Modulating β-arrestin 2 recruitment at the δ- And μ-opioid receptors using peptidomimetic ligands

Article abstract of DOI:10.1039/d1md00025j

μ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional μ/δ opioid agonists, such a

Full text of DOI:10.1039/d1md00025j

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This article can be cited before page numbers have been issued, to do this please use: K. K. Sharma, R. J.
Cassell, Y. J. Meqbil, H. Su, A. Blaine, B. R. Cummins, K. Mores, D. K. Johnson, R. van Rijn and R. A. Altman,
RSC Med. Chem., 2021, DOI: 10.1039/D1MD00025J.
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Modulating  Arrestin-2 Recruitment at the - and -Opioid Receptors Using
Peptidomimetic Ligands
Krishna K. Sharma,1,† Robert J. Cassell,2,† Yazan J. Meqbil,2,7,† Hongyu Su,2 Arryn T. Blaine,2,6
Benjamin R. Cummins,3 Kendall L. Mores,2 David K. Johnson,8 Richard M. van Rijn,2,4,5,‡ Ryan
A. Altman2,3‡
1Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
2
University, Department of Medicinal Chemistry and Molecular Pharmacology, College of
3
4
Pharmacy, Purdue University, Department of Chemistry, Purdue University, Purdue Institute
5
for Drug Discovery, Purdue University, Purdue Institute for Integrative Neuroscience, Purdue
6
University, Purdue Interdisciplinary Life Science Graduate Program, Purdue University,
8
7Computational Interdisciplinary Graduate Program (CIGP), Purdue University, Computational
Chemical Biology Core and Molecular Graphics and Modeling Laboratory, The University of
Kansas
†Contributed equally to this work
‡Corresponding Authors: Ryan A. Altman (raaltman@purdue.edu) and Richard M. van Rijn
(rvanrijn@purdue.edu)
%Electronic Supporting Information (ESI) is available: Experimental details for the synthesis of,
characterization of, and determination of purity for compounds 1b–1g, detailed pharmacological
1

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procedures, additional pharmacological characterization, stability data, computational
procedures, additional computational data and figures.
Keywords: Leu-enkephalin; delta opioid receptor; mu opioid receptor; beta-arrestin; biased
signaling
ORCID: Krishna K. Sharma: 0000-0003-4927-745X, Robert J. Cassell: 0000-0003-4902-3062,
Yazan J. Meqbil: 0000-0001-5801-1608, Kendall Mores: 0000-0002-4605-1722, Arryn T Blaine:
0000-0002-8678-753, Richard van Rijn: 0000-0002-9957-1633, Ryan Altman: 0000-0002-8724-
1098
2

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Abstract
µ Opioid receptors agonists provide potent and effective acute analgesia; however, their
therapeutic window narrows considerably upon repeated administration, such as required for
treating chronic pain. In contrast, bifunctional µ/ opioid agonists, such as the endogenous
enkephalins, have potential for treating both acute and chronic pain. However, enkephalins
recruit β-arrestins, which correlate with certain adverse effects at µ- and -opioid receptors.
Herein, we identify the C-terminus of Tyr-ψ[(Z)CF=CH]-Gly-Leu-enkephalin, a stable
enkephalin derivative, as a key site to regulate bias of both δ- and µ-opioid receptors. Using in
vitro assays, substitution of the Leu5 carboxylate with amides (NHEt, NMe2, NCyPr) reduced β-
arrestin recruitment efficacy through both the δ- and µ-opioid, while retaining affinity and cAMP
potency. For this series, computational studies suggest key ligand-receptor interactions that
might influence bias. These findings should enable discovery of a range of tool compounds with
previously unexplored biased µ/ opioid agonist pharmacological profiles.
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Introduction
Though µOR (μ-opioid receptor) agonists are highly effective analgesics, particularly in
acute and subacute peri-operative settings, they are not recommended for treating chronic pain
due to concerns about analgesic tolerance, as well as an increase in likelihood and severity of
adverse µOR-mediated side effects, including constipation, dependence and respiratory
depression.1 Additionally, activation of the µOR is less able to overcome the adaptive changes
that occur in patients suffering from chronic pain. In sharp contrast to µOR agonism, δOR (δ-
opioid receptor) agonism less effectively induces acute analgesia,2 though δOR agonists display
utility in chronic pain settings, including migraine, inflammatory and neuropathic pain.2–5
However, there are currently no FDA-approved δOR agonists, in part because δOR agonists have
the potential for inducing seizures.6 If the µOR and δOR adverse effects could be reduced or
avoided, a bifunctional µOR/δOR agonist could produce analgesia for acute and chronic pain.7
Such an agonist could be useful in patients suffering from chronic pain, particularly for patients
with cancer or arthritis who experience episodes of breakthrough pain8–10
.
The exact mechanisms for the described µOR and δOR-related adverse effects have been
an issue of debate. First, recruitment of β-arrestin (-Arr) 2 has been hypothesized as possible
underlying cause for both δOR-induced seizures, as well as µOR-induced respiratory depression,
constipation, and the development of analgesic tolerance.11–13 -Arr 2 KO mice have been
reported to have diminished µOR side effects14 or no impact,15 while phosphorylation-deficient
mutant µORs also still exhibit µOR side effects, with the exception of tolerance.16 The
therapeutic window for µOR agonists has been positively correlated with G-protein bias, 17,18 but
a recent study argued that the correlation was driven by G-protein signaling efficacy rather than
bias.19 This study has spurned an alternate hypothesis that gives more significance to partial
4

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agonism especially when receptor reserve is limited.20,21 A third hypothesis emphasizes
intracellular Golgi signaling as a contributor to µOR-induced adverse effects, which is
minimized with peptide-based agents.22 According to these concepts, the creation of peptidic
opioids, that do not readily activate intracellular ORs and that exhibit reduced efficacy for both
-Arr 2 and G-protein signaling, may provide antinociception with improved therapeutic
windows. Some µOR/δOR bifunctional agonists indeed display antinociception with reduced
tolerance, dependence, locomotor activation and self-administration relative to classical
morphinans.7,23–25 Though thus far, development of these µOR/δOR dual agonists has largely
ignored β-Arr 2 recruitment, which makes it impossible to predict the contribution of low β-Arr
2 recruitment to the reduced side effect profile in the context of dual agonism of µOR/δOR. In a
single exception, UFP-505, a µOR/δOR dual agonist, activates β-Arr 2 through the µOR, but
underrecruits β-Arr 2 at δOR, and also only exhibits partial agonist G-protein activity at δOR.26
Thus, µOR/δOR dual agonists with a range of well-characterized β-Arr 2 profiles are essential to
validate µOR/δOR dual agonism as a desired pharmacological profile.
Peptides have historically served as ligands for studying opioid pharmacology, and in
many cases the rapid and modular synthesis of peptides has enabled the delivery of analogs with
novel profiles. We recently showed that small modifications of Phe4 can alter arrestin
recruitment and µOR/δOR potency and selectivity of Leu5-enkephalin (Leu-Enk, YGGFL), an
endogenous δOR opioid peptide,27 while other δOR pentapeptides exist that display G-protein
biased signaling profiles albeit with low potency.28 As such, derivatization of peptides can
facilitate the study of biased-signaling in relation to desired µOR and/or δOR-mediated
antinociception and undesired adverse effects. Herein, we derivatize the carboxyl-terminal region
of previously reported Leu5-Enk pepidomimetics29,30 with the goal of delivering a set of opioid
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peptides with varying degrees of β-Arr 2 recruitment, in particular with limited µOR β-Arr 2
recruitment, as such compounds remain unidentified. Further, computational modeling points to
key ligand-target interactions that regulate β-Arr 2 recruitment at both receptors, which provides
insight for designing next-generation analogs with precisely tuned pharmacological profiles for
studying antinociceptive potency and adverse effect profiles signal-biased µ/δ opioids.
Figure 1. Designing Leu-Enk Analogs with Decreased β-Arr 2 Recruitment. (A) According to
the classical “Message-Address” model for opioid action, the C-terminal residue regulates opioid
selectivity. By extension of this model, modifications to this position might also regulate bias at
the δOR (cAMP vs. -Arr 2). (B) The present work exploits C-terminal modifications in the
“Address” domain to deliver biased δOR agonists with low -Arr recruitment at both the δOR
and µOR.
Design Considerations: To deliver a series of peptide-based signal-biased δOR/µOR
agonists, we initially explored Leu-Enk, which acts at the OR with 1–5-fold higher binding
affinity over µOR and >1000-fold over OR,31,32 and that has served as a starting point for
decades worth of medicinal chemistry efforts to study OR pharmacology. In a seminal paper
6

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from 1981, Chavkin and Goldstein introduced the “message-address” concept of opioid peptide
binding to opioid receptors.33 According to this model, Tyr1-Gly2-Gly3-Phe4, the common
backbone of Leu5-Enk, Met5-Enk and dynorphin constitute the “message” that to recognizes and
binds to opioid receptors, and that amino acids at the fifth position and beyond contribute to the
“address” portion of the peptide that confers potency and receptor selectivity (Figure 1A).33
Though this hypothesis was developed prior to recognition of opioid-induced β-Arr signaling, we
speculated that the message-address model might apply to the concept of biased ligands,
specifically that C-terminal modifications of Leu5-Enk might reduce β-Arr recruitment potency
at ORs (Figure 1B). In support of this hypothesis, replacement of Leu5 with aza-β-homoleucine
or cycloleucine residues biases signaling toward G-protein coupling at the δOR (2–5 fold bias
factor), though these ligands still overrecruit β-Arr though the µOR,34 which may lead to
undesired adverse effects. Nonetheless, we envisioned that alternate modifications near the C-
terminus might further regulate bias at both the µOR and δOR, specifically by weakening
charged interactions between the anionic C-terminus of the ligand and cationic residues in
receptor and by increasing steric bulk in this region (Figure 2). To explore this hypothesis, we
initiated studies using Leu-enk derivatives bearing the Tyr-ψ[(Z)CF=CH]-Gly substitution that
improves stability, physicochemical and distribution properties relative to the parent peptide,
while still delivering a single digit nanomolar δOR agonist activity (Figure 2).29,30
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Figure 2. C-Terminal Substituted Analogs Synthesized and Pharmacologically Characterized.
Synthesis of Analogs: Analogs were generally prepared using microwave-assisted solution
phase coupling chemistry using a Boc-protection strategy that has previously been demonstrated
to deliver peptides in high purity (Scheme 1).35 C-terminal functionalized tripeptides (3b–d)
were accessed from the corresponding methyl esters (2a). To access compounds 3b–d, reaction
of the amine with the corresponding ester afforded the tripeptides in suitable yields, though these
conditions did not afford bulkier intermediates 3e–f. Thus to access 3e–f, we reacted the amines
with the corresponding acid (2b) using coupling with N,N’-diisopropylcarbodiimide (DIC) and
N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) under microwave (MW)
irradiation. These conditions also effectively coupled Boc–Gly–Phe–OH (4) with N-Piperidine-
4-N(Ph)(COEt) (6) to afford 3g. These tripeptides were deprotected using HCl in 1,4-dioxane,
then coupled on to Tyr-ψ[(Z)CF=CH]-Gly–OH29,30 using DIC/HONB with N,N-
diisopropylethylamine (DIEA) under MW irradiation and subsequently deprotected (Scheme
1B). Purification by reversed phase HPLC provided analytically pure samples for
pharmacological evaluation.
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A) Preparation of C-Terminal Tripeptide
iPr
iPr
O
O
O
H
N
(a)
H
OMe
N
X
BocHN
N
H
BocHN
N
H
Amines: NH4OH,
CH3NH2, C2H5NH2
O
O
O
Ph
Ph
2
3b (X = NH2)
3c (X = NHMe)
3d (X = NHEt)
iPr
O
iPr
O
O
O
H
N
(b)
H
N
OH
X
BocHN
N
H
BocHN
N
H
Amines: NHMe2,
Cyclopropylamine
O
O
Ph
Ph
2
3e (X = NMe2)
3f (X = NHCyPr)
O
H
Me
O
Me
O
H
N
N
(c)
O
BocHN
N
BocHN
OH
+
O
N
N
O
HN
Ph
Ph
Ph
4
Ph
3g
6
B) Coupling of C-Terminal and N-Terminal Fragments
1a (X = OH)
1b (X = NH2)
OH
(d), 3b–g (e), then (d)
1c (X = NHMe)
1d (X = NHEt)
1e (X = NMe2)
1f (X = NHCyPr)
1g [N-Piperidine-4-
N(Ph)(COEt)]
CO2H
BocHN
F
7
Scheme 1. Synthesis of Analogs 1a–g. Reagents and Conditions: (a) Amine : MeOH (1:1), rt, 14
h; (b) Amine, DIC, HONB, DMF, 60 °C, 30 min, MW; (c) DIC, HONB, DMF, 60 °C, 30 min,
MW; (d) 4N-HCl in 1,4-Dioxane, 15 °C, 30 min; (e) DIC, HONB, DIEA, DMF, 60 °C, 30 min,
MW.
Results and Discussion
C-terminal substitution of Tyr-ψ[(Z)CF=CH]-Gly-Leu-Enk with various alkyl amides (Figure
2) delivered a series of compounds with sub-µM binding affinities at both OR and µOR (Table
1), G-protein coupling activities comparable to the parent carboxylate 1a (Figure 3A,C), and
interestingly demonstrating a range of -Arr recruitment activities with clear structure-function
trends (Figure 3B,D).
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Table 1. Binding Affinities at δOR and µOR for C-Terminal Analogs of Tyr-ψ[(Z)CF=CH]-Gly-
Leu-Enk
pKi±SEM
(δOR)
Ki (nM)
pKi±SEM
(µOR)
Ki (nM)
Binding Selectivity
(δOR vs µOR)
Compound
1a (O–)
1b (NH2)
1c (NHMe)
1d (NHEt)
7.59±0.2
7.03±0.2
7.25±0.1
7.26±0.1
6.59±0.1
6.99±0.1
6.43±0.1
8.95±0.1
25.6
94.4
55.9
7.37±0.1
8.15±0.1
8.00±0.2
7.70±0.1
7.07±0.1
7.58±0.2
6.43±0.1
8.69±0.1
42.7
7.07
9.92
20.0
85.4
26.1
368.1
2.07
1.7
0.1
0.2
0.4
0.3
0.3
1.0
1.8
54.7
1e (NMe2)
255.1
103.5
372.4
1.12
1f (NHCyPr)
1g [Pip-N(Ph)(COEt)]
Leu5-Enk
Using standard competition radioligand binding assays and [3H]DPDPE or [3H]DAMGO as
radioligands, C-terminal substituted analogs 1b–f engaged both the OR and µOR within an
order of magnitude of parent compound 1a, with bulky analog 1g binding with slightly lower
affinities (Table 1). However, a clear trend emerged with analogs bearing at least one H-bond
donor-acceptor pair (e.g. NH2, NHMe, NHEt, NHCyPr; 1b–d, f) possessing better binding
affinities relative to analogs bearing bulky NMe2 and Pip-N(Ph)(COEt) (1e, 1g) substituents.
Further, analogs 1b–1f bearing C-terminal amides preferentially bound to the µOR (selectivities:
0.1–0.4), which contrasts the parent analogs and Leu-Enk that preferentially bound to the OR
(selectivities: 1.7–1.8), or analog 1g that bound to the two receptors with equal affinities (1.0–
1.2).
Despite these binding trends, analogs 1b–g activated both the OR and µOR with within an
order of magnitude of the potency as the parent using the GloSensor assay (Table 2). In general,
the potency for the peptides to recruit -Arr 2 at OR was 10-fold lower than for the peptides to
inhibit cAMP at OR (Table 2), which matches previous findings.27 Despite their similar binding
profiles (Table 1) and potencies inhibiting cAMP (Figure 3A,C), the bulky C-terminal
substituted enkephalin peptides weakly recruited -Arr 2 at OR and µOR (Table 2, Figure
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3B,D). Most notably, increasing bulk at the C-terminus decreased -Arr 2 recruitment efficacies
(EMax) at OR, specifically ~70% for NHEt (1d) and NHCyPr (1f), and 62% for NMe2 (1e).
Strikingly, this decrease was even more pronounced at µOR than at OR 1d (47%), 1f (27%), 1e
(26%). Yet larger substituents, such as Pip-4-N(Ph)(COnPr) (1g), which previously provided a
potent and selective analog of Leu-Enk,36 followed the same trend, and actually delivered an
analog with no detectable -Arr 2 efficacy at µOR (Figure 3D, Table 2). Such decreases in -Arr
2 efficacy may have beneficial in vivo properties, because low arrestin efficacy, especially when
paired with partial agonism at the G-protein pathway should provide consistently low in vivo
adverse effects.17,19 More so, such low -Arr 2 efficacy should be preferred relative to calculated
bias factors (Table 2), because in vitro-determined bias factors are linked to context (e.g. cell and
assay systems/endpoints), overvalue the contribution of potency in their calculation, and are
difficult to translate to in vivo outcomes as bias scores do not factor in pharmacokinetic or
pharmacodynamic (particularly ligand residence time) parameters.37,38 Overall, these structure-
function trends clearly indicate that peptides can effectively separate G-protein coupling and -
Arr 2 recruitment at both OR and µOR through shifts in efficacy, which can facilitate discovery
of tool compounds to investigate optimal biased pharmacology for bifunctional OR/µOR
agonists.
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Figure 3. C-Terminal Modifications Delivered OR agonists with Varying Levels of -Arr 2
Recruitment at δOR and µOR. (A) Inhibition of cAMP Production at OR; (B) -Arr 2
Recruitment at δOR; (C) Inhibition of cAMP Production at µOR; (D) -Arr 2 Recruitment at
µOR. Legend: o = Leu-Enk (control); ▲ = 1a (O–), ● = 1d (NHEt), ■ = 1f (NHCyPr), ▼ = 1g
[N-Pip-N(Ph)(COEt)]. Each concentration was tested as technical triplicate (cAMP) or duplicate
(-Arr 2), and a minimum of three biological independent replicate dose-response curves were
produced for each agonist. Data is normalized to Leu-Enk and for each agonist a composite was
produced from the average of the dose response curves of the replicate assays. The error bars
depict the standard error of the mean.
Table 2. G-protein Coupling Activities and -Arr Recruitment Profiles for C-Terminal Analogs
of Tyr-ψ[(Z)CF=CH]-Gly-Leu-Enk.
δOR
-Arr 2
µOR
-Arr
cAM
P
IC50
(nM)
cAMP
pIC50
SEM
cAMP
IC50
(nM)
cAMP
pIC50
SEM
-Arr 2
pEC50
SEM
-Arr 2
EMax
%±SEM
-Arr 2
pEC50
SEM
-Arr 2
EMax
%+SEM
Compound
Bias
Bias
±
±
±
EC50
(nM)
±
EC50
(nM)
Factor*
Factor*
–
7.47±0.2
7.33±0.1
7.30±0.3
33.7
46.6
50.2
6.12±0.1
6.02±0.1
6.18±0.1
764
959
667
102±4
84±14
90±13
1.2
3.6
1
6.40±0.1
6.73±0.1
7.37±0.2
363
186
42.7
4.49±0.1
5.58±0.1
5.14±0.2
31999
2644
7215
90±10
92±7
92±2
0.4
0.2
0.9
1a (O )
1b (NH2)
1c (NHMe)
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1d (NHEt)
1e (NMe2)
1f (NHCyPr)
7.18±0.1
6.37±0.1
6.82±0.2
6.87±0.2
8.97±0.1
66.2
425
152
134
1.07
6.16±0.1
5.42±0.1
6.16±0.2
5.41±0.1
7.99±0.1
695
3817
693
70±6
63±7
69±10
73±3
100
3.9
0.6
1.4
1.5
1
6.75±0.2
6.00±0.2
6.90±0.2
6.74±0.2
7.70±0.2
178
1000
126
5.65±0.1
4.75±0.1
5.37±0.2
ND
2265
17640
4256
ND
48±5
26±4
27±1
ND
0.6
2.2
1.9
ND
1
1g [Pip-
N(Ph)(COEt)]
3926
10.2
183
Leu5-Enk
20.0
5.89±0.1
1274
100
ND = Not Detected. * A bias factor > 1 indicates that a compound is G-protein biased, while a
bias factor < 1 indicates that a compound is β-arrestin biased. Except for 1g, which displayed
partial agonism at µOR, all compounds displayed full agonist activity at OR and µOR.
Modeling at the OR: The recently published OR crystal structures in their active-like
conformations (peptide-bound: 6PT2 and small molecule-bound: 6PT3)39 provide a good starting
point to better understand possible binding modes of the reported ligands and how they might
engage the ligand binding pocket of the OR. However, first, the relatively low-resolution of the
structures as well as the presence of nine thermostabilizing mutations necessitated advanced
preparation of the model structures (see SI 1 for details). Nonetheless, with appropriate model
optimization, these OR crystal structures were used to further understand the conformational
changes associated with biased agonism at OR. Molecular modeling was performed using the
Schrödinger Suite (Schrödinger, Inc., NY, USA; See SI 1 for details). Molecular docking based
on the 2.8Å crystal structure of the peptide agonist-bound OR (PDB: 6PT2)39 though the
thermostabilizing mutation D108(2.63) was reverted to the WT K108(2.63), as this mutation
minimally effected cAMP inhibition and binding affinity of the crystallized peptide, KGCHM07,
but decreased β-Arr 2 recruitment.39 Moreover, based on preliminary modeling (not shown), we
predicted that the existence of potential interactions involving the C-terminus with the
hydrophobic pocket formed between residues in TM2, TM3 and ECL2, and Phe4 with residues in
ECL3, TM6 and TM7, necessitated the use of the WT K108(2.63). Other thermostabilizing
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mutations in the crystal structure were embedded deeper into the orthosteric binding site of OR
near the sodium allosteric binding site, so they were not expected to directly engage in binding
interactions with the C-terminus of Leu-Enkephalin analogs.39 The tyraminium moiety of the
peptide agonist KGCHM0739 was used as a common scaffold for the initial docking of the Leu-
Enkephalin analogs (Figure 4A). Additionally, our model retained two crystallized water
molecules that maintain a polar network involving Y129(3.33), K214(5.40), H278(6.52), and which
enabled the tyraminium portion of the analogs to retain key interactions deep in the binding
pocket, while allowing for the flexible docking near the C-terminal modifications of the docked
analogs (Figure 4B).
In general, most docked analogs retained a similar alignment of the Gly2–Gly3 backbone,
featured hydrogen bonding interactions with K108(2.63) and R291ECL3, and engaged in -stacking
interactions between Phe4 and W284(6.58) (Figures 4B,C, SI-1 Figures 1 & 3). These docked
poses were further supported by an all-atom, 200 ns MD simulation using Desmond (see SI-1 for
further details). Analog 1a (O–) bound to the model OR structure in a fashion that retained most
of the reported interactions with residues K108(2.63), R291(ECL3) and W284(6.58) (Figure SI-5).39
However, the Phe4 residue of analog 1a (O–) formed a CAryl-H interaction with W284(6.58) instead
of the expected - interaction largely due to the insufficient rotation of the side chain of
W284(6.58) (Figure 4B).39 Analogs 1b–1g showed -stacking interactions for which the rotation
of the W284(6.58) side chain enabled the benzyl moiety to access a hydrophobic pocket between
TM6, TM7 and ECL3 (SI-1 Figures SI-1 & SI-3). Furthermore, Leu-Enkephalin analog 1a (O–)
was embedded further into the binding pocket relative to the crystallized peptide, KGCHM07,
while forming two - interactions with W274(6.48) and Y308(7.42), possibly due to the presence
of the thermostabilizing mutation S131(3.35) in the sodium binding site (Figure SI-6). Near the C-
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terminus of compound 1a, R291ECL3 appeared to facilitate an interaction between Phe4 and
W284(6.58) via a - interaction, as well as a water-mediated H-bond with C198(45.50). Future
studies using optimized and dynamic model structures of the OR will further deduce the role
mediated by the C-terminal modifications of peptide agonists at OR.
Based on our docking model at the OR, C-terminal modifications of peptide agonists may
underrecruit β-Arr 2 through two potential interactions. First, in the C-terminal groups of the
docked analogs mainly interacted with K108(2.63), W284(6.58), R291ECL3. Of the three residues,
R291ECL3 has been reported to act as a mediator for peptide selectivity at opioid receptors, by
helping position W284(6.58) to engage naltrindole with a - interaction.40 Similarly,
modifications of the C-termini of analogs 1a–1g modulated the interactions of peptide agonists
with K108(2.63)/W284(6.58)/R291ECL3, and specifically, we speculate that the perturbation of this
interaction network may induce conformational changes in TM6/7 and ECL3 with implications
on arrestin recruitment, through biophysical and pharmacological experiments to probe this
hypothesis are beyond the scope of the present manuscript. Second, the Leu5 side chain fits
within a narrow hydrophobic pocket in the δOR involving K108(2.63) and W114(23.50), and in this
region, analogs with decreased β-Arr 2 efficacies have poor overlap with the docked pose of 1a
(Figure 4C). We hypothesize that the different orientations of the Leu5 side chain might arise
from increased steric bulk at the C-terminus that pushes the side chain out of its energetically
favorable orientation, which is also supported by previous studies in which substitution of the
Leu5 side chain also modulates β-Arr 2 efficacy.34
Modeling at the µOR: Further modeling of peptide-µOR interactions using morphinan
agonist BU72 bound 2.1 Å mouse µOR crystal structure (PDB: 5C1M)41 provided possible
interactions that could rationalize the decreased β-Arr 2 recruitment efficacy imparted by C-
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terminal modifications (Figure 4D; See SI for details on modeling methodology). However, the
flexibility of the docked peptides in this model, especially in the presence of the crystallized
water molecules, could be addressed in future studies using dynamic structures to further probe
key interactions. In the docked pose of compound 1a, the ligand engages multiple residues on
TM3, while the C-terminal carboxylate engages both K233(5.40) and K303(6.58) in favorable
charged interactions (Figures 4E). Near the C terminus, the Phe4 side chain resides in a
hydrophobic pocket composed of L219ECL2 and F221ECL2 and the Leu5 side chain presents
towards solvent. Using enhanced sampling modeling of analogs 1a–g, the conversion of the
ligand’s charged C-terminus to neutral amides resulted in similar binding poses, with different
interactions with between the Leu5-amide and K303(6.58) (Figure 4F, also See SI 1). Notably,
though some C-terminal amides interact with K303(6.58), none of the analogs engage the
TM2/ECL3 region of the receptor, which make key interactions in the docked models of the
OR. Moreover, no systematic major conformational differences within the binding pocket or
rotational differences around the Phe-piperidine amide bond correlated with changes to β-Arr 2
or cAMP efficacies.42–44
Considering this model, the interactions between the ligand’s C-terminus and K233(5.40) and
K303(6.58) might be critical to modulating β-Arr 2 recruitment, as molecular modeling of a
macrocyclic peptide with strong β-Arr 2 recruitment was shown to move TM5 and TM6 inward,
whereas G-protein biased peptides did not encourage an inward movement of these μOR
helices.43 Further, though several ligands, including BU72, morphine, DAMGO, and fentanyl
make strong interactions with D147(3.32), Y148(3.33), and Y326(7.43) 42,43,45–47
,
these interactions
don’t seem to correlate with ligand bias. Hence, the design of peptide agonists that gain affinity
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from interactions with TM3, TM7, but that perturb the positioning of TM5 and TM6 could lead
to the modulation of β-arrestin bias.
Figure 4. Molecular Docking into a Model Based on δOR (PDB: 6PT2) and µOR (PDB: 5C1M).
(A) Selected docked pose of analog 1a (yellow) aligned on the crystal pose of KGCHM07
(orange); PDB 6PT2 crystal structure (cyan ribon) and docked structure starting with 6PT2
(yellow ribbons). The red circle encompasses the common scaffold selected for initial docking.
(B) At the C-terminal, analog 1a (O–) forms hydrogen bonds with K108(2.63) and R291(ECL3) and
CAryl–H- interaction with W284(6.58). R291(ECL3) appears to mediate the key interactions between
the C-termini of the Leu-Enk analogs and δOR. (C) Larger C-terminal substituents push the
respective Leu5 side chains or N-Pip-N(Ph) moiety further into a narrow hydrophobic pocket.
Notably, for 1h, the N(Ph) group presents toward L200(45.52) and F202(ECL2) (the pocket in which
the Leu5 side chains reside) and the COEt group presents towards unoccupied space and does not
make constructive interactions with the receptor. (D) Selected docked pose of analog 1a aligned
on the crystal pose of BU70 (green), PDB 5C1M crystal structure (green). and docked structure
starting with 5C1M (yellow ribbons). The red circle encompasses the common scaffold selected
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for initial docking. (E) In the µOR model, analog 1a forms a hydrogen bond with K303(6.58), but
does not appear to interact with N127(2.63) or with residues in ECL3. Furthermore, the pocket is
more open near the C-terminal region and the Leu5 side chain of the analogs. (F) Aligned poses
of analogs docked into µOR. See SI for more details. Compounds Depicted: 1a (O–) yellow, 1d
(NHEt) purple, 1f (NHCyPr) cyan, 1g [N-Pip-N(Ph)(COEt)] pink. Receptor side chains in panels
B, E are depicted in white.
Conclusion
Overall, the experimental data and computational modelling identify the Leu5 C-terminus of
Leu-Enk as a key site to regulate -Arr 2 recruitment through both the δOR and µOR, which
provides an important benchmark for µ/δOR agonists for which data for -Arr 2 recruitment is
generally unavailable. Nonetheless, no previous analogs have been reported that display
decreased efficacy at -Arr 2 at both the δOR and µOR, and thus future in vivo characterization
of improved Leu5 analogs will provide broader understanding of how biased signaling at
µOR/δOR cooperatively impact nociception and side effect profiles. Considering the excellent
stability imparted by the Tyr-ψ[(Z)CF=CH]-Gly substitution29 (See SI), these C-terminal
substituted Leu-Enk analogs provide excellent leads for further optimization to deliver biased
ligands for the δOR for treating pain. By combining such C-terminal modifications with other
structural modifications that improve δOR/µOR potency and/or selectivity, it should be possible
to develop a range of tool compounds for thoroughly investigating δOR/µOR dual agonists and
δOR-selective agonists with low -Arr recruitment efficacies. Testing such future analogs with
well-defined -Arr profiles side-by-side in models of chronic pain, particularly in a design that
includes repeated administration, may help validate the utility of metabolically stabile signal-
biased δOR/µOR agonists.
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EXPERIMENTAL PROCEDURES
Synthesis of Peptides: Analogs were synthesized using a microwave synthesizer using a
solution-phase protocol using Boc chemistry and 4N HCl in 1,4-dioxane for deprotection.35
Purification and determination of the purity of final compounds was conducted using reversed
phase chromatography using appropriate gradients.
Pharmacological Characterization: As previously described,27 we assessed binding affinity
using a competition radioligand binding assay, G protein potency and efficacy using a cAMP
GloSensor assay, and β-Arr 2 recruitment via PathHunter assays at both δOR and µOR, using
Leu-Enk as the reference compound. A minimum of three independent values were obtained for
each compound in each of the cellular assays. Bias factors were calculated using the operational
model equation in Prism 8 [Log R (τ/KA)] as previously described48,49 using Leu5-Enk as
reference compound.
Conflict of Interests
The authors declare no competing financial interests.
Acknowledgments
R.M.vR. is supported by funds provided by the National Institute on Alcohol Abuse and
Alcoholism (AA025368, AA026949, AA026675) and Drug Abuse (DA045897) of the National
Institutes of Health, a National Center for Advancing Translational Sciences ASPIRE Reduction-
to-Practice Challenge prize, the Purdue Institute for Drug Discovery and the Department of
Medicinal Chemistry and Molecular Pharmacology. R.A.A. acknowledges the National Institute
on Drug Abuse (DA036730) and National Institute of General Medical Sciences (GM124661)
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for support. NMR Instrumentation was provided by the National Institutes of Health
(S10OD016360, S10RR024664, P20GM103418), and National Science Foundation (9977422
and 0320648). The content herein is the sole responsibility of the authors and does not
necessarily represent the official views of the US National Institutes of Health. We thank Dr.
Suvajit Koley for analytical analysis of the peptides.
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