Novel Dimethyltyrosine-Tetrahydroisoquinoline Peptidomimetics with Aromatic Tetrahydroisoquinoline Substitutions Show in Vitro Kappa and Mu Opioid Receptor Agonism

Article abstract of DOI:10.1021/acschemneuro.9b00250

The dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold was originally developed in the production of selective delta opioid receptor (DOR) antagonists. Installation of a 7-benzyl pendant on the tetrahydroisoquinoline core of this classic opioid scaffold introduced kappa opioid receptor (KOR) agonism. Further modification of this pendant resulted in retention of KOR agonism and the addition of mu opioid receptor (MOR) partial agonism, a bifunctional profile with potential to be used in the treatment of cocaine addiction.

Full text of DOI:10.1021/acschemneuro.9b00250

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Article
Novel Dimethyltyrosine-Tetrahydroisoquinoline Peptidomimetics
with Aromatic Tetrahydroisoquinoline Substitutions
Show In Vitro Kappa and Mu Opioid Receptor Agonism
Deanna Montgomery, Jessica P. Anand, Nicholas Griggs, Thomas J. Fernandez, Joshua G
Hartman, Ashley A Sanchez-Santiago, Irina D. Pogozheva, John R. Traynor, and Henry I. Mosberg
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00250 • Publication Date (Web): 14 Jun 2019
Downloaded from http://pubs.acs.org on June 15, 2019
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Novel Dimethyltyrosine-Tetrahydroisoquinoline Peptidomimetics with Aromatic
Tetrahydroisoquinoline Substitutions Show In Vitro Kappa and Mu Opioid Receptor
Agonism
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Deanna Montgomery†, Jessica P. Anand‡,§, Nicholas W. Griggs‡, Thomas J. Fernandez‡, Joshua
G. Hartman‡, Ashley A. Sánchez-Santiago‡, Irina D. Pogozheva†, John R. Traynor‡,§, Henry I.
Mosberg†,§,*
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†Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan
48109 United States
‡Department of Pharmacology, Medical School, University of Michigan, Ann Arbor, Michigan 48109 United
States
§Edward F. Domino Research Center, Medical School, University of Michigan, Ann Arbor, Michigan 48109
United States
*To whom correspondence should be addressed
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Abstract
The dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold was originally developed
in the production of selective delta opioid receptor (DOR) antagonists. Installation of a 7-benzyl
pendant on the tetrahydroisoquinoline core of this classic opioid scaffold introduced kappa opioid
receptor (KOR) agonism. Further modification of this pendant resulted in retention of KOR
agonism and the addition of mu opioid receptor (MOR) partial agonism, a bifunctional profile with
potential to be used in the treatment of cocaine addiction.
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Keywords: opioids, multifunctional ligands, peptidomimetics, cocaine addiction, synthesis,
dimethyltyrosine-tetrahydroisoquinoline
Introduction
The opioid receptors and their associated endogenous ligands were discovered as a result
of early medicinal and recreational use of opium. These receptors are ubiquitously expressed
throughout the brain and peripheral and central nervous systems and are known to be involved
in a variety of biological and physiological processes. Among other functions, the three major
types of opioid receptors – kappa (KOR), mu (MOR), and delta (DOR) - modulate pain,1–3 mood,4–6
reward,7–9 stress,4,10 respiration,11,12 and gastrointestinal function.12,13 Endogenous and
exogenous opioids elicit effects on many of these activities, giving opioids great potential as
therapeutic agents for a wide assortment of indications. As a result, the opioid system has been
intensely studied for several decades, and many semi-synthetic and synthetic opioid ligands have
been developed as therapeutics and chemical tools. The endogenous opioid ligands, discovered
in the 1970s, are peptides, and their structure-activity relationships (SAR) have been well-studied
through the synthesis of numerous peptide and peptidomimetic analogues.1,14–18 As a result of
this work, many novel opioid ligands have been developed. Early progress in the discovery of
synthetic opioids focused on the development of selective ligands for each receptor type, following
the traditional drug discovery convention of using selectivity to minimize unwanted off-target side
effects and creating tools to further elucidate the functions of each opioid receptor.19
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Development of selective DOR antagonists stemmed from modification of the endogenous
dermorphins (MOR agonists) and deltorphins (DOR agonists), the most selective opioid peptides
known in the mid-1990s. Two residues – Tyr1 and Phe3 – of the common N-terminal tripeptide
sequence (Tyr-D-Xaa-Phe) were identified as essential for interaction with the opioid receptors.
Subsequent SAR studies led to the development of ligands without Phe3 that showed opioid
activity. The first of these ligands, including Tyr-Tic-NH2, contained 1,2,3,4-tetrahydroisoquinoline
carboxylate (Tic) and exhibited DOR antagonism.20 Replacement of the tyrosine residue with 2’,6’-
dimethyltyrosine (Dmt) resulted in exceptionally potent and selective DOR antagonists.21 Further
work resulted in the creation of many new DOR antagonists based on the Dimethyltyrosine-
Tetrahydroisoquinoline carboxylic acid (Dmt-Tic) peptide scaffold.22–24
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More recently, the opioid field has shifted from synthesis of selective agonists and
antagonists to multifunctional ligands, leaving room for the development of new generations of
opioids. Because of the complex pharmacology of the opioid system, unwanted effects often
result from the same interaction of an opioid ligand with its target that causes the desired effect.
The development of multifunctional ligands which simultaneously act at multiple opioid receptors
represents one strategy for overcoming such undesired on-target effects.25–28 The development
of multifunctional opioid ligands has recently been reviewed.27 Since relatively minor structural
modifications often result in significant changes in the pharmacological profiles of opioid ligands,
selective scaffolds represent an important starting point for the creation and discovery of
multifunctional compounds.
Through this strategy, Dmt-Tic ligands with other pharmacological profiles have been
developed from this originally DOR antagonist selective scaffold.29–37 The evaluation of Dmt-Tic
ligands with modified C-termini suggests that the DOR selectivity observed in this series is
primarily imparted by the negative charge of the C-terminal carboxylate.38 The removal or
modification of this moiety results in a Dimethyltyrosine-Tetrahydroisoquinoline (Dmt-Tiq) core
useful in the development of multifunctional ligands (Figure 1).
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Figure 1. Dimethyltyrosine -
Tetrahydroisoquinoline (DMT-Tiq)
scaffold with carbon numbering on
the tetrahydroisoquinoline.
Despite the plethora of Dmt-Tiq compounds that have been reported, modifications to this
scaffold have been limited by the constraints of traditional peptide synthesis. As a result,
substitution on the tetrahydroisoquinoline (Tiq) aromatic ring has been largely underexplored. In
2000, Page et. al. reported a series of DOR antagonist Dmt-Tic compounds with substitutions at
the 6-, 7-, and 8-positions of Tiq ranging from hydroxy and methoxy substituents to aryl rings.24
In the same year, Santagada et. al. published Dmt-Tiq compounds with small substituents at the
7-position of Tiq which showed a similar DOR antagonist profile to the standard peptide.39 Overall,
a severely limited range of substitutions has been reported at these positions, and for these
compounds there remain gaps in the knowledge of their binding and efficacy.
Given this gap, we set out to explore the structure-activity relationships between the
aromatic region of Tiq within the Dmt-Tiq scaffold and its opioid profile by synthesizing novel Dmt-
Tiq analogues with substitution on the Tiq aromatic ring.
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Results and Discussion
To investigate the SAR of the tetrahydroisoquinoline aromatic region of the Dmt-Tiq
scaffold, we synthesized novel Dmt-Tiq analogues and investigated their binding and efficacy
profiles at KOR, MOR, and DOR. Binding at each receptor was evaluated by competitive
displacement of [3H]diprenorphine, and efficacy and potency were determined by a [35S]GTPγS
binding assay. Reported percent stimulation values are from comparison to a standard agonist
for each receptor and are used as a surrogate for efficacy. Initial exploration began with synthesis
of analogues with substituents larger than those previously reported.
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A benzyl pendant was installed onto each available carbon of the Tiq aromatic ring of the
DOR antagonist Dmt-Tiq scaffold. Each of these analogues 4a-4d (Table 1) showed single to
triple digit nanomolar binding at each of the opioid receptors. At both KOR and MOR, 4c bound
tightest, followed by 4b and 4d, with 4a having the lowest affinity for these two receptors. The
binding profiles of these compounds at DOR differed from the other receptors, with all analogues
displaying similar single digit nanomolar binding except for 4d, which had much lower affinity for
DOR. None of these compounds displayed agonism at MOR or DOR; however, the efficacy profile
at KOR differed widely. While 4a and 4b displayed no KOR agonism, 4c showed potent, full (85%)
agonism at KOR, and 4d showed weak, partial (46%) agonism at KOR. The compounds which
bind best at KOR, 4c and 4d, also show the highest efficacy at this receptor.
Table 1: Binding, Potency, and Efficacy Data for Benzyl Pendant Dmt -Tiq Analogues 4a-
4d
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O
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N
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Bn
Ki (nM)
EC50 (nM)
% Stimulation
NH2
7
OH
KOR MOR DOR KOR MOR DOR KOR MOR DOR
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4a
4b
4c
4d
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6-Bn
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8-Bn
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(13)
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(5)
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(0.2)
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(5)
9.9
(1.6)
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(0.6)
-
dns
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(0.8)
2.7
(0.8)
6.1
(2.1)
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(6)
85
(8)
26
(5)
4.1
(0.4)
185
(28)
319
(45)
46
(6)
Table 1. Binding affinity (Ki) values determined by competitive displacement of [3H]diprenorphine in membrane
preparations from rat C6 or human CHO cells expressing KOR, MOR, or DOR. Potency (EC50) and efficacy values
determined by [35S]GTPγS binding in the same membrane preparations. Efficacy expressed as percent stimulation
versus standard agonist - U69,593 (KOR), DAMGO (MOR), or DPDPE (DOR). All values expressed as mean (SEM)
of three or more separate assays run in duplicate. Bn = benzyl; dns = does not stimulate, average maximal
stimulation <10% at concentrations up to 10 µM
To our knowledge, 4c is the first reported compound in the classically DOR antagonist
selective Dmt-Tiq series which exhibits high KOR agonist activity. Based on this interesting profile
– potent, full KOR agonism and high opioid receptor binding affinity – we decided to use 4c as a
starting point to further explore the SAR of this series. To investigate steric and electronic effects
of 7-benzyl pendant substitutions on opioid binding and activity, we synthesized analogues of 4c
with various functional groups installed at each position of the phenyl ring. We introduced a methyl
group at each position to test the steric effects of para, meta, and ortho substitutions. To further
probe electronic effects at these positions, we used a nitro group (electron withdrawing, hydrogen
bond accepting) and an amino group (electron donating, hydrogen bond donating). Table 2 shows
binding and efficacy data for substituted 7-benzyl pendant Dmt-Tiq analogues 10a-10i.
Table 2: Binding, Potency, and Efficacy Data for Substituted 7-Benzyl Pendant Dmt -Tiq
Analogues 10a-10i
O
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MOR
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(0.8)
EC50 (nM)
% Stimulation
R
H
KOR
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(0.8)
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(2.1)
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p-Me
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10d
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10f
p-NO2
p-NH2
m-Me
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(4)
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(0.5)
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(10)
3.1
(0.6)
3.1
(0.8)
9.7
(2.4)
2.5
(0.3)
10
(2)
2.1
(0.1)
2.9
(0.3)
3.1
(0.4)
3.0
(0.8)
2.5
(0.7)
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(0.9)
5.8
424
(84)
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(8)
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dns
dns
dns
dns
dns
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(7)
2.7
(0.3)
2.5
(0.5)
28
80
(25)
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(30)
405
(65)
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(7)
32
(6)
471
(56)
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(13)
-
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(5)
76
(6)
24
(3)
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(9)
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(2)
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30
(2)
dns
m-NO2
m-NH2
o-Me
-
dns
(4)
10g
10h
10i
1.2
(0.2)
2.6
(1.0)
25
48
(16)
-
21
(4)
dns
(1.1)
4.7
(1.5)
9.0
o-NO2
o-NH2
116
(40)
27
(4)
(5)
(2.2)
(11)
Table 2. Binding affinity (Ki) values determined by competitive displacement of [3H]diprenorphine in membrane
preparations from rat C6 or human CHO cells expressing KOR, MOR, or DOR. Potency (EC50) and efficacy values
determined by [35S]GTPγS binding in the same membrane preparations. Efficacy expressed as percent stimulation
versus standard agonist - U69,593 (KOR), DAMGO (MOR), or DPDPE (DOR). All values expressed as mean
(SEM) of three or more separate assays run in duplicate. Bn = benzyl; dns = does not stimulate, average maximal
stimulation <10% at concentrations up to 10 µM
Para substitutions (10a-10c) decreased KOR binding and resulted in a loss of KOR
efficacy versus lead compound 4c with no significant gain in MOR or DOR binding or agonism.
Analogues with a meta or ortho substituent (10d-10i) displayed binding profiles similar to that of
parent 4c, mostly in the single digit nanomolar range at KOR, MOR, and DOR, with the notable
exception of meta and ortho amino analogues (10f, 10i) which showed an approximately tenfold
decrease in binding affinity at KOR versus 4c. Overall, however, small meta and ortho
substitutions are accommodated by the orthosteric site of each opioid receptor. Meta substitutions
(10d-10f) all retained some degree of KOR agonism. Methyl and nitro substitutions at the meta
position (10d, 10e) retained high (>75%) KOR efficacy while the amino analogue (10f) displayed
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only partial (24%) KOR agonism. The meta methyl analogue (10d) also displayed partial (30%)
MOR agonism, while the nitro and amino analogues did not. Ortho substitutions (10g-10i) all
retained high (>75%) KOR efficacy, suggesting that small substituents at this position are allowed
by the active conformation of KOR. Methyl (10g) and amino (10i) analogues not only displayed
higher KOR efficacy than lead compound 4c but also introduced partial (21-27%) MOR agonism.
Overall, no strong trend is seen with electron withdrawing or electron donating groups on
the 7-benzyl pendant. As such, the pharmacological profiles of these compounds are likely more
influenced by steric, space-filling effects in the opioid receptor binding sites than specific,
electronic interactions. To further investigate interactions of this series of Dmt-Tiq compounds
with the opioid receptors, we employed computational models similar to those previously
developed40,41 and used42,43 by our group. As expected, for each compound docked, the
protonated nitrogen of Dmt is shown to interact with Asp138 of KOR. Figure 3 shows each of the
methyl-substituted analogues (10a, 10d, 10g) docked in the recent crystal structure of KOR in the
active conformation (PDB ID: 6b73).44 The drastic decrease in KOR binding affinity and efficacy
for para substituted analogues compared to lead compound 4c is likely due to a lack of space in
the KOR binding site. As shown, the para methyl group of 10a causes a steric clash with V118
and W124 in the active configuration of KOR, suggesting that para substitutions are unfavorable
for KOR agonism and binding. However, the meta and ortho methyl groups of 10d and 10g
respectively are directed towards a different area of the binding site where there is space for them
to be accommodated.
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Figure 3. 10a (cyan), 10d (yellow), and 10g (magenta) docked in the orthosteric site of the active conformation
of KOR. Dashed yellow lines represent distance, labeled in angstroms. The para methyl group of 10a sterically
clashes with V118 and W124 of KOR.
Several of the compounds in this series display a multifunctional KOR agonist/MOR partial
agonist profile. Evidence suggests that a ligand with this profile may be useful in the treatment of
cocaine addiction.45–47 It has previously been demonstrated that KOR agonists have the potential
to reduce cocaine self-administration in non-human primates.48,49 However, negative side effects
such as dysphoria are associated with activation of KOR, so the clinical utility of selective KOR
ligands is limited. Since MOR agonists cause euphoria, it has been suggested that partial MOR
agonism may mitigate dysphoria associated with KOR agonism, increasing the therapeutic
potential of a KOR agonist.46,50–52 This work represents progress towards the development of
multifunctional KOR/MOR ligands which can be investigated for this purpose.
Three compounds from this series (10d, 10g, 10i) display this multifunctional KOR/MOR
profile. Notably, the KOR potency and MOR potency of 10g and 10d are higher than that of the
10i, making them the most promising KOR/MOR ligands in this series. These compounds show
little to no selectivity for KOR and MOR over DOR; however, there is evidence from our group
and others to suggest that DOR antagonist activity may help mitigate the addictive potential of
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MOR agonists.53–59 Since none of the compounds reported here show agonism at DOR, this lack
of selectivity may be beneficial for the development of a therapeutic.
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Future efforts will explore the incorporation of additional and larger ortho and meta
substituents and modification of the methylene linker between the 7-position pendant and the Tiq
core with the primary goals of increasing MOR efficacy and improving the drug-like properties of
these compounds. In addition, promising compounds from this series will be evaluated in vivo for
opioid activity.
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In conclusion, we have repurposed a selective DOR antagonist scaffold to develop a
series of novel multifunctional opioid peptidomimetics by investigating previously unexplored
chemical space. The addition of a 7-benzyl pendant to the Tiq aromatic ring of Dmt-Tiq introduced
strong KOR agonism. The introduction of ortho and meta substituents on the 7-benzyl pendant
resulted in retention of KOR agonism and addition of partial MOR agonism, a profile which has
shown promise for the treatment of cocaine addiction.
Methods
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Synthesis
Unless otherwise noted, all reagents and solvents were purchased from commercial
sources and used without additional purification. DiBoc-Dmt was prepared from commercially
available Dmt according to standard procedures.43,60 Microwave reactions were performed in a
CEM Discover SP microwave synthesizer in a closed vessel with maximum power input of 300
W. Column chromatography was carried out on silica gel cartridges using a Biotage Isolera One
flash purification system. Before chromatographic purification, crude reaction mixtures were
analyzed by thin layer chromatography in hexanes/ethyl acetate. Purification of final compounds
was performed using a Waters semipreparative HPLC with a Vydac protein and peptide C18
reverse phase column using a linear gradient of 100% solvent A (water with 0.1% TFA) to 100%
solvent B (acetonitrile with 0.1% TFA) at a rate of 1% per minute with UV absorbance monitored
at 230 nm. Purity of final compounds was determined on a Waters Alliance 2690 analytical HPLC
with a Vydac protein and peptide C18 reverse phase column using the same gradient with UV
absorbance monitored at 230 nm. Purity of final compounds used for testing was ≥95% as
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determined by HPLC. H NMR data for intermediates and final compounds in CDCl3 or CD3OD
was obtained on a 400 MHz or 500 MHz Varian spectrometer. LC-MS data was obtained using
an Agilent 6130 LC-MS in positive ion mode. HRMS data was obtained using an Agilent QTOF
HPLC-MS in positive ion mode. Detailed synthetic procedures and characterization data can be
found in the Supporting Information.
Synthesis of Benzyl Pendant Dmt-Tiq Analogues 4a-d. Analogues 4a-4d were
prepared according to the synthetic route shown in Scheme 1. Commercially available bromo-
substituted tetrahydroisoquinolines 1a-1d were Boc-protected, and the resulting bromides 2a-2d
were coupled to benzylboronic acid pinacol ester via Suzuki reaction to produce intermediates
3a-3d. Deprotection of the tetrahydroisoquinoline nitrogen with hydrochloric acid, peptide
coupling with diBoc-protected dimethyltyrosine, and subsequent deprotection with trifluoroacetic
acid yielded peptidomimetics 4a-4d.
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Scheme 1: Synthesis of Benzyl Pendant Dmt-Tiq Analogues 4a-4d
O
5
6
Boc
Boc
ii
NH
i
iii, iv, v
N
N
N
7
8
9
NH2
Br
Br
1a 2a
Bn
OH
Bn
1a-d
2a-d
3a-d
4a-d
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,
, R = 5-Br
, R = 6-Br
, R = 7-Br
, R = 8-Br
3a 4a
,
, R = 5-Bn
, R = 6-Bn
, R = 7-Bn
, R = 8-Bn
1b 2b
,
3b 4b
,
1c 2c
,
3c 4c
,
1d 2d
,
3d 4d
,
(i) Boc2O; (ii) Benzylboronic acid pinacol ester, Pd(dppf)Cl2, K2CO3, acetone/water; (iii) HCl, 1,4-
dioxane; (iv) diBoc-Dmt, PyBOP, 6Cl-HOBt, DIEA, DMF; (v) TFA, DCM
Synthesis of Substituted 7-Benzyl Pendant Dmt-Tiq Analogues 10g and 10i.
Analogues 10g and 10i were prepared according to the first synthetic route shown in Scheme 2.
Commercially available 5 was reduced with borane dimethylsulfide to secondary alcohol 6 which
was then converted to the corresponding secondary bromide 7 via Appel reaction. Suzuki
coupling of 7 with 2-methylphenylboronic acid or 2-aminophenylboronic acid produced
intermediate 9e or 9g. Deprotection of the tetrahydroisoquinoline nitrogen with hydrochloric acid,
peptide coupling with diBoc-protected dimethyltyrosine, and subsequent deprotection with
trifluoroacetic acid yielded peptidomimetic 10g or 10i.
Synthesis of Substituted 7-Benzyl Pendant Dmt-Tiq Analogues 10a-10f, 10h.
Analogues 10a-10f and 10h were prepared according to the second synthetic route shown in
Scheme 2. Common intermediate 8 was prepared from 2c. Suzuki coupling of 8 with various
substituted benzyl bromides yielded intermediates 9a-9d and 9f. Deprotection of the
tetrahydroisoquinoline nitrogen with hydrochloric acid, peptide coupling with diBoc-protected
dimethyltyrosine, and subsequent deprotection with trifluoroacetic acid yielded peptidomimetics
10a, 10b, 10d, 10e, and 10h. For peptidomimetics 10c and 10f, reduction of the nitro group with
zinc dust and ammonium chloride in acetone and water was performed prior to final deprotection.
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Scheme 2: Synthesis of Substituted 7-Benzyl Pendant Dmt-Tiq Analogues 10a-10i
O
Boc
Boc
Boc
i
HO
N
ii
HO
N
Br
N
7
8
9
6
7
5
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iii
O
Boc
Br
Boc
B
Boc
iv
N
v
N
O
N
R
2c
8
9a-g
vi, vii, viii, ix
OR
9a
9b
9c
9d
9e
, R = p-Me
, R = p-NO2
, R = m-Me
, R = m-NO2
, R = o-Me
10a
10b
10c
10d
10e
10f
10g
10h
10i
, R = p-Me
, R = p-NO2
, R = p-NH2
, R = m-Me
, R = m-NO2
, R = m-NH2
, R = o-Me
, R = o-NO2
, R = o-NH2
vi, vii, ix
O
9f
9g
, R = o-NO2
, R = o-NH2
N
NH2
OH
R
10a-i
(i) BH3 SMe2, THF; (ii) CBr4, PPh3, DCM (iii) substituted benzyl bromide, Pd(dppf)Cl2, K2CO3,
acetone/water; (iv) bis(pinacolato)diboron, Pd(dppf)Cl2, CH3CO2K, DMSO; (v) substituted aryl
boronic acid, Pd(dppf)Cl2, K2CO3, 3:1 acetone:water; (vi) HCl, 1,4-dioxane; (vii) diBoc-Dmt,
PyBOP, 6Cl-HOBt, DIEA, DMF; (viii) Zn, NH4Cl, acetone/water; (ix) TFA, DCM
In Vitro Characterization
Unless otherwise noted, all tissue culture reagents and radiolabeled ligands were
purchased from commercial sources.
Cell Lines and Membrane Preparations. Membranes prepared from transfected C6 rat glioma
cells stably expressing rat MOR or rat DOR or Chinese Hamster Ovary cells stably expressing
human KOR were used for all assays. Cells were grown to confluence at 37 °C in 5% CO2 in
Dulbecco’s Modified Eagle’s Medium containing 10% v/v fetal bovine serum and 5% v/v
penicillin/streptomycin. Membranes were prepared by washing confluent cells three times with ice
cold phosphate-buffered saline (0.9% NaCl, 0.61 mM Na2HPO4, 0.38 mM KH2PO4, pH 7.4). Cells
were detached from the plates by incubation in warm harvesting buffer (20 mM HEPES, 150 mM
NaCl, 0.68 mM EDTA, pH 7.4) and pelleted by centrifugation at 200xg for 3 minutes. The cell
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pellet was suspended in ice-cold 50 mM Tris-HCl buffer, pH 7.4 and homogenized with a Tissue
Tearor (Biospec Products, Inc) for 20 seconds at setting 4. The homogenate was centrifuged at
20,000xg for 20 minutes at 4 °C, and the pellet was rehomogenized in 50 mM Tris-HCl pH 7.4
with a Tissue Tearor for 10 seconds at setting 2, followed by recentrifugation. The final pellet was
resuspended in 50 mM Tris-HCl pH 7.4 and frozen in aliquots at -80 °C. Protein concentration
was determined via Pierce BCA protein assay kit using bovine serum albumin as the standard.
Binding Affinity. Binding affinities for peptidomimetics 4a-4d and 10a-10i at KOR, MOR, and
DOR were determined by competitive displacement of [3H]diprenorphine as previously
reported.43,61–63 In a 96-well plate format, cell membranes (20 µg of protein) and [3H]diprenorphine
(0.2 nM) were incubated in Tris-HCl buffer (50 mM, pH 7.4) with various concentrations of test
compound at 25 °C for 1 hour, allowing the mixture to reach equilibrium. Nonspecific binding was
determined using the opioid antagonist naloxone (10 µM), and total binding was determined using
vehicle in the absence of competitive ligand. After incubation, membranes were filtered through
Whatman GF/C 1.2 micron glass fiber filters and washed with 50 mM Tris-HCl buffer. The
radioactivity remaining on the filters was then quantified by liquid scintillation counting after
saturation with EcoLume liquid scintillation cocktail in a Perkin-Elmer Microbeta 2450. Binding
affinity (Ki) values were calculated using the Cheng-Prusoff equation via nonlinear regression
analysis using GraphPad Prism software from at least three separate binding assays performed
in duplicate.
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Stimulation of [35S]GTPγS Binding. Agonist stimulation of KOR, MOR, and DOR by
peptidomimetics 4a-4d and 10a-10i was determined by [35S]guanosine 5′-O-[γ-thio]triphosphate
([35S]GTPγS) binding assays as previously reported.43,61–63 In a 96-well plate format, membranes
from cells expressing opioid receptors as described above (10 µg of protein), [35S]GTPγS (0.1
nM), and guanosine diphosphate (30 µM) were incubated in GTPγS buffer (50 mM Tris-HCl, 100
mM NaCl, 5 mM MgCl2, 1 mM EDTA, pH 7.4) with various concentrations of test compound at 25
°C for 1 hour. Basal stimulation was determined by incubation in the absence of any ligand. After
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incubation, membranes were filtered through Whatman GF/C 1.2 micron glass fiber filters and
washed with GTPγS buffer with no EDTA. The radioactivity remaining on the filters was then
quantified by liquid scintillation counting after saturation with EcoLume liquid scintillation cocktail
in a Perkin-Elmer Microbeta 2450. Data are reported as percent stimulation compared to the
effects of 10 µM standard agonist - U69,593 (KOR), DAMGO (MOR), or DPDPE (DOR). Percent
stimulation and EC50 values were determined via nonlinear regression analysis using GraphPad
Prism software from at least three separate assays performed in duplicate. Efficacy is expressed
as percent stimulation relative to standard agonist.
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Computational Modeling
Modeling of three-dimensional (3D) structures of receptor-ligand complexes was based
on the available X-ray structure of the human KOR in the active conformation (PDB ID: 6b73).44
Structures of peptidomimetic ligands were generated using the 3D-Builder Application of
QUANTA (Accelrys, Inc.) followed by Conformational Search included in the program package.
Low-energy ligand conformations (within 2 kcal/mol) that demonstrated the best superposition of
aromatic substituents of the tetrahydroisoquinoline core with the pharmacophore elements (Tyr1
and Phe3) of receptor-bound conformations of cyclic tetrapeptides64 were selected for docking
into the receptor binding pocket. Ligands were positioned inside the receptor binding cavity to
reproduce the binding modes of cyclic tetrapeptides and co-crystalized ligands in MOR and
KOR X-ray structures. Two conformations of peptidomimetics were tested: with the peptide
bond in cis (=0°) and trans (=180°) configurations. Though ligands with a cis peptide group
have slightly higher conformational energy (by ~1 kcal/mol), they fit better in the ligand binding
pockets of the receptors, overlapping with ligands co-crystalized with KOR and MOR. The
docking pose of each ligand was subsequently refined using the solid docking module of
QUANTA. Models of opioid ligand-receptor complexes are available upon request.
Abbreviations
6Cl-HOBt, 6-chlorohydroxybenzotriazole;
Boc2O, di-tert-butyl dicarbonate;
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DCM, dichloromethane;
DiBoc-Dmt, tert-Butoxycarbonyl-protected-L-2,6-dimethyltyrosine;
DIEA, diisopropylethylamine;
DMF, dimethylformamide;
DMSO, dimethylsulfoxide;
Dmt-Tic, dimethyltyrosine-tetrahydroisoquinoline carboxylic acid;
Dmt-Tiq, dimethyltyrosine-tetrahydroisoquinoline;
DOR, delta opioid receptor;
GTPγS, guanosine 5’-O-[γ-thio]triphosphate;
KOR, kappa opioid receptor;
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MOR, mu opioid receptor;
PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate;
SAR, structure-activity relationships;
TFA, trifluoroacetic acid;
THF, tetrahydrofuran;
Tic, tetrahydroisoquinoline carboxylate;
Tiq, tetrahydroisoquinoline
Author Information
Corresponding Author Information:
Phone: 734-764-8117
Fax: 734-763-5595
Email: him@umich.edu
Author Contributions
DM designed and synthesized novel compounds and wrote the manuscript under the direction
of HIM. JPA, NWG, JGH, and TJF collected in vitro data under the direction of JRT. IDP
performed modeling studies.
Funding Sources
This study was supported by NIH grant DA003910. Resources were provided by the Edward F.
Domino Research Center. DM was supported by an ACS MEDI Pre-doctoral Fellowship. NWG
was supported by the NIDA Training Program in Neuroscience (NTPiN) DA007281-22. IP was
supported by NSF grant DBI-1458002.
Supporting Information
Detailed experimental methods and characterization data for compounds 4a-4d, 10a-10i, and
related intermediates
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For Table of Contents Use Only
Novel Dimethyltyrosine-Tetrahydroisoquinoline Peptidomimetics with Aromatic
Tetrahydroisoquinoline Substitutions Show In Vitro Kappa and Mu Opioid Receptor
Agonism
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Deanna Montgomery†, Jessica P. Anand‡,§, Nicholas W. Griggs‡, Thomas J. Fernandez‡, Joshua
G. Hartman‡, Ashley A. Sánchez-Santiago‡, Irina D. Pogozheva†, John R. Traynor‡,§, Henry I.
Mosberg†,§,*
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O
O
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N
N
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NH2
COOH
NH2
OH
OH
R
KOR agonist/
MOR agonist
DOR antagonist
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