Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208

Article abstract of DOI:10.1002/cmdc.201100113

An alanine scan was performed on the novel κopioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent invivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combi

Full text of DOI:10.1002/cmdc.201100113

DOI: 10.1002/cmdc.201100113
Unexpected Opioid Activity Profiles of Analogues of the
Novel Peptide Kappa Opioid Receptor Ligand CJ-15,208
Jane V. Aldrich,*[a] Santosh S. Kulkarni,[a] Sanjeewa N. Senadheera,[a] Nicolette C. Ross,[b]
Kate J. Reilley,[b] Shainnel O. Eans,[b] Michelle L. Ganno,[b] Thomas F. Murray,[c] and
Jay P. McLaughlin[b]
An alanine scan was performed on the novel k opioid receptor
(KOR) peptide ligand CJ-15,208 to determine which residues
contribute to the potent in vivo agonist activity observed for
the parent peptide. These cyclic tetrapeptides were synthe-
sized by a combination of solid-phase peptide synthesis of the
linear precursors, followed by cyclization in solution. Like the
parent peptide, each of the analogues exhibited agonist activi-
ty and KOR antagonist activity in an antinociceptive assay
in vivo. Unlike the parent peptide, the agonist activity of the
potent analogues was mediated predominantly, if not exclu-
sively, by m opioid receptors (MOR). Thus analogues 2 and 4, in
which one of the phenylalanine residues was replaced by ala-
nine, exhibited both potent MOR agonist activity and KOR an-
tagonist activity in vivo. These peptides represent novel lead
compounds for the development of peptide-based opioid an-
algesics.
Introduction
Recent reports suggest that k opioid receptor (KOR) antago-
nists could have potential therapeutic application in the treat-
ment of mood disorders and drug abuse.[1] Pretreatment with
the nonpeptide KOR-selective antagonists nor-binaltorphimine
(nor-BNI) or JDTic decrease immobility in the forced swim
assay similar to antidepressants,[2] and have been shown to
reduce behavioral measures of anxiety in rats.[3] Pretreatment
with these antagonists also prevents stress-induced reinstate-
ment of extinguished cocaine-seeking behavior.[2c,4] Likewise,
heroin-dependent patients treated with a “functional KOR an-
tagonist” (buprenorphine plus naltrexone to block m opioid re-
ceptors (MOR)) for 12 weeks showed significantly improved
drug abstinence relative to patients treated only with naltrex-
one.[5]
caine-conditioned place preference after subcutaneous admin-
istration.[9]
The cyclic tetrapeptide CJ-15,208 was reported to preferen-
tially bind to KOR and antagonize the activity of a KOR agonist
in the rabbit vas deferens smooth muscle preparation,[10] but
the stereochemistry of the Trp residue in this natural product
was not determined. We therefore undertook the synthesis of
both tryptophan isomers of this cyclic tetrapeptide,[11] and
found that the optical rotation of the l-Trp isomer 1 is consis-
tent with that reported for the natural product (Figure 1a). Al-
though the l-Trp peptide did not exhibit any agonist activity
at either KOR or MOR in vitro,[12] it unexpectedly exhibited
robust agonist activity in vivo in the 558C warm-water tail-
withdrawal antinociceptive assay in addition to KOR-selective
antagonist activity.[12a]
These nonpeptide antagonists demonstrate notably pro-
longed durations of activity,[1,6] antagonizing KOR for weeks
after a single dose.[7] This unusual pharmacological profile can
complicate their use as pharmacological tools and could con-
ceivably slow their development for clinical use, sparking inter-
est in shorter-acting KOR-selective antagonists.
Therefore we undertook structure–activity relationship (SAR)
studies of CJ-15,208, first performing an alanine scan (Fig-
ure 1b) of the peptide to determine which amino acid side
chains are important for interaction with opioid receptors and
[a] Prof. J. V. Aldrich, Dr. S. S. Kulkarni,+ Dr. S. N. Senadheera
Department of Medicinal Chemistry, University of Kansas
1251 Wescoe Hall Dr., Lawrence, KS 66045 (USA)
Fax: (+1)785-864-5326
We have a long-standing interest in peptide ligands for KOR,
particularly those that demonstrate KOR-selective antagonism.
A number of analogues of the endogenous opioid peptide dy-
norphin A have demonstrated KOR antagonism (for example,
see reference [8]). Modifications to linear peptides can de-
crease proteolytic cleavage so that the peptide’s activity is pre-
served after systemic administration. This was demonstrated
for the KOR-selective peptide antagonist zyklophin ([N-benzyl-
Tyr1,cyclo(d-Asp5,Dap8)]dynorphin A-(1–11)NH2) developed in
our research group,[8e] which exhibits KOR-selective antagonist
activity following systemic (subcutaneous) administration,[9]
and prevents stress-induced reinstatement of extinguished co-
E-mail: jaldrich@ku.edu
[b] Dr. N. C. Ross, K. J. Reilley, S. O. Eans, M. L. Ganno, Dr. J. P. McLaughlin
Torrey Pines Institute for Molecular Studies
11350 SW Village Parkway, Port St. Lucie, FL 34987 (USA)
[c] Prof. T. F. Murray
Creighton University School of Medicine
2500 California Plaza, Omaha, NE 68178 (USA)
[+] Current address: Syngene Int. Ltd.
Biocon Park, Bangalore (India)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100113.
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performed by slow addition of the linear peptide precursor to
the coupling reagent HATU and DIEA in DMF; under these con-
ditions, the formation of the dimeric cyclic octapeptide is mini-
mal.[11] The peptides were purified by reversed-phase HPLC.
In vitro pharmacological characterization
The peptides were evaluated for opioid receptor affinity in
radioligand binding assays using cloned opioid receptors.[13]
Substitution of alanine had a large effect on affinities for KOR
with generally less effect on affinities for MOR (Table 1). Unex-
Table 1. Opioid receptor affinities of alanine analogues of CJ-15,208.
Figure 1. Structures of a) the cyclic tetrapeptide CJ-15,208 (1) and b) alanine
analogues 2–5. The residues are numbered 1–4, arbitrarily starting with the
Phe C-terminal to the Trp residue.
Ki [nm]ÆSEM
Selectivity
KOR/MOR/DOR
Peptide
KOR
MOR
DOR
2
3
4
5
1[a]
8.03Æ1.67
32.1Æ3.9
140Æ9
8680Æ1270
1370Æ70
1/4.0/1080
1/1.2/12
1.2/1/>15
2.3/1/>14
1/17.5/117
113 Æ23
the pharmacological activity observed in vivo. Herein we
report the results of these initial studies, including both basic
in vitro and in vivo characterization of these analogues of CJ-
15,208.
663Æ220
1550Æ290
35.4Æ3.6
533Æ28
687Æ81
619Æ87
>10000
>10000
4150Æ3020
[a] Data from reference [12a].
pectedly, substitution of Phe1 (see Figure 1 for notation) in-
creased both KOR and MOR affinities by 4.4- and 19-fold, re-
spectively. In contrast, substitution of the other three residues
in the cyclic tetrapeptide with alanine decreased KOR affinity
from 3- to 44-fold (Table 1), with the largest decrease occurring
if the Trp residue is replaced by Ala, followed by substitution
of Phe3 (see Figure 1). These results suggest that Trp and Phe3
are important for KOR affinity. These substitutions, however,
did not decrease MOR affinity, but in one case (replacement of
d-Pro by NMe-d-Ala in 3) increased MOR affinity 4.4-fold, re-
sulting in these three analogues exhibiting negligible selectivi-
ty for KOR over MOR. All of the peptides exhibited very low af-
finity for d opioid receptors (DOR) in the binding assays.
Results and Discussion
Synthesis
The cyclic tetrapeptides were synthesized by a combination of
solid-phase synthesis of the linear tetrapeptide precursors, fol-
lowed by cyclization in solution (the synthesis of cyclo[Ala-d-
Pro-Phe-Trp] (2) is shown in Scheme 1) using the optimized
procedure described for CJ-15,208.[11] The cyclizations were
The ligand-stimulated [35S]GTPgS binding assay was used to
assess the efficacy and potency of the cyclic tetrapeptides.
None of the peptides exhibited appreciable stimulation of
[35S]GTPgS binding via either KOR or MOR, consistent with the
lack of agonist activity of the parent peptide 1 in this assay.[12]
The Ala analogue 2 is a reasonably potent antagonist of both
dynorphin A-(1–13)NH2 at KOR (KB =2.6Æ0.8 nm) and [d-
Ala2,NMePhe4,glycol]enkephalin (DAMGO) at MOR (KB =7.3Æ
1.6 nm), consistent with its affinities for KOR and MOR. Notably,
it is 25-fold more potent as a KOR antagonist than the parent
peptide 1 (KB =65.2Æ1.6 nm), while retaining MOR antagonist
potency similar to 1 (KB =10.2Æ1.7 nm).
In vivo pharmacological characterization
Scheme 1. Synthesis of peptide 2. Reagents and conditions: a) Fmoc-AA,
DIEA, CH2Cl2/DMF (4:1), 6 h, RT; b) piperidine/DMF (1:4), 2ꢁ20 min, RT;
c) Fmoc-AA, PyBOP, HOBt, DIEA, CH2Cl2/DMF (1:1), 2–4 h, RT; d) 1% TFA in
CH2Cl2, 10ꢁ2 min, RT; e) HATU, DIEA, DMF, 12 h addition + 12–24 h reac-
tion, RT; f) TFA/CH2Cl2 (1:1), 30 min, RT. Black spheres: 2-chlorotrityl chloride
resin.
The opioid activity of the cyclic tetrapeptides was determined
in vivo by using C57Bl/6J mice in the 558C warm-water tail-
withdrawal assay following intracerebroventricular (i.c.v.) ad-
ministration. This initial evaluation was done following central
administration to measure the inherent pharmacological activi-
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Opioid Activity Profiles of CJ-15,208 Analogues
ty of the analogues in vivo without the complications associat-
ed with distribution (i.e., blood–brain barrier penetration) that
could affect activity following systemic administration. Like CJ-
15,208, each of the alanine analogues exhibited antinocicep-
tive activity in vivo, albeit with varying potencies (Figure 2).
Figure 3. Cyclic tetrapeptide-induced antinociception is opioid receptor
mediated. Peak antinociceptive activity of peptides 2 (3 nmol), 3 (10 nmol),
4 (0.3 nmol), and 5 (30 nmol) was determined in the 558C warm-water tail-
withdrawal assay after i.c.v. administration to C57Bl/6J mice (open bars). Na-
loxone pretreatment (15 nmol, i.c.v., striped bars) 25 min prior to peptide
administration significantly antagonized the effect of each cyclic tetrapep-
tide. Tail-withdrawal latencies were measured 30 min after injection of the
cyclic tetrapeptide. Data represent average percent antinociception ÆSEM
Figure 2. The antinociceptive activity of the cyclic tetrapeptides was as-
sessed in vivo following i.c.v. administration in the 558C warm-water tail-
withdrawal assay in C57Bl/6J mice. All points represent antinociception at
peak response, which was 20 min (for 4), 30 min (for peptides 1, 2, and 5) or
40 min (peptide 3). All points represent average percent antinociception
ÆSEM from 7–8 mice. Data for 1 from reference [12a].
from 7–8 mice. : Significantly different from response of matching adminis-
*
tered compound alone (p<0.05), one-way ANOVA followed by Tukey’s HSD
post-hoc test.
Analogues 2 and 3 exhibited similar antinociceptive potencies
to CJ-15,208 in this assay, with ED50 (and 95% confidence inter-
val) values of 1.49 (0.39–7.41) and 2.43 (0.71–8.85) nmol for 2
and 3, respectively, versus 1.74 (0.62–4.82) nmol for CJ-15,208.
Interestingly, analogue 4 (ED50 =0.10 (0.03–0.35) nmol) is 17-
fold more potent than the parent peptide. Peptide 5, in which
the Trp residue is substituted by Ala, proved to be the least
potent analogue, with an ED50 value of 6.97 (1.02–47.4) nmol,
fourfold lower than the parent peptide.
While consistent with the in vivo activity of the parent pep-
tide CJ-15,208, the antinociceptive activity of these analogues
was surprising, given their lack of agonist activity in the
[35S]GTPgS assay in vitro and the relatively low opioid receptor
affinities of analogues 3–5. Therefore we evaluated whether
the antinociceptive activity is mediated through opioid recep-
tors. In our initial testing, the antinociceptive activity of each
peptide was completely blocked by pretreatment with the
nonselective opioid antagonist naloxone (Figure 3), verifying
opioid receptor involvement. We subsequently examined
which opioid receptors are involved in the antinociceptive ac-
tivity by pretreating test subjects with antagonists selective for
MOR, KOR, and DOR. The antinociceptive activity of each of
the alanine analogues 2–5, administered at a dose producing
50–80% antinociception, was almost completely blocked by
pretreatment with the MOR-selective irreversible antagonist b-
funaltrexamine (b-FNA, Figure 4). Of interest, pretreatment
with the KOR-selective antagonist nor-BNI produced differing
effects, significantly antagonizing the antinociceptive activity
of only peptide 5, with no effect on 3 or 4 (Figure 4). Pretreat-
ment with nor-BNI decreased the antinociceptive activity of an-
alogue 2 by >30%, but the difference in the presence versus
absence of nor-BNI did not reach statistical significance. The
Figure 4. Opioid receptor-selective agonism by the cyclic tetrapeptides. The
antinociceptive activity of peptides 2 (3 nmol), 3 (3 nmol), 4 (0.1 nmol), and
5 (30 nmol) was determined in the 558C warm-water tail-withdrawal assay
after i.c.v. administration to C57Bl/6J mice (solid bars). Antinociception was
also assessed 24 h after administration in mice pretreated with b-FNA
(5 mgkgÀ1, s.c.; diagonally striped bars) or nor-BNI (10 mgkgÀ1, i.p.; wave-
filled bars). Additional mice were pretreated with naltrindole (20 mgkgÀ1
,
i.p., À15 min; hatched bars) before administration of one of the cyclic tetra-
peptides. Tail-withdrawal latencies were measured in the mouse 558C warm-
water tail-withdrawal test 30 min after injection of the cyclic tetrapeptides 2,
4, and 5, or 40 min after peptide 3. Data represent average percent antinoci-
ception ÆSEM from 8–16 mice. : Significantly different from response of
*
matching administered compound alone (p<0.05), one-way ANOVA fol-
lowed by Tukey’s HSD post-hoc test.
DOR-selective antagonist naltrindole significantly decreased
the antinociceptive activity of only peptide 5. Together, these
results suggest that the antinociception induced by peptides
2–4 is mediated almost exclusively by MOR, although it is pos-
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sible that KOR contributes to the antinociceptive activity of 2.
In contrast, all three receptors appear to contribute to the anti-
nociceptive activity of 5. These results are in contrast to the
antinociceptive activity of 1, which appears to be predomi-
nantly mediated by KOR, with a lesser contribution by MOR.[12a]
We next examined the peptides for antagonist activity fol-
lowing dissipation of the agonist activity. For the parent pep-
tide 1 significant agonist activity was detected for up to
100 min after administration of the highest dose (10 nmol),
whereas for analogues 2–5 significant agonist activity was de-
tected for 70–80 min. To ensure that there was no residual ag-
onist activity, the peptides were evaluated for antagonist activ-
ity 3 h after pretreatment. All of the analogues dose-depend-
ently antagonized the antinociceptive effects of the KOR-selec-
tive agonist U50,488 (Figure 5). Peptides 2 and 4 appeared to
Figure 6. Duration of cyclic tetrapeptide-mediated antagonism of U50,488-
induced antinociception in the mouse 558C warm-water tail-withdrawal test.
Antinociception of U50,488 (10 mgkgÀ1, i.p.; thatched bar) was determined
in mice pretreated 3, 6, 18, or 24 h with peptides 2–5, and for peptide 1 at
8, 18, and 24 h.[12a] Tail-withdrawal latencies were determined 40 min after
agonist administration. Data represent average percent antinociception
ÆSEM from eight mice per point. : Significantly different from response of
*
U50,488 (p<0.05), one-way ANOVA followed by Tukey’s HSD post-hoc test.
Figure 5. Dose-dependent antagonism of U50,488-induced antinociception
by tested cyclic tetrapeptides. The antinociceptive effects of U50,488
(10 mgkgÀ1, i.p.; thatched bar) were determined 40 min after administration
in mice pretreated 3 h with peptides 1–5 in the 558C warm-water tail-with-
drawal assay after i.c.v. administration. Data represent average percent anti-
nociception ÆSEM from eight mice. : Significantly different from response
*
of U50,488 (p<0.05), one-way ANOVA followed by Tukey’s HSD post-hoc
Figure 7. Receptor selectivity of antagonism by cyclic tetrapeptides in the
mouse 558C warm-water tail-withdrawal test. Antinociceptive activity of
morphine (10 mgkgÀ1, i.p.; left group) was not decreased by a 3 h pretreat-
ment of the mice with the indicated cyclic tetrapeptides 2 (10 nmol), 3
(1 nmol), 4 (1 nmol), or 5 (10 nmol). However, the antinociceptive effect of
U50,488 (10 mgkgÀ1, i.p.; center group) was significantly antagonized by
pretreatment of the mice with any of the cyclic tetrapeptides. (For this set
of tests peptide 2 was administered at 3 nmol, i.c.v.). In contrast, the antino-
ciceptive effect of SNC-80 (100 nmol, i.c.v., right group) was significantly pre-
vented only by pretreatment with peptides 3 and 5. Tail-withdrawal laten-
cies were measured in the mouse 558C warm-water tail-withdrawal test
40 min after injection of the known selective agonists. Data represent aver-
test. Data for 1 from reference [12a].
be somewhat more potent than the parent peptide 1, whereas
peptides 3 and 5 are less potent as KOR antagonists than 1.
Importantly, the duration of the KOR antagonist activity for
each of the peptides was relatively brief (<18 h, Figure 6), sub-
stantially shorter than the duration of activity of the parent
peptide 1, which exhibits significant KOR antagonist activity
for at least 24 h.[12a] The reason for the shorter duration of an-
tagonist activity of the analogues relative to the parent pep-
tide is unclear, but could be due to differences in hydrophobic-
ity, with the more hydrophobic parent peptide being retained
longer in the tissue.
age percent antinociception ÆSEM from 8–16 mice. : Significantly different
*
from response of matching administered agonist alone (p<0.05), one-way
ANOVA followed by Tukey’s HSD post-hoc test.
This DOR antagonist activity was unexpected given the low af-
finity of these cyclic tetrapeptides for DOR.
The selectivity of the antagonist activity was next evaluated
by examining the ability of the peptides to antagonize the an-
tinociception produced by the MOR-preferring agonist mor-
phine or the DOR-selective agonist SNC-80 (Figure 7). None of
the peptides antagonized morphine. While peptide 2 did not
significantly antagonize SNC-80, surprisingly, peptides 3 and 5
did antagonize this agonist (the decrease in the antinocicep-
tion of SNC-80 by pretreatment with 4 was not significant).
Conclusions
The alanine analogues of CJ-15,208 exhibited in vivo pharma-
cological profiles that were unexpected based on their opioid
receptor affinities and lack of agonist activity in the [35S]GTPgS
assay in vitro. All of the analogues exhibited antinociceptive
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Opioid Activity Profiles of CJ-15,208 Analogues
activity in the 558C warm-water tail-withdrawal assay, with an-
alogue 4 exhibiting particularly potent agonist activity. This an-
tinociceptive activity involves opioid receptors, as it is blocked
by the nonselective opioid antagonist naloxone. Further exami-
nation with selective antagonists suggests that the antinoci-
ceptive activity of the more potent analogues 2–4 is predomi-
nantly if not entirely mediated through MOR activation, which
contrasts with the antinociceptive activity of the parent pep-
tide 1,[12a] which appears to be predominantly mediated
through activity at KOR with a smaller contribution by MOR.
These alanine analogues also exhibited antagonist activity at
KOR after dissipation of the agonist activity, which was unex-
pected, especially in the case of the potent antagonist 4, given
its low KOR affinity. The DOR antagonist activity of at least two
of the analogues was also unexpected, given the very low
DOR affinities of these compounds. Clearly, these analogues
have different opioid activity profiles in vivo from what was ex-
pected from the in vitro assays and also from the results for
the parent peptide 1.
The in vivo data suggest that there are different structural
requirements for agonist versus antagonist activity mediated
by KOR and for the activation of KOR versus MOR. All three of
the aromatic residues appear to contribute to agonist activity
mediated by KOR. Interestingly, only the antinociceptive activi-
ty of the lower-potency agonist 5, in which the Trp residue is
replaced by Ala, was significantly antagonized by nor-BNI. All
of the peptides, however, antagonized KOR in vivo, although
peptide 5 also exhibited relatively low potency as an antago-
nist. All of the analogues exhibited agonist activity that was
mediated predominantly by MOR, suggesting that only two of
the aromatic residues are sufficient for activation of MOR. The
analogues in which one of the Phe residues is replaced with
alanine exhibited high potency both as MOR agonists and as
KOR antagonists in vivo. These analogues are undergoing addi-
tional evaluation in vivo as lead compounds for potential de-
velopment as analgesics.
In conclusion, these unusual ligands represent valuable com-
pounds for further study and as novel lead compounds, partic-
ularly analogues 2 and 4, for the development of peptide-
based opioid analgesics. These studies are ongoing in our lab-
oratories.
Experimental Section
Materials: Reagents for peptide synthesis were obtained from the
following sources: Fmoc (fluorenylmethoxycarbonyl)-protected
amino acids (Novabiochem (EMD), San Diego, CA, USA), 2-chlorotri-
tyl chloride resin (1.4 mmolgÀ1, Novabiochem), coupling reagents
HATU
(2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate, Novabiochem), PyBOP (benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate, Novabiochem)
and HOBt (1-hydroxybenzotriazole, Fluka, Milwaukee, WI, USA),
DIEA (N,N-diisopropylethylamine, Fluka), TFA (trifluoroacetic acid,
Pierce, Rockford, IL, USA), and HPLC-grade solvents (Fisher Scientif-
ic, Pittsburg, PA, USA). The other solvents and routine chemicals
were obtained from Fisher Scientific. HPLC analyses and purifica-
tions were performed on Vydac 218TP C18 reversed-phase columns
(Grace Davison, 4.6ꢁ50 mm, 5 mm, and 22ꢁ250 mm, 10 mm, re-
spectively).
Peptide synthesis:[11]
Solid-phase synthesis of linear peptide precursors. The linear peptide
precursors were synthesized using Fmoc-protected amino acids on
a 2-chlorotrityl chloride resin and a custom-made manual peptide
synthesizer (CHOIR)[17] constructed in house. Following swelling of
the resin in CH2Cl2 (2ꢁ10 min), the C-terminal Fmoc-protected
amino acid (2 equiv) and (DIEA, 5 equiv) in CH2Cl2/N,N-dimethyl-
formamide (DMF) (4:1, 5 mL per 0.5 g resin) were added to the
resin, and the reaction gently agitated with N2 gas for 6 h. Addi-
tional CH2Cl2 was added every 30 min to maintain the solvent
volume, and additional DIEA (5 equiv) was added to the reaction
every 2 h. The resin was washed with CH2Cl2/DMF (1:1, 5ꢁ), and
quantitative Fmoc analysis[18] was then used to determine loading
efficiency. A capping step was then performed using 15% MeOH
and 5% DIEA in CH2Cl2 (2ꢁ10 min), and the resin was washed with
CH2Cl2/DMF (1:1, 5ꢁ). The Fmoc group was then removed with
20% piperidine in DMF (2ꢁ20 min), and the resin was washed
with CH2Cl2/DMF (1:1, 5ꢁ) and CH2Cl2 (5ꢁ).
While these peptides produce antinociception and antago-
nist activity that is clearly mediated through opioid receptors,
the marked differences between the activity profiles in the in
vitro versus in vivo assays suggest that these compounds elicit
their opioid activity through more complex mechanisms than
typical opioid receptor ligands. Notably, similar differences
have been found between in vitro and in vivo opioid activity
for other novel peptide-based antinociceptive compounds that
are structurally distinct from these peptides.[14] Involvement of
additional mechanisms in analgesic activity has been reported
for other opioid peptides. For example, the indirect activation
of opioid receptors by release of endogenous opioid peptides
has been reported for several opioid peptides,[15] most notably
for the potent and selective MOR peptides Dmt-DALDA (Dmt-
d-Arg-Phe-Lys-NH2, Dmt=2’,6’-dimethyltyrosine) and endomor-
phin-2. A non-opioid mechanism (inhibition of norepinephrine
uptake) was also reported to contribute to the antinociceptive
effects of Dmt-DALDA.[16] Additional studies are being conduct-
ed to explore possible mechanisms for the observed antinoci-
ceptive activity of these cyclic tetrapeptides.
Fmoc-protected amino acids (4 equiv) were coupled to the resin
using PyBOP (4 equiv), HOBt (4 equiv), and DIEA (8 equiv) in
CH2Cl2/DMF (1:1) for 2–4 h. The resin was washed after the cou-
pling reactions with CH2Cl2/DMF (1:1, 5ꢁ) and CH2Cl2 (5ꢁ). The re-
actions were monitored to determine completion using the Kaiser
test for primary amines or the chloranil test for the secondary
amine of Pro. The Fmoc group was then removed as described
above, and the deprotection/coupling cycle repeated to assemble
the linear tetrapeptides. Finally, the resin was washed with CH2Cl2/
DMF (1:1, 5ꢁ), CH2Cl2 (10ꢁ), iPrOH (2ꢁ), hexane (2ꢁ), CH2Cl2 (2ꢁ),
MeOH (2ꢁ), and finally CH2Cl2 (2ꢁ).
The peptides were cleaved from the resin using 1% TFA in CH2Cl2.
Following swelling of the resin in CH2Cl2, the TFA solution was
mixed with the resin (5 mL ꢁ 2 min ꢁ 10), and the cleavage solu-
tion was drained into a round-bottom flask. This procedure was re-
peated until all of the cleavage solution was collected in the flask.
Following cleavage, the resin was washed with CH2Cl2 (2ꢁ) and
MeOH (2ꢁ). The combined solutions were evaporated to give the
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crude linear tetrapeptides, which were used in the cyclizations
without purification.
100 mm NaCl, 1 mgmLÀ1 bovine serum albumin, and ~100000 dis-
integrations per min (dpm) [35S]GTPgS (0.1–0.2 nm). Approximately
10 mg KOR- or MOR-expressing CHO cell membrane protein was
used per tube. Following 90 min incubation at 228C, the assay was
terminated by filtration under vacuum on a Brandel (Gaithersburg,
MD, USA) model M-48R cell harvester using Schleicher and Schuell
Inc. (Keene, NH, USA) number 32 glass fiber filters. The filters were
rinsed with 4ꢁ4 mL washes of ice-cold 50 mm Tris-HCl, pH 7.4,
5 mm MgCl, at 58C to remove unbound [35S]GTPgS. Filter disks
were then placed into counting vials to which 8 mL Biocount scin-
tillation fluid (Research Products International Corp., Mount Pros-
pect, IL, USA) was added. Filter-bound radioactivity was deter-
mined by liquid scintillation spectrometry (Beckman Instruments,
Fullerton, CA, USA) following overnight extraction at room temper-
ature. The amount of radioligand bound was <10% of the total
added in all experiments. Specific binding is defined as total bind-
ing minus that occurring in the presence of 3 mm unlabeled GTPgS.
Nonspecific binding was ~1% of the total binding at 0.1 nm
[35S]GTPgS.
Cyclization reaction and final deprotection. The linear peptides were
cyclized as follows: The crude linear peptide (0.5 equiv) in DMF (5–
10 mL) was added dropwise at a rate of 1.6 mLhÀ1 (using a KD Sci-
entific single infusion syringe pump and a 10 mL syringe) to a solu-
tion of HATU (0.75 equiv, 1 mm) and DIEA (8 equiv) in DMF over
6 h. After 6 h a second portion of HATU (0.75 equiv) was added to
the reaction in one portion, and a second portion of linear peptide
(0.5 mmol) in DMF (5–10 mL) was added dropwise at a rate of
1.6 mLhÀ1 as described above. The reaction was then allowed to
stir for an additional 12–24 h. Following removal of the solvent
under reduced pressure, the residue was dissolved in EtOAc/Et2O
(4:1) or CH2Cl2 and the solution washed with 1n citric acid (2ꢁ),
saturated bicarbonate (2ꢁ) and brine (2ꢁ). The organic layer was
separated, dried (Na2SO4), and the solvent was removed under re-
duced pressure to give the crude cyclic peptide. This workup was
not performed for peptide 5 because of its water solubility; in-
stead, following removal of the DMF the crude peptide was dis-
solved in water and lyophilized.
To evaluate the peptides for agonist activity, the membranes were
incubated with ten different concentrations of peptide (0.01 nm to
1 mm). The antagonist activity of the peptides was determined by
measuring the EC50 of an agonist (dynorphin A-(1–13)NH2 for KOR
and DAMGO for MOR) in the absence or presence of four different
concentrations (10 nm to 3 mm) of the peptide. The pA2 was deter-
mined by Schild analysis,[21] and the results are reported as KB
values.
The Boc (tert-butyloxycarbonyl) group on the indole group of Trp
in the cyclic precursors of 2–4 was then removed by treating a so-
lution of the cyclic peptide in CH2Cl2 (1 mL) with 50% TFA in
CH2Cl2 (2 mL) for 30 min. The solution was then evaporated, and
the peptide triturated with 10% aqueous AcOH; the peptide was
then dried by lyophilization.
Purification and characterization. The cyclic peptides were purified
by reversed-phase HPLC (30–70% aqueous MeOH over 40 min,
except for peptide 5, for which the gradient was 20–60% aqueous
MeOH over 40 min). The cyclic peptides were characterized by re-
versed-phase HPLC and electrospray ionization mass spectrometry
(see Supporting Information).
In vivo pharmacological evaluation
Animals. 317 adult male C57Bl/6J mice weighing 20–25 g were ob-
tained from Jackson Labs (Bar Harbor, ME, USA), and were housed
and cared for in accordance with the 2002 National Institutes of
Health Guide for the Care and Use of Laboratory Animals and as
approved by the Torrey Pines Institute for Molecular Studies Insti-
tutional Animal Care Committee, operating under the OLAW ap-
proval number A4618-01. All mice were group housed, four to a
cage, in self-standing plastic cages within the animal care facility.
The colony room was illuminated on a 12 h light–dark cycle, with
the lights on at 7:00 each morning. Food pellets and distilled
water were available ad libitum. Note that C57Bl/6J mice were se-
lected for this study because of their established responses to ther-
mal noxious stimuli and antinociceptive testing.[22] All compounds
other than the peptides were obtained from Sigma (St. Louis, MO,
USA).
In vitro pharmacological evaluation
Radioligand binding assays. Opioid receptor affinities were deter-
mined in radioligand binding assays using membranes from Chi-
nese hamster ovary (CHO) cells stably expressing KOR, MOR, or
DOR as previously described.[13] Incubations with isolated mem-
brane protein were performed in triplicate with 12 different con-
centrations from 0.1 nm to 10 mm of the cyclic tetrapeptides for
90 min in 50 mm Tris, pH 7.4, at 228C using [3H]diprenorphine,
[3H]DAMGO, and [3H]DPDPE as the respective radioligands for KOR,
MOR, and DOR. Nonspecific binding was determined in the pres-
ence of 10 mm unlabeled dynorphin A-(1–13)NH2, DAMGO, and
DPDPE for KOR, MOR, and DOR, respectively. Reactions were termi-
nated by rapid filtration over Whatman GF/B fiber filters using a
Brandel M24-R cell harvester, and the filters were counted in 4 mL
Cytocint (ICN Radiochemicals) using a Beckman LS6800 scintillation
counter. IC50 values were determined by nonlinear regression anal-
ysis to fit a logistic equation to the competition data using Prism
software (GraphPad Software, La Jolla, CA, USA). Ki values were cal-
culated from the IC50 values by the Cheng and Prusoff equation[19]
using KD values of 0.45, 0.49, and 1.76 nm for [3H]diprenorphine,
[3H]DAMGO, and [3H]DPDPE, respectively. The results presented are
the mean ÆSEM from at least three separate assays.
Intracerebroventricular administration technique. Intracerebroventric-
ular (i.c.v.) injections were made directly into the lateral ventricle
according to the modified method of Haley and McCormick.[23] The
volume of all i.c.v. injections was 5 mL, using a 10 mL Hamilton mi-
croliter syringe. The mouse was lightly anesthetized with isoflur-
ane, an incision was made in the scalp, and the injection was
made 2 mm lateral and 2 mm caudal to bregma at a depth of
3 mm.
Antinociceptive testing. The 558C warm-water tail-withdrawal assay
was performed in C57Bl/6J mice as previously described.[24] Briefly,
warm (558C) water in a 2 L heated water bath was used as the
thermal nociceptive stimulus, with the latency of the mouse to
withdraw its tail from the water taken as the endpoint. After deter-
mining baseline tail-withdrawal latencies, mice were administered
a graded dose of compound though the i.c.v. route. Intracerebro-
ventricular injections (5 mL, using a 10 mL Hamilton syringe) were
performed as described above; the cyclic tetrapeptides were ad-
GTPgS assays. The binding of the GTP analogue [35S]GTPgS to
membranes was assayed following the method described by Siebe-
nallar and Murray.[20] Binding was determined in a volume of
500 mL. The assay mixture contained 50 mm HEPES, pH 7.4, 1 mm
EDTA, 5 mm magnesium acetate, 1 mm GDP, 1 mm dithiothreitol,
1744
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1739 – 1745

Opioid Activity Profiles of CJ-15,208 Analogues
[1] J. V. Aldrich, J. P. McLaughlin, AAPS J. 2009, 11, 312–322.
[2] a) S. D. Mague, A. M. Pliakas, M. S. Todtenkopf, H. C. Tomasiewicz, Y.
Zhang, W. C. J. Stevens, R. M. Jones, P. S. Portoghese, W. A. J. Carlezon, J.
Pharmacol. Exp. Ther. 2003, 305, 323–330; b) J. P. McLaughlin, M.
Marton-Popovici, C. Chavkin, J. Neurosci. 2003, 23, 5674–5683; c) P. M.
Beardsley, J. L. Howard, K. L. Shelton, F. I. Carroll, Psychopharmacology
(Berlin) 2005, 183, 118–126.
ministered in 50% DMSO/50% sterile saline (0.9%). To determine
agonist activity, the tail-withdrawal latency was determined every
10 min following administration of a cyclic tetrapeptide for 3 h, or
until latencies returned to baseline values.
A cutoff time of 15 s was used in this study; if the mouse failed to
display a tail-withdrawal response during that time, the tail was re-
moved from the water, and the animal was assigned a maximal an-
tinociceptive score of 100%. At each time point, antinociception
was calculated according to the following formula: percent antino-
ciception=100ꢁ(test latencyÀcontrol latency)/(15Àcontrol laten-
cy).
[3] A. T. Knoll, E. G. Meloni, J. B. Thomas, F. I. Carroll, W. A. Carlezon, Jr., J.
Pharmacol. Exp. Ther. 2007, 323, 838–845.
[4] V. A. Redila, C. Chavkin, Psychopharmacology (Berlin) 2008, 200, 59–70.
[5] a) R. B. Rothman, D. A. Gorelick, S. J. Heishman, P. R. Eichmiller, B. H. Hill,
J. Norbeck, J. G. Liberto, J. Subst. Abuse Treat. 2000, 18, 277–281; b) G.
Gerra, A. Fantoma, A. Zaimovic, J. Psychopharmacol. 2006, 20, 806–814.
[6] M. D. Metcalf, A. Coop, AAPS J. 2005, 7, E704–722.
To determine the opioid receptor selectivity of the agonist activity
of peptides 2–5, mice were pretreated with a single dose of b-FNA
(5 mgkgÀ1, s.c.) or nor-BNI (10 mgkgÀ1, i.p.) 23.3 h in advance of
administration of a graded dose of a cyclic tetrapeptide compound
(0.1–30 nmol, i.c.v.). Additional mice were pretreated prior to the
administration of a cyclic tetrapeptide compound with the nonse-
lective opioid receptor antagonist naloxone (15 nmol, i.c.v.,
À25 min), or naltrindole (20 mgkgÀ1, i.p., À15 min), with antinoci-
ceptive testing 40 min later. Reference agonists and antagonists
were administered using sterile saline (0.9%) as the vehicle, except
for SNC-80, which was dissolved in 35% DMSO/65% saline.
[7] a) P. Horan, J. Taylor, H. I. Yamamura, F. Porreca, J. Pharmacol. Exp. Ther.
1992, 260, 1237–1243; b) F. I. Carroll, L. S. Harris, M. D. Aceto, Eur. J.
Pharmacol. 2005, 524, 89–94.
[8] a) Q. Wan, T. F. Murray, J. V. Aldrich, J. Med. Chem. 1999, 42, 3011–3013;
b) Y. Lu, T. M.-D. Nguyen, G. Weltrowska, I. Berezowska, C. Lemieux, N. N.
Chung, P. W. Schiller, J. Med. Chem. 2001, 44, 3048–3053; c) M. A. Ben-
nett, T. F. Murray, J. V. Aldrich, J. Med. Chem. 2002, 45, 5617–5619;
d) B. S. Vig, T. F. Murray, J. V. Aldrich, J. Med. Chem. 2003, 46, 1279–
1282; e) K. A. Patkar, X. Yan, T. F. Murray, J. V. Aldrich, J. Med. Chem.
2005, 48, 4500–4503.
[9] J. V. Aldrich, K. A. Patkar, J. P. McLaughlin, Proc. Natl. Acad. Sci. USA
2009, 106, 18396–18401.
[10] T. Saito, H. Hirai, Y.-J. Kim, Y. Kojima, Y. Matsunaga, H. Nishida, T. Sakaki-
bara, O. Suga, T. Sujaku, N. Kojima, J. Antibiot. 2002, 55, 847–854.
[11] a) S. S. Kulkarni, N. C. Ross, J. P. McLaughlin, J. V. Aldrich, Adv. Exp. Med.
Biol. 2009, 611, 269–270; b) N. C. Ross, S. S. Kulkarni, J. P. McLaughlin,
J. V. Aldrich, Tetrahedron Lett. 2010, 51, 5020–5023.
[12] a) N. C. Ross, K. J. Reilley, T. F. Murray, J. V. Aldrich, J. P. McLaughlin, Br. J.
Pharmacol., DOI: 10.1111/j.1476-5381.2011.01544.x; b) R. E. Dolle, M. Mi-
chaut, B. Martinez-Teipel, P. R. Seida, C. W. Ajello, A. L. Muller, R. N. De-
Haven, P. J. Carroll, Bioorg. Med. Chem. Lett. 2009, 19, 3647–3650.
[13] S. Arttamangkul, J. E. Ishmael, T. F. Murray, D. K. Grandy, G. E. DeLander,
B. L. Kieffer, J. V. Aldrich, J. Med. Chem. 1997, 40, 1211–1218.
[14] K. J. Reilley, M. Giulianotti, C. T. Dooley, A. Nefzi, J. P. McLaughlin, R. A.
Houghten, AAPS J. 2010, 12, 318–329.
[15] a) M. Ohsawa, H. Mizoguchi, M. Narita, H. Nagase, J. P. Kampine, L. F.
Tseng, J. Pharmacol. Exp. Ther. 2001, 298, 592–597; b) M. Ohsawa, M.
Shiraki, H. Mizoguchi, M. Narita, K. Kawai, H. Nagase, E. Y. Cheng, L. F.
Tseng, Neurosci. Lett. 2001, 316, 1–4; c) S. Sakurada, T. Hayashi, M.
Yuhki, T. Orito, J. E. Zadina, A. J. Kastin, T. Fujimura, K. Murayama, C. Sa-
kurada, T. Sakurada, M. Narita, T. Suzuki, K. Tan-no, L. F. Tseng, Eur. J.
Pharmacol. 2001, 427, 203–210; d) H. H. Szeto, Y. Soong, D. Wu, X.
Qian, G. M. Zhao, J. Pharmacol. Exp. Ther. 2003, 305, 696–702; e) H. Miz-
oguchi, G. Bagetta, T. Sakurada, S. Sakurada, Peptides 2011, 32, 421–
427.
To determine antagonist activity, mice were pretreated with a
cyclic tetrapeptide 150 min prior to administration of the MOR-pre-
ferring agonist morphine (10 mgkgÀ1, i.p.), the KOR-selective ago-
nist U50,488 (10 mgkgÀ1, i.p.) or the DOR-selective agonist SNC-80
(100 nmol, i.c.v.). Antinociception produced by these established
agonists was then measured 30 min after administration. Addition-
ally, to determine the duration of KOR antagonist activity, addition-
al mice were pretreated for 7.3, 17.3, or 23.3 h prior to administra-
tion of U50,488 as described above.
Statistical analysis. Radioligand binding results represent the mean
ÆSEM obtained from 3–5 independent experiments, each per-
formed in triplicate. IC50 values were calculated by least-squares fit
to a logarithm–probit analysis. The Ki values of unlabeled com-
pounds were calculated from the equation Ki =IC50/(1+S), where
S=(concentration of radioligand)/(KD of radioligand),[19] and report-
ed as the mean ÆSEM of at least three independent experiments.
KB values from the GTPgS assay represent the mean ÆSEM from
2–4 experiments.
All tail-withdrawal data points shown are the means of 7–16 mice,
with SEM represented by error bars. Data for antinociception ex-
periments were analyzed with ANOVA using the Prism 5.0 software
package (GraphPad, La Jolla, CA, USA). Analyses examined the
main effect of baseline and post-treatment tail-withdrawal laten-
cies to determine statistical significance for all tail-withdrawal data.
Significant effects were further analyzed using Tukey’s HSD post-
hoc testing. All data are presented as mean ÆSEM, with signifi-
cance set at p<0.05.
[16] M. Shimoyama, N. Shimoyama, G. M. Zhao, P. W. Schiller, H. H. Szeto, J.
Pharmacol. Exp. Ther. 2001, 297, 364–371.
[17] B. S. Vig, J. V. Aldrich, Aldrichimica Acta 2004, 37, 2.
[18] W. C. Chan, P. D. White, Fmoc Solid Phase Peptide Synthesis: A Practical
Approach, Oxford University Press, New York, 2000, pp. 41–76.
[19] Y. C. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099–3108.
[20] J. F. Siebenaller, T. F. Murray, Biol. Bull.-US 1999, 197, 388–394.
[21] H. O. Schild, Br. J. Pharmacol. Chemother. 1947, 2, 189–206.
[22] a) J. S. Mogil, B. Kest, B. Sadowski, J. K. Belknap, J. Pharmacol. Exp. Ther.
1996, 276, 532–544; b) B. Kest, E. Hopkins, C. A. Palmese, M. Adler, J. S.
Mogil, Pharmacol. Biochem. Behav. 2002, 73, 821–828.
Acknowledgements
[23] T. J. Haley, W. G. McCormick, Br. J. Pharmacol. Chemother. 1957, 12, 12–
15.
This research was supported by grants from the National Institute
on Drug Abuse R01 DA018832 and R01 DA023924 and funds
from the State of Florida.
[24] J. P. McLaughlin, K. P. Hill, Q. Jiang, A. Sebastian, S. Archer, J. M. Bidlack,
J. Pharmacol. Exp. Ther. 1999, 289, 304–311.
Received: February 23, 2011
Revised: June 7, 2011
Keywords: analgesics
·
k opioid receptor antagonists
·
peptides · structure–activity relationships
Published online on July 14, 2011
ChemMedChem 2011, 6, 1739 – 1745
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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