a good understanding of the structure-activity relationship of EMs.11, 12 The function of the key residues in the molecules can be determined using chemically modified peptides, particularly those containing non-natural amino acids.13, 14 As a result, thousands of analogs have been designed and reported in order to study the structure-activity relationships of opioid peptides. For example, the Tyr1 residue has been shown to play an important role in maintaining the physiological activity of opioid peptides, and the methylation of this tyrosine to 2,6-dimethyltyrosine (Dmt) effectively enhance the affinity of EMs for opioid receptors.15, 16 The Pro2 residue of EMs is considered to be a linkage with stereochemical characteristics, and its main role is to limit the relative position of the pharmacophore in the peptide. The study of the degradation pathway of EMs has shown that Pro2 is also an important restriction site.17-20 Insertion of the noncyclic amino acid N-methyl-D-alanine (D-NMeAla) is a transformation method that can reduce the stacking effect between peptide side-chain groups to provide a more potent peptide molecule.21, 22 Phe4 can contribute to stabilizing the dominant conformation of polypeptide molecules. Furthermore, it not only plays an important role in maintaining the affinity of the peptides, but also has a considerable effect on maintaining its selectivity.23, 24 Thus, modifying the key residues in the structures of the EMs is a feasible approach to developing analogs with improved properties.25, 26 Recently, our research group constructed a new series of unnatural β-amino acids known as α-methylene-β-amino acids (Map).27 We found that when the C terminal 4


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Phe4 residue in an EM was substituted with a Map, the interaction between the analog and MOR was enhanced. In the present study, we performed a multi-site combinatorial modification by introducing a variety of non-natural amino acid blocks into the sequence of EMs. Dmt, D-NMeAla, (Ph)Map, and (2-furyl)Map were used as the chemical building blocks in the present study. The resulting compounds were designated the MEL-N16 series. The structures of the EM analogs, which were modified with unnatural amino acids, are shown in Figure 1. The opioid receptor affinities and selectivities of the MEL-N16 compounds were determined by radioligand binding and in vitro pharmacological activity experiments. The biological functionality of the compounds was assessed using cAMP accumulation and isolated tissue assays. The degradation rates of the compounds were then evaluated in the presence of mouse brain homogenate and serum. We further investigated the biological activity of the MEL-N16 series in a selection of analgesic models after central or peripheral administration, in order to detect their activity in reducing acute and inflammatory pain. Finally, the side effects of the compounds were evaluated in vivo.

5



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Figure 1. Structures of EM analogs of MEL-N16 series

Results and Discussion Chemistry All of the analogs were obtained via a segment-coupling peptide synthesis strategy using solution-phase methods. All of the standard amino acids, Dmt, and D-NMeAla were commercially available, and the chiral 2-methylene-3-amino propanoic acids (Map) was prepared as described previously.27 Synthesis of the analogs was conducted

by

performing

an

active

ester

reaction,

and

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and hydroxybenzotrazole (HOBt) were used as coupling agents (see the Supporting Information for experimental details). The established peptides were then purified using semi-preparative

reversed

phase

high-performance

liquid

chromatography

(RP-HPLC), and characterized by RP-HPLC, thin layer chromatography (TLC), electrospray ionization-time-of-flight mass spectrometry (ESI-TOF MS), and melting point measurements. The purities of the compounds were characterized by analytical 6


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RP-HPLC, and determined to be >95%. The detailed analytical properties of the synthetic analogs are provided in Table 1.

Table 1. Analytical Data for EMs Analogs. TOF MS [M+H]+ no.

RP HPLCa

sequence calcd

found

(min)

mp (oC)

purityb (%)

MEL-N1601

H-Tyr-D-NMeAla-Trp-(Ph)Map-NH2

611

611.2957

17.8

162-165

97

MEL-N1602

H-Tyr-D-NMeAla-Trp-(2-furyl)Map-NH2

601

601.2701

24.8

152-155

97

MEL-N1603

H-Tyr-D-NMeAla-Phe-(Ph)Map-NH2

572

572.4621

16.9

150-153

99

MEL-N1604

H-Tyr-D-NMeAla-Phe-(2-furyl)Map-NH2

562

562.4396

16.7

151-153

98

MEL-N1605

H-Dmt-D-NMeAla-Trp-(Ph)Map-NH2

639

639.3227

21.9

165-168

98

MEL-N1606

H-Dmt-D-NMeAla-Trp-(2-furyl)Map-NH2

629

629.3100

26.1

160-163

98

MEL-N1607

H-Dmt-D-NMeAla-Phe-(Ph)Map-NH2

600

600.3119

24.5

155-158

96

MEL-N1608

H-Dmt-D-NMeAla-Phe-(2-furyl)Map-NH2

590

590.2910

25.1

156-158

97

a

tR 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. bPurity determination based on analytical RP-HPLC.

Binding affinity A radioligand binding assay was employed to examine the binding affinity and receptor selectivity of the synthesized peptides in whole cell preparations from HEK293 cells. [3H]DAMGO, [3H]DPDPE, and [3H]U69,593 were used as the radioligand for MOR, DOR, and KOR, respectively. The results are summarized in Table 2. Introduction of both D-NMeAla2 and (Ph)Map4 into EM-1 to give MEL-N1601 greatly enhanced the binding affinity and MOR selectivity of the compound. The subsequent substitution of position 4 with (2-furyl)Map4 afforded MEL-N1602, which showed an ~8.58-fold increase in MOR affinity. Both MEL-N1601 and MEL-N1602 exhibited dramatically increased selectivity toward the MOR over the DOR. The EM-2 analogs MEL-N1603 and MEL-N1604, which bear a Phe3 residue, showed decreased affinity for MOR compared to that shown by MEL-N1601 and MEL-N1602, but they had similar MOR selectivities as their 7



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corresponding EM-1 analogs. In MEL-N1605 and MEL-N1606, the replacement of Tyr1 with Dmt1 greatly improved the activity, and the two analogs showed very high MOR affinities in the two-digit picomolar range. MEL-N1606 exhibited the highest MOR affinity of 10.9 pM. MEL-N1605 and MEL-N1606 also showed decreased selectivity toward the MOR compared with those shown by MEL-N1601 and MEL-N1602. Co-application of Dmt1, D-NMeAla2 and (Ph)Map4/(2-furyl)Map4 into EM-2 resulted in MEL-N1607 and MEL-N1608 which also exhibited MOR affinities in the picomolar range. The DOR binding affinity of MEL-N1607 and MEL-N1608 was comparable to that of MEL-N1605 and MEL-N1606, respectively. MEL-N1601 to MEL-N1604 displayed no detectable KOR affinity, MEL-N1605 to MEL-N1608 had very low affinity for KOR. Collectively, the analogs behaved as MOR-selective ligands.

Table 2. Opioid Receptor Binding Affinities of EMs and Analogs.

No.

sequence

Kiµ (nM)a,d

Kiδ(nM)b,d

Kiκ (nM) c,d

Ki ratio µ/δ/κ

EM-1

H-Tyr-Pro-Trp-Phe-NH2

2.60 ± 0.21

6080 ± 640

>10000

1/2338/-

EM-2

H-Tyr-Pro- Phe-Phe-NH2

3.20 ± 0.13

6420 ± 330

>10000

1/2006/-

MEL-N1601


0.575 ± 0.023

3990 ± 220

>10000

1/6939/-

MEL-N1602


0.303 ± 0.022

3100 ± 140

>10000

1/10231/-

MEL-N1603


0.612 ± 0.17

4280 ± 210

>10000

1/6993/-

0.447 ± 0.15

4510 ± 160

>10000

1/10089/-

MEL-N1604


MEL-N1605


0.0319 ± 0.0039

37.2 ± 1.7

8620 ± 920

1/1166/270219

MEL-N1606


0.0109 ± 0.0015

33.2 ± 5.1

6700 ± 870

1/3046/614678

MEL-N1607


0.0511 ± 0.0023

55.7 ± 6.9

>10000

1/1090/-

MEL-N1608


0.0140 ± 0.0018

47.2 ± 5.5

8310 ± 890

1/3371/593571

a

Displacement of [3H]DAMGO (Kd = 0.6 nM, µ-selective). b Displacement [3H]DPDPE (Kd = 2.8 nM, δ-selective). c Displacement [3H]U69,593 (Kd = 2.9 nM, κ-selective). d Displacement was done using whole cell preparations from transfected HEK293 cells expressing µ-opioid receptor, δ-opioid receptor or κ-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. Data are expressed as the mean ± SEM, each performed in triplicate. 8


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cAMP accumulation assay The functional activity of the peptides was evaluated using a cAMP accumulation assay in HEK293 cells expressing opioid receptors. As shown in Table 3, all of the tested

analogs

inhibited

forskolin-stimulated

cAMP

production

in

a

concentration-dependent manner. All of the analogs displayed potencies higher than that of the EMs. Analogs MEL-N1601 to MEL-N1604, which contained a Tyr1 residue, displayed an MOR potency in the range of 1.45 to 4.39 nM. MEL-N1605 to MEL-N1608, which contained a Dmt1 residue, showed even better potency in the low picomolar range. MEL-N1606 and MEL-N1608 were the two most potent compounds with EC50 values for the MOR of 1.77 and 2.01 pM, respectively. DAMGO is regarded as a full agonist of the MOR; morphine and endomorphins have lower efficacies than DAMGO and behave as partial agonists, and MEL-N1605 to MEL-N1608 showed similar efficacies to DAMGO, indicating that these peptides acted as full agonists.28, 29 MEL-N1601 to MEL-N1604 displayed similar efficacies to their parent peptides, suggesting that these peptides acted as partial agonists.

Table 3. Functional Activity of EMs and Analogsa.

no.

µ-OR

sequence EC50(nM)

Emax (%)

EM-1


14.4 ± 0.6

83.1 ± 4.1

EM-2


11.8 ± 0.2

82.7 ± 4.5

MEL-N1601


2.27 ± 0.11

88.3 ± 5.1

MEL-N1602


1.45 ± 0.23

90.6 ± 6.6

MEL-N1603


4.39 ± 0.57

80.4 ± 4.2

MEL-N1604


4.11 ± 0.28

87.7 ± 6.2

MEL-N1605


0.00395 ± 0.00014

98.1 ± 6.7

MEL-N1606


0.00177 ± 0.00009

98.3 ± 3.3

MEL-N1607


0.00515 ± 0.00033

95.1 ± 2.9

MEL-N1608


0.00201 ± 0.00006

96.9 ± 6.8

3.04 ± 0.32

98.1 ± 6.1

12.6 ± 0.9

77.2 ± 5.5

DAMGO

Tyr-D-Ala-Gly-NMe-Phe-Gly-ol

morphine 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 9



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peptides. EC50 and Emax values were calculated by using the GraphPad Prism software. Data are expressed as the mean ± SEM, each performed in triplicate.

β-arrestin-2 recruitment DAMGO, morphine, MEL-N1606, and MEL-N1608 were evaluated for their potential stimulatory effects on the β-arrestin-2 signaling pathway using the PathHunter enzyme complementation assay (DiscoveRx). To determine whether the test compounds are biased agonists, their cAMP accumulation versus β-arrestin-2 recruitment potencies were compared according to previously reported methods.30, 31 DAMGO was the reference compound and was considered as the “balanced agonist”. As shown in Table 4, morphine displayed no significantly biased agonistic profile compared with that of DAMGO, which was in agreement with previous reports.30, 32 The tested compounds stimulated the β-arrestin-2 signaling pathway in a concentration-dependent manner. The two analogs displayed potencies that were much higher than that of DAMGO. The calculated “bias factor” suggested that the two analogs displayed obvious cAMP pathway bias relative to that of DAMGO, indicating that the analogs preferentially activated the downstream G-protein signaling pathways over that involving β-arrestin-2 recruitment.

Table 4. Potency and efficacy of test compounds in cAMP and β-arrestin-2 recruitment assays. a cAMP (nM)

cAMP/β-arrestin-2

β-arrestin-2 (nM)

No. EC50

Emax

∆logR

EC50

Emax

∆logR

∆∆logR

95%CI

DAMGO

3.04 ± 0.32

98.1 ± 6.1

0

2590 ± 120

99.8 ± 3.3

0

0

morphine

12.6 ± 1.01

77.2 ± 4.2

-0.732 ± 0.014

7670 ± 220

36.1 ± 2.9

-0.922 ± 0.015

0.198

0.168-0.211

MEL-N1606

0.00177 ± 0.00009

98.3 ± 3.3

3.32 ± 0.19

7.50 ± 0.33

52.7 ± 3.7

1.26 ± 0.02

1.97

1.71-2.33

MEL-N1608

0.00201 ± 0.00006

96.9 ± 6.8

3.17 ± 0.22

2.36 ± 0.12

105.4 ± 6.1

2.06 ± 0.05

1.11

0.86-1.41

a

The parameter estimates are presented as mean ± SEM. R=Emax/EC50, the ∆∆LogR for each bias calculation is presented ± SEM along with 95% confidence interval (95% CI). Each performed in triplicate.

In vitro pharmacological activity Guinea pig ileum (GPI) and mouse vas deferens (MVD) can be used to assess a peptide’s ability to inhibit electrically evoked neurotransmitter release, and 10


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subsequent muscle contraction. The GPI assay is generally considered to contain predominantly MOR, but also KOR, and all of the test analogs exhibited increased or similar potencies compared to those shown by the EMs (Table 5). In the MVD assay, the predominant receptor subtype is DOR, and MEL-N1601 to MEL-N1604 exhibited similar potencies in this assay. In agreement with the radioligand binding results, the Dmt1 substitution in MEL-N1605 to MEL-N1608 gave these analogs subnanomolar potencies ranging from 0.401 to 0.818 nM in the GPI assay. A specific, highly potent, and selective DOR antagonist, naltrindole, was used to examine the MVD activity of the analogs. The results showed that the MVD activity of the tested analogs was only partly inhibited by naltrindole, suggesting that this activity was, for the most part, elicited via the MOR, which is coexpressed with other receptors in MVD tissues. These results were in agreement with previous reports.33, 34 Table 5. In Vitro Pharmacological Activity of EMs and Analogs. IC50 (nM) No.

sequence GPI

MVD

ratio

EM-1


14.08 ± 0.23

30.4 ± 2.6

1/2.2

EM-2


9.33 ± 1.22

21.6 ± 3.4

1/2.3

MEL-N1601


8.82 ± 0.92

11.4 ± 1.17

1/1.3

MEL-N1602


5.81 ± 0.72

7.21 ± 0.55

1/1.2

MEL-N1603


9.7 ± 3.3

25.1 ± 5.7

1/2.6

MEL-N1604


11.8 ± 4.2

20.4 ± 3.3

1/1.8

MEL-N1605


0.433 ± 0.02

1.89 ± 0.28

1/4.4

MEL-N1606


0.401 ± 0.03

2.76 ± 0.15

1/6.9

MEL-N1607


0.818 ± 0.12

3.01 ± 0.33

1/3.7

MEL-N1608


0.693 ± 0.08

2.13 ± 0.23

1/3.1

Metabolic stability The metabolic stability of the EMs and the analogs was assessed in mouse brain homogenate and blood serum. Table 6 summarizes the in vitro half-lives of the EMs, as well as peptides MEL-N1605 to MEL-N1608. EM-1 and EM-2 disappeared rapidly 11



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in the mouse brain homogenate, with a half-life of 16.9 and 12.4 min, respectively. The synthesized peptides displayed significantly improved stability with the in vitro half-lives all exceeding 300 minutes. In the blood serum, EM-1 and EM-2 also demonstrated short half-lives of 10.9 and 12.7 minutes, respectively. MEL-N1605 and MEL-N1607 exhibited long serum half-lives. Furthermore, although MEL-N1606 and MEL-N1608 were less stable in the serum than they were in the brain homogenates, they were far more stable than the parent peptides were.

Table 6. Half-lives of EMs and its analogs in mouse-brain membrane homogenate and blood serum. a no

sequence

brain homogenate

blood serum

EM-1


16.9 ± 2.1

10.9 ± 2.1

EM-2


12.4 ± 1.4

12.7 ± 1.5

MEL-N1605


>300

>300

MEL-N1606


>300

246 ± 47

MEL-N1607


>300

>300

MEL-N1608


>300

291 ± 31

a

Values are arithmetic mean of at least three individual experiments ± SEM. The protein content of the brain homogenate was 2.3 mg/mL. Half-lives were calculated on the basis of pseudo-first-order kinetics of the disappearance of the analogs.

Warm water tail-flick assay MEL-N1606 and MEL-N1608 were selected for the subsequent in vivo examinations, as they displayed the highest binding affinities and functional activities in the MEL-N16 series. EMs were employed as the control compound. The compounds were assessed in a mouse tail-flick test after intracerebroventricular (i.c.v.) administration, and the percentage of the maximum possible effect (%MPE) was calculated to represent the antinociceptive response. As shown in Figure 2A and 2B, EM-1 and EM-2 displayed similar antinociceptive activity with ED50 values of 15.2 (13.1−19.3) and 22.6 (16.9−29.3) nmol/kg, respectively. The synthesized analogs exhibited time- and dose-dependent inhibition of heat-induced pain. The maximum antinociceptive effect was produced 10-20 min after administration, and the %MPE 12


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for MEL-N1606 and MEL-N1608 was 95.8% and 94.9%, respectively (Figure 2C and 2D). MEL-N1606 and MEL-N1608 both showed very potent activity with an ED50 value of 0.695 (0.396–1.017) and 0.732 (0.449–1.033) nmol/kg, respectively. Dose-response curves were calculated over a period of 0-30 min to illustrate the change in response (Figure 2E). As shown in Figure 2F, the antinociceptive effects induced by the analogs at 20 nmol/kg were significantly antagonized when 10 mg/kg naloxone (NLX) was administered intraperitoneally 10 min before administration of the analog.

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Figure 2. (A-D) Time curves of antinociceptive effect and AUC data from 0 to 30 min in mouse tail-flick test of EM-1, EM-2, MEL-N1606, and MEL-N1608 after i.c.v. administration. (E) Dose-response curves for analgesic effects at different doses were presented as AUC data from 0 to 30 min. (F) A antinociceptive effects of analogs at 20 nmol/kg were significantly antagonized by naloxone. Doses used are shown in the figure and values are means ± standard error of the mean (SEM) of 8−12 mice. The asterisk indicates that response was significantly different from control (p < 0.05).

To determine whether the analogs could cross the blood–brain barrier, four different doses (0.3, 1, 3 and 10 mg/kg) of MEL-N1606 and MEL-N1608 were injected intravenously (i.v.), and their antinociceptive effect was again examined using a mouse tail-flick test. Morphine was employed as a positive control at a dose of 4 mg/kg. Figure 3A and 3B show the time course of the antinociceptive activity of the peptides over a period of 60 minutes after i.v. injection. The analogs displayed timeand concentration-dependent antinociception. EM-1 and EM-2 failed to induce any obvious antinociceptive effect at a dose of 10 mg/kg (data shown in the Supporting Information). In contrast, MEL-N1606 and MEL-N1608 exhibited strong analgesic activity, with ED50 values of 0.382 (0.252–0.517) and 0.489 (0.296–0.595) mg/kg, respectively. The tested analogs acted rapidly, reaching their peak of antinociception 10–20 minutes after i.v. administration. The analgesic effect of the analog then slowly declined after 40 minutes. At a dose of 10 mg/kg, the two analogs showed 79 and 86 %MPE at the end of the test, respectively. Even at the lowest dose of 0.3 mg/kg, the analogs still displayed ~50 %MPE after 10 min. Dose-response curves were calculated over a period of 0−30 min to illustrate the change in response at different doses (Figure 3C). Furthermore, naloxone was used to investigate the mechanism of action of the analogs. Injection of naloxone (10 mg/kg) intraperitoneally greatly suppressed the antinociceptive effect of the analogs (Fig 3D), confirming that the analgesic effect of the analogs was mainly elicited through opioid receptors. The i.p. injection of naloxone methiodide (10 mg/kg) did not markedly inhibit the activity of the analogs, but i.c.v. injection of naloxone methiodide (4 nM) significantly reduced the analgesic 14


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effect of the analogs (Figure 3E). The specific MOR antagonist, β-funaltrexamine (β-FNA, 8 nM), markedly attenuated the antinociception of the analogs, whereas naltrindole (NTI, DOR antagonist, 5 nM) and nor-binaltorphimine (nor-BNI, KOR antagonist, 5 nM) had no obvious antagonizing effect (Figure 3F). These results are in agreement with those of the radioligand binding assays, indicating that the antinociceptive effect of these analogs is primarily regulated through the MOR system.

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Figure 3. (A and B) Time curves and AUC from 0 to 30 min of antinociceptive effect in mouse tail-flick test of MEL-N1606, MEL-N1608, and morphine after i.v. administration. (C) Dose-response curves of analgesic effects at different doses were presented as AUC from 0 to 30 min. Antinociceptive effect induced by analogs after (D) i.p. injection of naloxone or (E) i.c.v./i.p. injection of naloxone methiodide. (F) Antinociceptive effect of analogs after i.c.v. injection of β-FNA, naltrindole, and nor-BNI. Doses used are shown in the figure and values are means ± SEM of 8−12 mice. Asterisk indicates response is significantly different from control (p < 0.05).

Formalin test The injection of formalin into the hind paw of mice induces a significant pain response, which is represented by the amount of time the mouse spends licking/biting the injected paw. As shown in Figure 4, the baseline value of the saline-treated group was about 38 seconds in phase I (0−5 minutes) and 214 seconds in phase II (15−30 minutes). The analogs significantly shortened the amount of time the mice spent on licking/biting the injured paw in both phases. MEL-N1606 was more effective in reducing the pain response than MEL-N1608. At a dose of 1 mg/kg, MEL-N1606 was approximately as effective as morphine at a dose of 4 mg/kg. The ED50 values of MEL-N1606 and MEL-N1608 in phase I were 0.108 (0.052–0.162) and 0.165 (0.057– 0.278) mg/kg, respectively. The ED50 values of the two analogs in phase II were 0.054 (0.03–0.089) and 0.135 (0.074–0.194) mg/kg, respectively.

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Figure 4. Dose-related analgesic effects in mouse formalin test of (A) MEL-N1606 and (B) MEL-N1608 after i.v. administration. Dose-response curves of analgesic effects at different doses in (C) phase I and (D) phase II. Value are means ± SEM of 6−8 mice. Asterisk indicates response was significantly different from saline-treated group (p < 0.05).

Writhing test Injection of acetic acid intraperitoneally (i.p.) results in abdominal constriction, which causes the mice to writhe. The baseline number of writhes in the saline-treated group was about 50. However, the i.v. administration of MEL-N1606 and MEL-N1608 resulted in dose-dependent antinociception, as shown in Figure 5. The tested compounds both showed great antinociception in reducing the number of writhes, and they exhibited similar activity in this test. The analgesic effect elicited by the analogs at a dose of 3 mg/kg was comparable to that of morphine at a dose of 4 mg/kg.

Figure 5. Dose-related analgesic effects in mouse writhing test of MEL-N1606, MEL-N1608, and morphine after i.v. administration. Test compounds were administered 5 min before i.p. injection of 1% acetic acid. Value are means ± SEM of 6−8 mice. Asterisk indicates response was significantly different from saline-treated group (p < 0.05).

Tolerance MEL-N1606 was injected into mice once daily for five consecutive days to examine whether the mice developed tolerance to the drug. After repeated administration, the analgesic effect was determined using a tail-flick assay. Morphine was used as a positive control, and equal analgesic doses were used for morphine and the analog. As shown in Figure 6A, on the first day of the examination, MEL-N1606 (6 mg/kg) displayed a similar antinociceptive effect to that of morphine (10 mg/kg). The analgesic activity of morphine was reduced dramatically after five days of repeated injections, and its potency was reduced to 56.4 %MPE. In contrast, on the fifth day of 17



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administration, MEL-N1606 still produced strong antinociception after repeated injection with a potency value of 85.5 %MPE. Colonic bead expulsion A colonic bead expulsion experiment was used to determine the influence of the compounds on the gastrointestinal system. The data are expressed as the %inhibition of colonic bead expulsion. Saline was used as a negative control, and it gave a base value of 2.74%. As shown in Figure 6B, MEL-N1606 inhibited colonic bead expulsion in a dose-dependent manner. At a dose of 6 mg/kg, the analog reduced the rate of expulsion with a mean %inhibition of 66.1%. Morphine showed a higher %inhibition value of 94.1% at the dose of 10 mg/kg. There was a significant difference between all of the results for MEL-N1606 and the result for morphine.

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Figure 6. (A) Tolerance development to MEL-N1606 and morphine after i.v. administration; values are means ± SEM of 8 mice. (B) Dose-related effects of colonic bead expulsion in mice after i.v. administration of MEL-N1606 and morphine; data are means ± SEM of 8–10 mice and are expressed as percentage inhibition compared with bead expulsion time of controls. (C) Place conditioning effects and time in the drug-paired compartment in mice after i.v. administration of MEL-N1606 and morphine; data are means ± SEM of 8–10 mice and are expressed as time spent in drug-associated compartment on post-conditioning day minus time spent in drug-associated compartment during 15 min pre-conditioning. (D) Locomotor activity in rotarod test effect after i.v. administrations of MEL-N1606 and morphine; data are mean ± SEM of 8–10 mice and are expressed as the AUC of MPI within 60 min and (E) endurance time. Asterisk indicates response was significantly different from control group (p < 0.05).

Conditioned place preference test A CPP test was performed to evaluate the potential of MEL-N1606 and morphine to cause drug seeking behavior. Equal effective antinociceptive doses of morphine and the analogs were used, producing > 95% MPE. As shown in Figure 6C, the saline treated mice did not display an obvious preference. In contrast, the animals that received morphine at the 10 mg/kg displayed a significant place preference and spent about 222 seconds in the drug-paired compartment. At a dose of 6 mg/kg, the mice in the analog group spent about 51 seconds in the drug-paired compartment. A lower dose was used to test whether the analog induced biphasic dose effect, at a dose of 3 mg/kg, the mice displayed no obvious preference after administration of the analog.

Locomotor activity In order to examine whether our analogs induced any psychotropic effects, the locomotor activity of the treated mice was evaluated using a rotarod test. The experiment was performed over the course of one hour, with tests being performed every 10 minutes. The data are expressed as the time (seconds) that the mice were able to maintain balance in the rotarod test, and as the AUC of the %maximum possible inhibition (MPI) after 60 minutes. The dosage of morphine and MEL-N1606 required to produce more than 95% MPE in a tail-flick test was used. As shown in Figure 6D and 6E, in the first 10 minutes, the morphine-treated group (10 mg/kg) spent less time on the rotating rod (nearly 183 seconds) compared to that spent by the control group, suggesting an impairment of locomotor activity. In contrast, the 19



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endurance time on the rod in the MEL-N1606-treated group (6 mg/kg) was 242 seconds in the first 10 min postinjection. After 30 min, MEL-N1606 did not decrease the endurance time in the mice.

The analogs showed improved bioavailability while retaining high activity Present data showed that the (2-furyl)Map4 (MEL-N1602, MEL-N1604, MEL-N1606, and MEL-N1608) substitution appeared to be more effective than the (Ph)Map4 substitution (MEL-N1601, MEL-N1603, MEL-N1605, and MEL-N1607) in improving the opioid activity of EMs. This finding is in agreement with our previous results.35 Dmt1 substitution enhanced the binding affinity of the peptides for opioid receptors relative to the Tyr1-containing analogs, but the Dmt1-containing analogs also displayed decreased MOR/DOR selectivity. Nevertheless, the least MOR-selective compound MEL-N1607 still displayed a robust binding preference for the MOR with a MOR/DOR selectivity ratio of 1090. The residue at position 2 of the EMs is regarded as a key stereochemical spacer.36 D-NMeAla was employed to mimic the conformation of proline, and some of the analogs containing D-NMeAla showed increased activity against the MOR compared to the parent peptides containing Pro2.22, 37

These data further confirm that D-NMeAla2 appears to be a favorable replacement

for improving MOR activity. The results of the functional activity experiments paralleled those of the binding assays, with all of the compounds acting as full or partial agonists of the MOR, and displaying high potencies. The poor stability of the endomorphins means it is difficult for them to reach the target system.17 The enzyme stability of our synthesized analogs was examined in brain homogenates and blood serum. The results showed that all of the analogs displayed significantly enhanced resistance to enzyme degradation. The incorporation of multiple unnatural amino acids probably impedes recognition of the cleavage site by proteases. As a result, the in vitro half-lives of our compounds all exceeded 200 minutes. The in vivo activity of the analogs demonstrates that these compounds are very potent, and exert their antinociceptive effect both supraspinally and systematically. The fact 20


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that naloxone reverses the analgesic of the analogs confirms that the activity of the analogs is mediated through the opioid system. The high antinociceptive activity after i.v. administration provides evidence that the synthesized compounds are able to penetrate the BBB. The in vivo analgesic effects of MEL-N1606 and MEL-N1608 were examined in mice using a tail-flick test. This test is sensitive to centrally acting analgesics, as the tail-flick response is considered to be primarily controlled at the spinal and supraspinal level.38 At a dose of 3 mg/kg MEL-N1606 and MEL-N1608 both displayed strong analgesic effects with a %MPE value of 86% and 79%, respectively. When the dose was lowered to 0.3 mg/kg, the analogs still gave high %MPE values and long-lasting analgesic effects. These results suggest that the antinociceptive effect of our analogs is mainly mediated through a central system. To further confirm the action site, naloxone methiodide, a peripherally restricted opioid antagonist, was employed to examine the analgesic mechanism of our analogs in the tail-flick test. Figure 3E shows that the pain-relief effect of MEL-N1606 and MEL-N1608 was significantly inhibited by central administration of naloxone methiodide, but not through peripheral administration. Collectively, these results indicate that the tested analogs are able to cross the highly selective BBB and exert their analgesic effect in the CNS. Considering that Dmt and D-NMeAla incorporation provide the peptide with limited CNS access, we proposed that the structurally constrained scaffold, Map, is the main factor in facilitating peptide transportation to the CNS.39 The prolonged in vitro half-lives of our analogs could also contribute to the BBB penetration, as this may increase the plasma concentration of the peptides leading to greater diffusion across the BBB.40 The in vivo antagonist examinations provided indirect evidence of the BBB penetration of our analog, and the quantitation or direct measurement of the BBB penetration by the compounds will be one of the priorities of our follow-up work. Opioid antagonists were used to investigate the role of opioid receptors in mediating the activity of the test compounds. Pretreatment with DOR and KOR antagonists (NTI and nor-BNI, respectively) was ineffective in blocking the effects of supraspinally administered analogs. However, inhibition of the tail-flick responses induced by the 21



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analogs was attenuated by the MOR antagonist β-FNA. Collectively, these results indicate that the in vivo analgesic activity of the test analogs was mainly regulated through the activation of the MOR, which is in agreement with the binding affinity assays. In the present work, a formalin test was also used to evaluate the analgesic effect of our analogs. The formalin model reflects distinct pain responses in a biphasic manner. The early phase is considered to reflect direct activation of nociceptive sensory afferents by formalin, resulting in a sharp increase in the spontaneous activity of C fibers. The late phase is regarded to be dependent on sensitization within the dorsal horn of the spinal cord and the brain.41 Our analogs produced a significant antinociceptive effect in both phases. MEL-N1606 was more potent than MEL-N1608 in this test. The %MPE value of MEL-N1606 at 1 mg/kg was similar to that of morphine at a dose of 4 mg/kg in both the first and second phases. The writhing test is a widely accepted visceral nociception model that is evoked by acetic acid, and is thought to be a clinically relevant model for intestinal pain in humans.42 The intraperitoneal injection of acetic acid leads to a writhing response in the mice. In this test, MEL-N1606 and MEL-N1608 significantly inhibited the number of writhes. Compared to morphine, both MEL-N1606 and MEL-N1608 showed potent analgesic effects even at lower doses. There are differences between the binding affinity and in vivo antinociception of opioid drugs as compared with other non-peptide opioid drugs. We assume that our peptide analogs probably have different absorption, distribution, metabolism, and excretion (ADME) profiles compared with organic molecules because the peptide-based structure may affect their in vivo activity. In addition, the mechanisms of activiation by opioid peptides may differ from those of non-peptide opioid drugs. For example, EMs are known to increase the release of dynorphins in the spinal cord while morphine dose not.43 The analogs showed favorable side-effect profile In order to characterize the side-effect profile of our analogs, pharmacology studies were carried out in mice. Opioid tolerance is a major health problem, and opioids 22


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often become ineffective after repeated administration.44-46 The development of tolerance means that increased opioid doses are required in order to achieve adequate analgesia. The present results indicate that after 5 days of repeated administration, MEL-N1606 still exerts a remarkable analgesic effect in the tail-flick test. On the 5th day, MEL-N1606 still inhibited the pain response behavior, with only a slight and insignificant decrease as compared with that on the 1st day, indicating that the analgesic potency of the compound is retained. In contrast, the analgesic effect of morphine was significantly reduced after chronic administration, only producing an 56.4 %MPE on the 5th day of injection. These result suggests that our compounds are less likely to induce opioid tolerance after repeated injection. The efficacy of the opioid agonist may be involved in regulating the tolerance development, and high-efficacy agonists tend to produce less tolerance than low-efficacy agonists at equi-effective doses. The fact that analog MEL-N1606 has higher efficacy than morphine may contribute to the lower development of tolerance to our compound. Constipation is a common and costly side effect of opioid treatment affecting patients’ quality of life. Opioid induced constipation may also limit effective pain therapy.47, 48 The binding of opioids to MORs in the gastrointestinal system affects gastrointestinal motility, leading to constipation.49, 50 The results showed that morphine significantly delayed the time of colonic bead expulsion, while our analog had limited influence on gastrointestinal transit. The lower tendency of MEL-N1606 to induce constipation may be due to its different pharmacokinetic/pharmacodynamic profile and tissue distribution to morphine. Opioid treatment can cause drug-seeking behavior, and induce corresponding behavior such as conditioned place preference. Our current work indicated that morphine induced a strong CPP at a dose of >95% MPE. In comparison, MEL-N1606 produced a lower CPP at the dose required to give >95% MPE, despite MEL-N1606 producing higher antinociceptive effect than morphine as indicated in the in vivo analgesic assays. These results demonstrate that although MEL-N1606 is not devoid of abuse-potential, it is still less likely than morphine to result in drug-seeking behavior. 23



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The locomotion activity of the mice was assessed using a rotarod test. The results reveal that MEL-N1606 does not have a large influence on the motor performance of the mice. In contrast, morphine significantly shortened the time for which the mice could maintain balance on the rotarod. These results demonstrated that this analog is less likely to result in sedation/depression than morphine. The MOR is well known to stimulate multiple downstream pathway signals, including G-protein coupling and β-arrestin recruitment. A growing body of evidence suggests that specific agonist could selectively stimulate one signaling pathway over another.51 52

These ligands with biased agonism create new possibilities for discovering novel

MOR agonists.53, 54 For instance, it has been reported that G-protein-biased opioid agonist tend to induce analgesic effect with few side effects.55 In addition, morphine has been observed to produce enhanced analgesia and an alleviated side effect profile in β-arrestin-2 knockout mice.56, 57 Therefore, MOR agonists may deviate from biased agonism toward G-protein-dependent signaling pathways over the β-arrestin-2 recruitment pathway. In the current study, the in vitro functional activity assays revealed that MEL-N1606 and MEL-N1608 exhibited biased agonism toward the cAMP pathway. Moreover, the subsequent in vivo assays confirmed that the synthesized analogs had a low tendency to cause side effects such as tolerance, which is in good agreement with previous findings. In a subsequent investigation, we will attempt to further study the pathway signaling mechanisms of the peptide because an understanding of the molecular mechanisms would facilitate further optimization of the analogs.

Conclusion Although endomorphins have many interesting properties as endogenous peptides, they are only active when they are administered directly into the central nervous system. The limited capacity of endomorphins to cross their blood–brain barrier and their poor metabolic resistance limits their pharmacological applications. We designed and synthesized a series of EMs analogs containing unnatural amino acid modifications, known as the MEL-N16 series. These analogs were shown to have 24


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excellent MOR affinity and selectivity. In particular, MEL-N1606 and MEL-N1608 showed biased activity for G-protein signaling pathway over β-arrestin-2 recruitment, the G-protein-biased MOR agonist was believed to produce analgesic effect with reduced side effects. Furthermore, the modification strategy used in the present study significantly increased the stability and BBB permeability of the analogs. The MEL-N16 compounds showed very good analgesic activity in a variety of analgesic models. In addition, MEL-N1606 was studied to determine whether it produced the adverse events common with most opioid drugs. The results revealed that compared to morphine, this analog had reduced motor impairment, drug seeking behavior and constipation. Notably, the analgesic effect of MEL-N1606 did not decrease significantly after repeated administration, which showed that this compound has the potential to be a safe and effective medication for long-term use. We believe that this new class of opioid lead compounds has potential value in clinical application.

Materials and Methods Animals Animals (Animal Center of Medical College of Lanzhou University, Gansu, People’s Republic of China) were housed in a temperature-controlled environment (22 ± 1 °C) under standard 12 h light/dark conditions and received food and water ad libitum. Animals were used only once and received good care and humane treatment. 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 the protocols in this study were approved by the Ethics Committee of Lanzhou University (permit number: SYXK Gan 2009–0005), China. All efforts were made to minimize animal suffering and to reduce the number of animals used. Chemicals EMs and their analogs were synthesized by manual solution-phase methods as described in our previous report.27 The crude products were purified by semipreparative RP-HPLC and were >95% pure as determined by analytical RP-HPLC. The molecular weight of the peptide was confirmed by an electrospray 25



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Page 26 of 38

ionization mass spectrometer (ESI-Q-TOF Maxis-4G, Bruker Daltonics, Germany). [3H]DAMGO (50 Ci/mmol), [3H]DPDPE (43 Ci/mmol), [3H]U69,593 (43.6 Ci/mmol) and [3H] cAMP (50 Ci/mmol) were purchased from Perkin-Elmer, Boston, MA. Protein kinase A, forskolin and IBMX were the products of Sigma-Aldrich (St. Louis, Missouri, USA). The radioactivities were measured by a Perkin Elmer precisely 2460 Microplate Counter. The scintillation cocktail was obtained from Perkin-Elmer, Boston, MA. Naloxone hydrochloride, naloxone methiodide, β-funaltrexamine hydrochloride

(β-FNA),

naltrindole

isothiocyanate

hydrochloride

(NTI),

nor-binaltorphimine hydrochloride (nor-BNI), and formalin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All compounds were dissolved in saline solution and stored at -20 °C. Radioligand Binding Assay The detailed description of competition binding experiments was described previously.58 In the experiments designed to define peptide specificity for µ-, δ- and κ-opioid receptors, HEK293 cells stably expressing MOR, DOR, or KOR (2.5– 3.5×106 cells/tube) were incubated with 1.7 nM [3H]DAMGO, 1.0 nM [3H]DPDPE or 2.0 nM [3H]U69,593 and 10-10–10-4 M unlabeled ligands for each experiments. The reaction was performed in 25°C for 1 h in freshly prepared binding buffer (25 mM Hepes, 5 mM MgCl2, 1 mM CaCl2, 0.4% bovine serum albumin [BSA], and 2.5 mM ethylenediaminetetraacetic acid [EDTA], pH 7.4). 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 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 Software Inc., San Diego, CA). The dissociation constant (Kdµ = 0.6 nM, Kdδ = 2.8 nM,

Kdκ = 2.9 nM) and the number of binding sites (Bmax) were calculated by iterative curve-fitting analysis using at least seven concentrations of [3H]DAMGO, [3H]DPDPE or [3H]U69,593 in a range of 0.085–8.5 nM, 0.10–5.05 nM, or 0.10–5.00 nM. Nonspecific binding was assessed in the presence of 10 µM naloxone, 10 µM 26


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naltrexone, or 10 µM nor-BNI. Measurements of cAMP Accumulation. In vitro cAMP assays were performed as described in our previous reports.27,

59

HEK293 cells which stably expressing the µ-opioid receptor were seeded in 24-well microtiter plates in the day before the assay. Before beginning the assay, cells were washed twice and warmed to 37 °C in serum-free medium containing 1 mM IBMX, vehicle, or various concentrations (10-10–10-4 M) of compounds and 50 µM forskolin for 0.5 h. After incubation, cells were lysed, and cAMP levels were assessed by a competition PKA binding assay. The concentrations of cAMP in lysates were calculated using the standard curve that was constructed using cAMP standard. Scintillation fluid was added, and radioactivity was quantified in a scintillation counter (liquid scintillation counter, PerkinElmer). Analysis of the data was performed using the Graph-Pad Prism software (version 5.0, San Diego, CA). β-Arrestin-2 Recruitment Assays

β-Arrestin recruitment was measured by the PathHunter enzyme complementation assay (DiscoveRx). Assays using DiscoveRx PathHunter eXpress OPRM1 CHO-K1 β –Arrestin-2 GPCR Assays were conducted as instructed by the manufacturer. Briefly, Cells were incubated with drug for 90 min at 37 °C and 10× dilutions of agonist (prepared in HBSS and 20 mM HEPES, pH 7.4) were added to the cells and incubated for 90 min. Next, the detection reagents were reconstituted, mixed at the appropriate ratio, and added to the cells. After 60 min, luminescence per well was measured on a plate counter FlexStation III. The bias factor was calculated according to previous report.30 In Vitro Assays on Isolated Tissue Preparation. In vitro opioid activities of peptides were tested in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. For the GPI assay, the myenteric plexus longitudinal muscle was obtained from guinea pig (300–350 g). For the MVD assay, the vas deferens of male Kunming strain mice (30–35 g) was prepared. 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°C and 36°C, respectively. Both tissues were used 27



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for field stimulation with bipolar rectangular pulses of supramaximal voltage. The parameter of rectangular pulse induced contraction is 1 Hz, 60 V, 0.5 ms pulse width for GPI assay; 1 Hz, 80 V, 1 ms pulse width for MVD assay. 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 with intervals of 15 min between each dose. The values were arithmetic means of 10–15 measurements. In order to measure whether δ-opioid receptor-mediated antagonism occurred in the MVD, the tissue preparation added naltrindole, a selective δ-receptor antagonist, after 5 min incubation, added the test analog at the IC50 dose value, and the percentage recovery (reversal rate) of electrically evoked contraction was then calculated. Metabolic Stability. The degradation studies of EMs and analogues were carried out using mouse brain homogenate and mouse serum. To obtain mouse serum, Male Kunming mice (30–35 g) were anesthetized and blood was collected from the carotid with a heparinized syringe. The blood was kept at 4°C overnight and then centrifuged for 20 min at 2,000 g in the same temperature. The supernatant was separated and stored at 80°C.60 The mouse brain homogenate was prepared as described previously.61 Protein content of the suspension was confirmed by BCA potein assay kit (Thermo, Rockford, IL, U.S.). A final protein concentration of 2.3 mg/mL in 50 mM Tris buffer, pH 7.4, was used for all incubations.61 RP-HPLC analysis determined the stability of peptides. Approximately 10 µL of peptide (10-2 M) stock solution was digested with 190 µL of rat brain homogenate at 37 °C in a final volume of 200 µL for incubation. 20 µL of the aliquots were withdrawn from the mixture at 0, 10, 30, 60, 120, 240, 480 min, and 90 µL acetonitrile was added immediately for precipitated proteins, placing the tube on ice for 5 min, added 90 µL of 0.5% acetic acid at the required time to prevent further enzymatic breakdown. The aliquots were centrifuged at 13,000 g 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 28


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mm, Milford, MA), using the solvent system of 0.1% TFA in acetonitrile (A) and 0.1% 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 1 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 seven time points. The rate constants obtained were used to establish the degradation half-lives (t1/2) as In2/k. Drug Administration I.c.v. administration was performed in conscious mice following the method previously described.62 The injection site was 1.5 mm from the middle, 1 mm from the bregma and 3 mm from the surface of the skull. All the drugs were delivered slowly in an injection volume of 4 µL. The opioid antagonists, naloxone and naltrindole were i.c.v. injected 10 min, nor-BNI were i.c.v. injected 30 min, and β-FNA were i.c.v. injected 4 h, prior to EMs analogs.

Tail-flick Test Male Kunming mice weighing 18−22 g were employed, various doses of analogs were injected intracerebroventricular (i.c.v.) or intravenous (i.v.), and the warm water tail-flick responses were measured at different times.63 Nociception was evoked by immersing the mouse tail in warm water (50 ± 0.2 oC) and measuring the latency to withdrawal. For the study involving the opioid antagonist, animals were pretreated with naloxone and naloxone methiodide before i.v. challenge with analogs. For each mouse, the tail-flick latency was recorded before treatment, and those with a latency of approximately 3−5 sec were selected. The latency to tail-flick was defined as the test latency, the corresponding cut-off time was set at 10 sec in order to minimize tissue damage. The antinociceptive response was expressed as the percent maximum possible effect (%MPE), calculated by the following equation: %MPE = 100 × (test latency – control latency) / (10 – control latency). Formalin Test The formalin test were performed as described in our previous reports.38 Male 29



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Kunming mice weighing 22−25 g were employed. The animals were acclimatized in a transparent acrylic observation chamber with a mirror positioned below the floor for a period of 15 min. Mice were pre-treated (i.v. injection) with test analogs 5 min before subcutaneous (s.c.) injection of 5% formalin solution (20 µL) into the right hind paw, and mice were immediately placed back in the observation chamber, and the time spent licking, shaking, and biting the injected paw at 0−5 min (first-phase) and 15−30 min (second-phase) was measured with a stopwatch. The nociceptive response was expressed as the second (sec), and 0.9 % saline was used as control. Writhing Test Male Kunming mice weighing 18−22 g were employed. Mice were pre-treated (i.v. injection) with test analogs 5 min before intraperitoneal (i.p.) injection of 1% acetic acid solution (10 ml/kg), which induce a contraction of the abdominal muscles together with a stretching of the hind limbs 64. Mice were immediately returned to a Plexiglas box (20 cm in height and 15 cm in diameter), and the number of writhes was cumulatively counted for 20 minutes. Development of Tolerance Male Kunming mice received i.v. injections of morphine and examined compounds once daily for five days, between 10 and 12 a.m.65 The development of tolerance was established by assessing the antinociception effect. Mice were tested by tail-flick before injections using the methods described above, and then injected assigned dose and tested at 5, 10, 15, 20, 30, 40, 50 and 60 min every testing day. %MPE was calculated by the following equation: %MPE = 100 × (test latency – control latency) / (10 – control latency). Colonic Bead Expulsion Male Kunming mice weighing 25−30 g were employed fasted 2 hour. Compounds were i.v. injected 5 min before insert a single glass bead (diameter 3 mm) 2 cm into the distal colon of each mouse under ether anesthesia.66 A glass rod, one end was fire-polished in order to be rendered atraumatic, inserted the glass bead. After that, mice were immediately placed into individual plastic cages. Before treatment, each mouse was tested the time required for expulsion of the glass bead, with 30 min as the 30


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cut-off time. Inhibition of colonic propulsion was measured as the increase in mean expulsion time of the glass bead, and expressed as the percent of increase of colonic bead expulsion, calculated in the following manner: %Inhibition = 100 × (mean expulsion time in drug-treated mice − mean expulsion time in saline-treated mice) / (30 − mean expulsion time in saline-treated mice). Conditioned Place Preference Test Male Kunming mice weighing 18−22 g were employed. The conditioned place preference (CPP) apparatus consists of two main compartments displaying each different features, the two compartments are linked by a corridor which doesn’t have different features.67 In order to preclude any interference from the smell of feces and urine, the compartments were thoroughly cleaned following each test. On day 1 (pre-conditioning), mice were placed in the corridor and allowed free access to the compartments for 15 minutes. The time spent in each compartment was recorded. On days 2–6 (conditioning phase) mice received an injection of compounds in the morning and were immediately confined into one of the two conditioning compartments for 45 minutes (drug pairing occurred in the least preferred compartment). In the afternoon, the mice received an injection of vehicle and were immediately confined into the other compartment which is different from morning. The interval between the injections is 6 hours. On day 7 (post-conditioning), mice were again allowed free access to both compartments for 15 minutes, the time mice spent in each compartment was recorded separately. A significant increase in the percentage

of

time spent

in

the

drug-paired

compartment

between

the

pre-conditioning session (day 1) and the post-conditioning session (day 7) indicates that the substance induces a place preference. The degree of drug induced conditioning place preference is calculated as the time spent during the post-conditioning phase (drug-paired compartment) – the time spent during the pre-conditioning phase in the white compartment. If the difference was positive, the drug has induced a preference while the opposite indicates the aversion. Rotarod Test Male Kunming mice weighing 18−22 g were employed. An automated apparatus was 31



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used to evaluate the ability of mice to maintain balance for up to 5 min on a rod rotating at a constant speed of 16 rpm.68 Prior to starting data collection, mice were trained for 2–3 days and were given three trials per day in order to achieve maximal performance in the rotarod test. The mice were frequently monitored by the researchers. Mice were given three trials with 2-min breaks between trials. After i.v. injection test analogs, the latency to fall from the rotating drum was measured in seconds. Statistical Analysis All 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.

Author Contributions Xin Liu, Long Zhao, Yuan Wang, Jingjing Zhou, Dan Wang, Yixin Zhang, Xianghui Zhang and Zhaojuan Wang performed the research. Xin Liu, Yuan Wang and Rui Wang designed the research study. Dongxu Yang, Lingyun Mou contributed essential reagents or tools. Xin Liu, Yuan Wang and Jingjing Zhou analyzed the data. Xin Liu, Yuan Wang and Rui Wang wrote the paper. Funding Sources We are grateful for the grants from the National Natural Science Foundation of China (Nos. 21432003, 81502904, 81473095, 21402076), 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-2017-131, lzujbky-2015-232, lzujbky-2017-132, lzujbky-2016-ct01). Conflict of interest The authors state no conflict of interest. Supporting information 32


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Instruments for mass spectra, Melting points, RP HPLC; the solvents for analytical HPLC; general procedure for synthesis of the Boc protected Map (Boc-Map-OH); Procedure for peptide synthesis; tail-flick results of EMs in i.v. administration; Copies of ESI TOF-MS spectra.

References 1. Christo, P. J., and Mazloomdoost, D. (2008) Cancer pain and analgesia, Ann N Y Acad Sci 1138, 278-298. 2. Parsells Kelly, J., Cook, S. F., Kaufman, D. W., Anderson, T., Rosenberg, L., and Mitchell, A. A. (2008) Prevalence and characteristics of opioid use in the US adult population, Pain 138, 507-513. 3. Dalal, S., and Bruera, E. (2013) Access to opioid analgesics and pain relief for patients with cancer, Nat Rev Clin Oncol 10, 108-116. 4. Pradhan, A. A., Smith, M. L., Kieffer, B. L., and Evans, C. J. (2012) Ligand-directed signalling within the opioid receptor family, Br J Pharmacol 167, 960-969. 5. Janecka, A., Perlikowska, R., Gach, K., Wyrebska, A., and Fichna, J. (2010) Development of opioid peptide analogs for pain relief, Curr Pharm Des 16, 1126-1135. 6. Holden, J. E., Jeong, Y., and Forrest, J. M. (2005) The endogenous opioid system and clinical pain management, AACN Clin Issues 16, 291-301. 7. Zadina, J. E., Hackler, L., Ge, L. J., and Kastin, A. J. (1997) A potent and selective endogenous agonist for the mu-opiate receptor, Nature 386, 499-502. 8. Fichna, J., Janecka, A., Costentin, J., and Do Rego, J. C. (2007) The endomorphin system and its evolving neurophysiological role, Pharmacol Rev 59, 88-123. 9. Harrison, C., McNulty, S., Smart, D., Rowbotham, D. J., Grandy, D. K., Devi, L. A., and Lambert, D. G. (1999) The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells, Br J Pharmacol 128, 472-478. 10. Aldrich, J. V., and McLaughlin, J. P. (2012) Opioid Peptides: Potential for Drug Development, Drug Discov Today Technol 9, e23-e31. 11. Liu, W. X., and Wang, R. (2012) Endomorphins: potential roles and therapeutic indications in the development of opioid peptide analgesic drugs, Med Res Rev 32, 536-580. 12. Borics, A., and Toth, G. (2010) Structural comparison of mu-opioid receptor selective peptides confirmed four parameters of bioactivity, J Mol Graph Model 28, 495-505. 13. Hruby, V. J., and Balse, P. M. (2000) Conformational and topographical considerations in designing agonist peptidomimetics from peptide leads, Curr Med Chem 7, 945-970. 14. Gentilucci, L., and Tolomelli, A. (2004) Recent advances in the investigation of the bioactive conformation of peptides active at the micro-opioid receptor. conformational analysis of endomorphins, Curr Top Med Chem 4, 105-121. 15. Koda, Y., Del Borgo, M., Wessling, S. T., Lazarus, L. H., Okada, Y., Toth, I., and Blanchfield, J. T. (2008) Synthesis and in vitro evaluation of a library of modified endomorphin 1 peptides, Bioorg Med Chem 16, 6286-6296. 33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

16. Fichna, J., Perlikowska, R., Wyrebska, A., Gach, K., Piekielna, J., do-Rego, J. C., Toth, G., Kluczyk, A., Janecki, T., and Janecka, A. (2011) Effect of 2',6'-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs, Bioorg Med Chem 19, 6977-6981. 17. Janecka, A., Staniszewska, R., Gach, K., and Fichna, J. (2008) Enzymatic degradation of endomorphins, Peptides 29, 2066-2073. 18. Paterlini, M. G., Avitabile, F., Ostrowski, B. G., Ferguson, D. M., and Portoghese, P. S. (2000) Stereochemical

requirements

for

receptor

recognition

of

the

mu-opioid

peptide

endomorphin-1, Biophys J 78, 590-599. 19. Keller, M., Boissard, C., Patiny, L., Chung, N. N., Lemieux, C., Mutter, M., and Schiller, P. W. (2001) Pseudoproline-containing analogues of morphiceptin and endomorphin-2: evidence for a cis Tyr-Pro amide bond in the bioactive conformation, J Med Chem 44, 3896-3903. 20. Perlikowska, R., and Janecka, A. (2014) Bioavailability of endomorphins and the blood-brain barrier--a review, Med Chem 10, 2-17. 21. Champion, H. C., and Kadowitz, P. J. (1999) Vasodepressor responses to [D-Ala2]-endomorphin 2 (TAPP) are mediated by an L-NAME-sensitive mechanism in the rat, J Cardiovasc Pharmacol 33, 280-284. 22. Adamska, A., Kolesinska, B., Kluczyk, A., Kaminski, Z. J., and Janecka, A. (2015) Synthesis of linear and cyclic opioid-based peptide analogs containing multiple N-methylated amino acid residues, J Pept Sci 21, 807-810. 23. Nadon, J. F., Rochon, K., Grastilleur, S., Langlois, G., Dao, T. T., Blais, V., Guerin, B., Gendron, L., and Dory, Y. L. (2017) Synthesis of Gly-psi[(Z)CF horizontal lineCH]-Phe, a Fluoroalkene Dipeptide Isostere, and Its Incorporation into a Leu-enkephalin Peptidomimetic, ACS Chem Neurosci 8, 40-49. 24. Rochon, K., Proteau-Gagne, A., Bourassa, P., Nadon, J. F., Cote, J., Bournival, V., Gobeil, F., Jr., Guerin, B., Dory, Y. L., and Gendron, L. (2013) Preparation and evaluation at the delta opioid receptor of a series of linear leu-enkephalin analogues obtained by systematic replacement of the amides, ACS Chem Neurosci 4, 1204-1216. 25. In, Y., Minoura, K., Tomoo, K., Sasaki, Y., Lazarus, L. H., Okada, Y., and Ishida, T. (2005) Structural function of C-terminal amidation of endomorphin. Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy, molecular calculation, and X-ray crystallography, FEBS J 272, 5079-5097. 26. De Marco, R., and Janecka, A. (2015) Strategies to Improve Bioavailability and In Vivo Efficacy of the Endogenous Opioid Peptides Endomorphin-1 and Endomorphin-2, Curr Top Med Chem 16, 141-155. 27. Wang, Y., Xing, Y., Liu, X., Ji, H., Kai, M., Chen, Z., Yu, J., Zhao, D., Ren, H., and Wang, R. (2012) A new class of highly potent and selective endomorphin-1 analogues containing alpha-methylene-beta-aminopropanoic acids (map), J Med Chem 55, 6224-6236. 28. Kelly, E. (2013) Efficacy and ligand bias at the mu-opioid receptor, Br J Pharmacol 169, 1430-1446. 29. Ronai, A. Z., Al-Khrasani, M., Benyhe, S., Lengyel, I., Kocsis, L., Orosz, G., Toth, G., Kato, E., and Tothfalusi, L. (2006) Partial and full agonism in endomorphin derivatives: comparison by null and operational model, Peptides 27, 1507-1513. 30. Winpenny, D., Clark, M., and Cawkill, D. (2016) Biased ligand quantification in drug discovery: 34


Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from theory to high throughput screening to identify new biased mu opioid receptor agonists, Br J Pharmacol 173, 1393-1403. 31. Malfacini, D., Ambrosio, C., Gro, M. C., Sbraccia, M., Trapella, C., Guerrini, R., Bonora, M., Pinton, P., Costa, T., and Calo, G. (2015) Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and beta-Arrestins 2, PLoS One 10, e0132865. 32. Thompson, G. L., Lane, J. R., Coudrat, T., Sexton, P. M., Christopoulos, A., and Canals, M. (2015) Biased Agonism of Endogenous Opioid Peptides at the mu-Opioid Receptor, Mol Pharmacol 88, 335-346. 33. Li, T., Fujita, Y., Tsuda, Y., Miyazaki, A., Ambo, A., Sasaki, Y., Jinsmaa, Y., Bryant, S. D., Lazarus, L. H., and Okada, Y. (2005) Development of potent mu-opioid receptor ligands using unique tyrosine analogues of endomorphin-2, J Med Chem 48, 586-592. 34. Sasaki, Y., Sasaki, A., Niizuma, H., Goto, H., and Ambo, A. (2003) Endomorphin 2 analogues containing Dmp residue as an aromatic amino acid surrogate with high mu-opioid receptor affinity and selectivity, Bioorg Med Chem 11, 675-678. 35. Liu, X., Wang, Y., Xing, Y., Yu, J., Ji, H., Kai, M., Wang, Z., Wang, D., Zhang, Y., Zhao, D., and Wang, R. (2013) Design, synthesis, and pharmacological characterization of novel endomorphin-1 analogues as extremely potent mu-opioid agonists, J Med Chem 56, 3102-3114. 36. Sakurada, S., Watanabe, H., Hayashi, T., Yuhki, M., Fujimura, T., Murayama, K., Sakurada, C., and Sakurada, T. (2002) Endomorphin analogues containing D-Pro2 discriminate different mu-opioid receptor mediated antinociception in mice, Br J Pharmacol 137, 1143-1146. 37. Keresztes, A., Borics, A., and Toth, G. (2010) Recent advances in endomorphin engineering, ChemMedChem 5, 1176-1196. 38. Le Bars, D., Gozariu, M., and Cadden, S. W. (2001) Animal models of nociception, Pharmacol Rev 53, 597-652. 39. Wang, Y., Liu, X., Wang, D., Yang, J., Zhao, L., Yu, J., and Wang, R. (2015) Endomorphin-1 analogues (MELs) penetrate the blood-brain barrier and exhibit good analgesic effects with minimal side effects, Neuropharmacology 97, 312-321. 40. Kastin, A. J., and Pan, W. (2010) Concepts for biologically active peptides, Curr Pharm Des 16, 3390-3400. 41. Higgs, J., Wasowski, C., Loscalzo, L. M., and Marder, M. (2013) In vitro binding affinities of a series of flavonoids for mu-opioid receptors. Antinociceptive effect of the synthetic flavonoid 3,3-dibromoflavanone in mice, Neuropharmacology 72, 9-19. 42. Reichert, J. A., Daughters, R. S., Rivard, R., and Simone, D. A. (2001) Peripheral and preemptive opioid antinociception in a mouse visceral pain model, Pain 89, 221-227. 43. Szeto, H. H., Soong, Y., Wu, D., Qian, X., and Zhao, G. M. (2003) Endogenous opioid peptides contribute to antinociceptive potency of intrathecal [Dmt1]DALDA, J Pharmacol Exp Ther 305, 696-702. 44. Collett, B. J. (1998) Opioid tolerance: the clinical perspective, Br J Anaesth 81, 58-68. 45. Schneider, J. P., and Kirsh, K. L. (2010) Defining clinical issues around tolerance, hyperalgesia, and addiction: a quantitative and qualitative outcome study of long-term opioid dosing in a chronic pain practice, J Opioid Manag 6, 385-395. 46. Zadina, J. E., Nilges, M. R., Morgenweck, J., Zhang, X., Hackler, L., and Fasold, M. B. (2016) Endomorphin analog analgesics with reduced abuse liability, respiratory depression, motor 35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

impairment, tolerance, and glial activation relative to morphine, Neuropharmacology 105, 215-227. 47. Swegle, J. M., and Logemann, C. (2006) Management of common opioid-induced adverse effects, Am Fam Physician 74, 1347-1354. 48. Benyamin, R., Trescot, A. M., Datta, S., Buenaventura, R., Adlaka, R., Sehgal, N., Glaser, S. E., and Vallejo, R. (2008) Opioid complications and side effects, Pain Physician 11, S105-120. 49. Bagnol, D., Mansour, A., Akil, H., and Watson, S. J. (1997) Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract, Neuroscience 81, 579-591. 50. Fickel, J., Bagnol, D., Watson, S. J., and Akil, H. (1997) Opioid receptor expression in the rat gastrointestinal tract: a quantitative study with comparison to the brain, Brain Res Mol Brain Res 46, 1-8. 51. Pupo, A. S., Duarte, D. A., Lima, V., Teixeira, L. B., Parreiras, E. S. L. T., and Costa-Neto, C. M. (2016) Recent updates on GPCR biased agonism, Pharmacol Res 112, 49-57. 52. Jacobson, K. A. (2015) New paradigms in GPCR drug discovery, Biochem Pharmacol 98, 541-555. 53. Thompson, G. L., Kelly, E., Christopoulos, A., and Canals, M. (2015) Novel GPCR paradigms at the mu-opioid receptor, Br J Pharmacol 172, 287-296. 54. Siuda, E. R., Carr, R., 3rd, Rominger, D. H., and Violin, J. D. (2017) Biased mu-opioid receptor ligands: a promising new generation of pain therapeutics, Curr Opin Pharmacol 32, 77-84. 55. Manglik, A., Lin, H., Aryal, D. K., McCorvy, J. D., Dengler, D., Corder, G., Levit, A., Kling, R. C., Bernat, V., Hubner, H., Huang, X. P., Sassano, M. F., Giguere, P. M., Lober, S., Da, D., Scherrer, G., Kobilka, B. K., Gmeiner, P., Roth, B. L., and Shoichet, B. K. (2016) Structure-based discovery of opioid analgesics with reduced side effects, Nature 537, 185-190. 56. Bohn, L. M., Gainetdinov, R. R., Lin, F. T., Lefkowitz, R. J., and Caron, M. G. (2000) Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence, Nature 408, 720-723. 57. Bohn, L. M., Lefkowitz, R. J., and Caron, M. G. (2002) Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice, J Neurosci 22, 10494-10500. 58. Pinyot, A., Nikolovski, Z., Bosch, J., Segura, J., and Gutierrez-Gallego, R. (2010) On the use of cells or membranes for receptor binding: growth hormone secretagogues, Anal Biochem 399, 174-181. 59. Gusovsky, F. (2001) Measurement of second messengers in signal transduction: cAMP and inositol phosphates, Curr Protoc Neurosci Chapter 7, Unit7 12. 60. Liu, H. M., Liu, X. F., Yao, J. L., Wang, C. L., Yu, Y., and Wang, R. (2006) Utilization of combined chemical modifications to enhance the blood-brain barrier permeability and pharmacological activity of endomorphin-1, J Pharmacol Exp Ther 319, 308-316. 61. Gillespie, T. J., Konings, P. N., Merrill, B. J., and Davis, T. P. (1992) A specific enzyme assay for aminopeptidase M in rat brain, Life Sci 51, 2097-2106. 62. Wang, Y., Zhou, J., Liu, X., Zhao, L., Wang, Z., Zhang, X., Wang, K., Wang, L., and Wang, R. (2017) Structure-constrained endomorphin analogs display differential antinociceptive mechanisms in mice after spinal administration, Peptides 91, 40-48. 63. Liu, X., Zhao, L., Wang, Y., Mou, L., Zhang, Y., Zhou, J., and Wang, R. (2016) Design and 36


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Synthesis of a Novel Series of Opioid Dipeptides and Evaluation of Their Analgesic Effect in Vivo, Acta Chim. Sinica 77, 44-48. 64. Suardiaz, M., Estivill-Torrus, G., Goicoechea, C., Bilbao, A., and Rodriguez de Fonseca, F. (2007) Analgesic properties of oleoylethanolamide (OEA) in visceral and inflammatory pain, Pain 133, 99-110. 65. Matsumoto, K., Takayama, H., Narita, M., Nakamura, A., Suzuki, M., Suzuki, T., Murayama, T., Wongseripipatana, S., Misawa, K., Kitajima, M., Tashima, K., and Horie, S. (2008) MGM-9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxy indolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate], a derivative of the indole alkaloid mitragynine: a novel dual-acting mu- and kappa-opioid agonist with potent antinociceptive and weak rewarding effects in mice, Neuropharmacology 55, 154-165. 66. Raffa, R. B., Mathiasen, J. R., and Jacoby, H. I. (1987) Colonic bead expulsion time in normal and mu-opioid receptor deficient (CXBK) mice following central (ICV) administration of mu- and delta-opioid agonists, Life Sci 41, 2229-2234. 67. McGeehan, A. J., and Olive, M. F. (2003) The anti-relapse compound acamprosate inhibits the development of a conditioned place preference to ethanol and cocaine but not morphine, Br J Pharmacol 138, 9-12. 68. Hamm, R. J., Pike, B. R., O'Dell, D. M., Lyeth, B. G., and Jenkins, L. W. (1994) The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury, J Neurotrauma 11, 187-196.

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Graphical Table of Contents

MEL-N1606 produces good analgesic activity and a favorable side effect profile

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MEL-N16: A Series of Novel Endomorphin Analogs with Good

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