Opioid activity profiles of oversimplified peptides lacking in the protonable N-terminus

Article abstract of DOI:10.1021/jm301213s

Recently, we described cyclopeptide opioid agonists containing the D-Trp-Phe sequence. To expand the scope of this atypical pharmacophore, we tested the activity profiles of the linear peptides Ac-Xaa-Phe-Yaa (Xaa = L/D-Trp, D-His/Lys/Arg; Yaa = H, GlyNH₂). Ac-D-Trp-PheNH₂ appeared to be the minimal binding sequence, while Ac-D-Trp-Phe-GlyNH ₂ emerged as the first noncationizable short peptide (partial) agonist with high μ-opioid receptor affinity and selectivity. Conformational analysis suggested that 5 adopts in solution a β-turn conformation.

Full text of DOI:10.1021/jm301213s

Brief Article
pubs.acs.org/jmc
Opioid Activity Profiles of Oversimplified Peptides Lacking in the
Protonable N‑Terminus
Rossella De Marco,† Alessandra Tolomelli,† Santi Spampinato,*,‡ Andrea Bedini,‡ and Luca Gentilucci*,†
†Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
‡Department of Pharmacology, University of Bologna, Via Irnerio 42, Bologna, Italy
S
* Supporting Information
ABSTRACT: Recently, we described cyclopeptide opioid agonists containing the D-Trp-Phe sequence. To expand the scope of
this atypical pharmacophore, we tested the activity profiles of the linear peptides Ac-Xaa-Phe-Yaa (Xaa = L/D-Trp, D-His/Lys/
Arg; Yaa = H, GlyNH2). Ac-D-Trp-PheNH2 appeared to be the minimal binding sequence, while Ac-D-Trp-Phe-GlyNH2 emerged
as the first noncationizable short peptide (partial) agonist with high μ-opioid receptor affinity and selectivity. Conformational
analysis suggested that 5 adopts in solution a β-turn conformation.
INTRODUCTION
■
It is generally accepted that the fundamental binding
interaction between the opioid receptors and their agonists is
represented by the ionic bridge between the conserved
Asp(3.32) of the receptor and a positively charged N-terminal
amine of the ligand.1 Indeed, the removal or derivatization of
this group normally gave inactive ligands or antagonists.2−7
Neverthelsess, there is recent evidence that some compounds
deprived of a protonable amine exhibit full or partial opioid
agonism: the diterpene salvinorin A8 and its many deriva-
tives;9,10 a bicyclic enkephalin mimetic;11 the cyclic analogue of
endomorphin-1 (EM1)12 c[Tyr-D-Pro-D-Trp-Phe-Gly] (1)13
and the correlated c[Tyr-Gly-D-Trp-Phe-Gly] and c[D-Asp-1-
amide-β-Ala-D-Trp-Phe] (2);14 a cyclic enkephalin analogue
containing 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid
(Dhp) in the place of Tyr;15 a “carba” analogue of fentanyl,
in which the classic piperidine nitrogen was replaced by a
carbon but equipped with a ionizable guanidino group.16
Figure 1. Structures of the cyclic EM1 analogues 1 and 2, of 3, and of
the Ac di- and tripeptides 4−12.
Compounds 1 and 2 represent the first examples of peptides
described in the literature whose μ-opioid receptor (MOR)
agonist activities reside in the aromatic side chains. Molecular
docking analysis revealed a inverse type II β-turn conformation
centered on D-Trp-Phe, relevant contacts with Trp318(7.35),
Trp293(6.48), and a fundamental binding interaction between
motif. Apparently, the stereochemistry and relative disposition
of Trp and Phe, the macrocycle size, and secondary structure
strongly impact affinity and selectivity and agonism vs
antagonism.
the indolic NH of D-Trp and the carboxylate of Asp147(3.32),
To determine the minimal bioactive sequence of 1 and 2 and
as sketched for 2 in Figure 1.
to expand the scope of the novel opioid pharmacophore, we
In the past few years, very similar sequences have been
designed (Figure 1) and tested the linear analogues Ac-D-Trp-
PheNH2 (4), Ac-D-Trp-Phe-GlyNH2 (5), the tripeptide 6
containing L-Trp, and the di- or tripeptides 7−12 containing
amino acids with a protonable side chain, D-His, D-Lys, and D-
Arg.
reproposed in the derivatives of the naturally occurring
cyclotetrapeptide c[D-Pro-Phe-Trp-Phe] (3, CJ-15,208),17
a
opioid ligand that preferentially binds to κ-opioid receptor
(KOR). The introduction of a D-configured Trp gave c[D-Pro-
Phe-D-Trp-Phe] and c[D-Pro-Phe-D-Trp-Ala], more potent than
the natural product.18 These compounds did not exhibit any
agonist activity in vitro; however, unexpected agonist activity in
vivo was observed for 3 and some analogues in the warm water
tail withdrawal antinociceptive assay, mediated predominantly
by MOR.19
RESULTS AND DISCUSSION
The peptides were prepared in solution under MW irradiation
from N-Ac amino acids and H-PheNH2 or H-Phe-GlyNH2,
■
These results support the hypothesis that the D-Trp-Phe
sequence constitutes an unusual kind of MOR pharmacophoric
Received: August 20, 2012
Published: September 20, 2012
© 2012 American Chemical Society
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Table 1. In Vitro Activity Profiles of the Ac Di- or Tripeptides 4−12 and of the Reference Compounds 2, DAMGO, DPDPE,
a
U50,488 toward the Cloned Human Opioid Receptors Expressed on HEK-293 Cells
purity
b
ESI-MS (m/z)
[M + 1] vs calcd
compd
sequence
[%]
Kiμ (nM)
1.5 0.1
Kiδ (nM)
Kiκ (nM)
nH
DAMGO
H-Tyr-D-Ala-Gly-NMePhe-glyol
DPDPE
H-Tyr-c[D-Pen-Gly-Phe-D-Pen]OH
3.30 0.05
U50,488
2.90 0.04
>105
c
2
c[D-Asp-1-amide-β-Ala-D-Trp-Phe]
Ac-D-Trp-PheNH2
5.9 0.1
15.5 0.5
5.6 0.2
(7.4 0.8) × 104
>105
>105
d
4
96
97
98
96
97
97
96
96
95
393.0/393.2
450.1/450.2
450.2/450.2
344.1/344.2
401.0/401.2
335.3/335.2
392.2/392.2
363.2/363.2
420.0/420.2
24
>105
9
>105
0.8 0.2
0.9 0.1
5
Ac-D-Trp-Phe-GlyNH2
Ac-Trp-Phe-GlyNH2
>105
d
6
12.1 0.4
>105
(3.3 0.8) × 104
>105
e
7
Ac-D-His-PheNH2
e
8
Ac-D-His-Phe-GlyNH2
>105
>105
>105
e
9
Ac-D-Lys-PheNH2
>105
>105
>105
e
10
11
12
Ac-D-Lys-Phe-GlyNH2
>105
>105
>105
e
Ac-D-Arg-PheNH2
>105
>105
>105
e
Ac-D-Arg-Phe-GlyNH2
>105
>105
>105
a
b
c
d
Mean of four to six determinations
SE. Determined by analytical RP-HPLC; see General Methods. Reference 14. [3H]Diprenorphine
e
displacement of <50%. TFA.
using hydroxybenzotriazole/O-benzotriazole-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate/diisopropylethylamine
(HOBt/HBTU/DIPEA) as coupling agents. H-Phe-GlyNH2
was obtained by coupling Boc-PheOH and H-GlyNH2; Boc
cleavage was performed with TFA. The peptides were obtained
pure (95−98%) after semipreparative reversed-phase (RP)
HPLC. The presence of traces of free amino groups at the N-
terminus was excluded on the basis of RP-HPLC/ESI-MS and
spectroscopic analyses.
The affinities toward the cloned human MOR, δ-opioid
receptor (DOR), and KOR, stably expressed on HEK-293 cells,
were determined by displacement binding assays. [3H]-
DAMGO, [3H]diprenorphine, and [3H]U69,593 were em-
ployed as specific radioligands for MOR, DOR, and KOR,
respectively. DAMGO, DPDPE, and U50,488 were chosen as
reference compounds for MOR, DOR, and KOR. The tested
peptides and the reference compounds were all used in the
10−12−10−4 M range; the calculated Ki values are reported in
Table 1.
exhibited low to moderate δ antagonist activity. The recently
reported tetrapeptides Ac-D-Pro-Phe-D-Trp-PheNH2 and Ac-
Phe-D-Trp-Phe-D-ProNH2, derived from 3 by systematic single
amide bond cleavage, displayed a modest 10−7 M potency at
the MOR.18
Given that 4 and 5 were the only peptides with a significant
affinity for MOR, their biological activities were measured by
evaluating their effects on forskolin-stimulated cAMP accumu-
lation in whole HEK-293 cells stably expressing MOR (Table
2). Morphine was assayed under the same experimental
Table 2. Inhibitory Effects of Morphine, 2, 4, 5 on Forskolin-
Induced cAMP Formation in HEK-293 Stably Expressing
a
MOR
b
c
compd
IC50 (nM)
4.3 0.4
Emax (% vehicle)
morphine
d
77
58
4
5
2
37
5
4
5
>105
200 30
52 3**
DAMGO,13,20 DPDPE,21 and U50,48822 all showed Ki in the
nanomolar range, in agreement with the literature. As for the
tested peptides, Ac-D-Trp-PheNH2 (4) and Ac-D-Trp-Phe-
GlyNH2 (5) displayed an easily measurable, concentration-
dependent displacement of [3H]DAMGO from MOR, with Kiμ
values of 15.5 × 10−9 and 5.6 × 10−9 M, respectively (Table 1),
comparable to that of cyclic 2.14 The Hill coefficients (nH) are
not significantly different from unity. Interestingly, 5 did not
show any relevant affinity toward DOR or KOR, as it does not
displace [3H]diprenorphine and [3H]U69,593. On the other
hand, 4 was less selective, showing a modest ability to displace
[3H]diprenorphine from DOR (Table 1). The tripeptide Ac-
Trp-Phe-GlyNH2 (6) displayed very poor affinity toward MOR
and KOR and displaced [3H]diprenorphine from DOR only
slightly (Table 1). Finally, compounds 7−12 did not show any
significant displacement of the radioligands employed in this
study (Table 1).
a
Mean
SE of five to six independent experiments performed in
triplicate; data were analyzed by one-way ANOVA followed by
Tukey’s test. IC50 is the half-maximal inhibitory concentration. Emax
is the maximal obtainable effect. Reference 14. ∗∗, P < 0.01 vs
morphine and 2.
b
c
d
conditions to compare the activity of the tested peptides to
this prototypical opioid analgesic drug. Furthermore, we report
the activity of cyclic 2, which we described in a previous
study.14
As expected, morphine significantly inhibited forskolin-
induced cAMP accumulation with IC50 = 4.3 nM and Emax
=
77%. The cyclic peptide 2 had IC50 = 37 nM and Emax = 58%
(Table 2). For peptide 5, it also significantly inhibited forskolin-
induced cAMP accumulation; however, it was less potent than
morphine or the cyclic 2, with IC50 = 200 nM and a Emax = 52%
(Table 2). This latter finding suggests that 5 behaves as a partial
agonist. The dipeptide 4 did not produce any significant effect,
thus appearing to lack agonist activity albeit it is capable of
binding to the MOR (Table 1).
To our knowledge, peptides 4 and 5, derived from the
cyclopeptides 1 and 2 and designed on the basis of molecular
docking insights of the bioactive conformation, represent the
first noncationizable linear peptides with nanomolar affinity for
MOR. The dipeptides c[Dmt-Tic] (Dmt, 2′,6′-dimethyl-Tyr)
and Ac-Dmt-Tic-NH2, previously described by Balboni et al.,23
The “partial” agonist activity of 5 makes sense, as EM1 and
EM2 have been reported to behave as partial agonists at
MOR,24,25 their full agonism being shown only in a few cases.26
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The tripeptide 5 is probably less effective in activating the
MOR compared to morphine as a consequence of its minimal
and more flexible structure, whereas the dipeptide 4, deprived
of the glycine, does not bind the MOR in the same way as 5
and lacks of MOR agonist activity on forskolin-induced cAMP
accumulation.
The significant receptor affinity of 4 indicates that the
dipeptide D-Trp-Phe represents the minimal bioactive sequence
for receptor binding but not for agonism. The opposite
configuration of Trp in 5 and 6 seems to be responsible for
tuning selectivity from exclusively binding MOR to some
affinity for DOR. A comparison between the different results of
4−6 and the other peptides highlighted the uniqueness of the
indole of D-Trp for efficient binding to MOR in this class of
atypical opioid peptides. Despite the presence of amino acids
with protonable side chains, 7−12 had no opioid activity.
The different profiles of the structurally correlated 4 and 5
prompted us to investigate the preferred conformations in
solution. Conformational analyses were performed by NMR
spectroscopy and molecular dynamics (MD) simulations.
Because of the negligible solubility in water, NMR spectra
were recorded in the biomimetic medium 8:2 DMSO-d6/
H2O.27 The higher ability of DMSO to mimic the environment
near the receptor with respect to pure water has been recently
commented.28
Figure 2. (Left and middle) Representative low-energy structures of 4
and 5, consistent with ROESY analysis, calculated by restrained MD,
and representative low-energy structure 5A (right) showing a well-
defined inverse β-turn, determined by unrestrained MD simulations.
β-turn centered on D-Trp-Phe. Generally, the formation of
inverse turns is favored by a D-residue in the position i + 1.29
Explicit H-bonds predicted by VT-NMR are not present
probably because of a fast equilibrium between different
geometries.
To investigate the dynamic behavior, the structures were
analyzed by unrestrained MD in a box of water molecules.
Besides the different random conformations, the analyses of the
trajectories of 5, but not 4, revealed the occurrence of well-
defined β-turn secondary structures stabilized by a clear H-
bond between D-TrpCO and GlyNH (Figure 2, 5A).
The predisposition to adopt the β-turn might be correlated
to the high affinity and selectivity of 5 and to the agonist
behavior. Nevertheless, the presence of the third residue
GlyNH2 can also strongly influence the binding mode by
interacting with an individual residue within the address locus
of the MOR, such as Glu229(5.35), in a similar way as
proposed for 2 (Figure 1).14
1
The H NMR of 4 and 5 revealed a single set of sharp
resonances suggestive of conformational homogeneity or a fast
equilibrium between conformers; gCOSY allowed the un-
ambiguous assignment of all the resonances.
VT-1H NMR experiments were used to deduce the presence
of H-bonds (Table 3). For 4, the Δδ/ΔT (ppb/K) of the
a
Table 3. Δδ/Δt [ppb/K] of Amide Protons for 4 and 5
CONCLUSIONS
compd
D-TrpNH
PheNH
YaaNH
CONH2
■
In this work we discussed a minilibrary of minimalist peptides
having the N-terminus acetylated. The peptides Ac-D-Trp-
PheNH2 (4) and Ac-D-Trp-Phe-GlyNH2 (5) turned out as
opioid ligands with nanomolar affinity. While 4 did not show
any agonist activity on MOR, 5 was a selective, partial MOR
agonist. The comparison with the structurally correlated 6−12
stressed the role of D-Trp and pointed out that the presence of
a basic amino group is not a sine qua non1,13,15 for a proper
interaction and activation of MOR. The peptides 4 and 5 might
represent interesting candidates for the development of a new
class of liphophilic MOR-active peptidomimetics.33 Conforma-
tional analysis showed that 4 and 5 adopt in solution similar
conformations; nevertheless, 5 tends to fold into a well-defined
β-turn, which might contribute to higher affinity and selectivity.
4
5
−6.6
−8.0
−10.1
−7.6
b
−1.1/−8.6
−2.4/−7.6
c
0.3
a
VT-NMR analysis in 8:2 DMSO-d6/H2O at 400 MHz over the range
b
c
298−348 K. Yaa = H. Yaa = GlyNH2.
PheCONH2 proton at 7.1 ppm was −1.1, comparatively lower
than that of the other amide protons, indicating some folded
population stabilized by a H-bond (|Δδ/Δt| ≲ 2).29 For 5, the
very low Δδ/ΔT of GlyNH of 0.3 is compatible with structures
in which the amide proton is involved in a strong H-bond,
supporting the speculation that 5 could be conformationally
more homogeneous and well-defined.
Molecular backbone conformations were investigated by 2D
ROESY; the intensities of the cross-peak were ranked to infer
plausible interproton distances (Tables S1 and S2). Structures
consistent with the spectroscopic analyses were obtained by
MD simulations30 in a box of explicit water molecules,31
starting with a set of random geometries. The structures were
subjected to high-temperature MD using the distances derived
from ROESY as constraints with a scaled force field, followed
by a simulation with full restraints. The system was gradually
cooled, and the structures were minimized with the AMBER32
force field. The results were clustered by the rmsd analysis of
the backbone atoms.
EXPERIMENTAL SECTION
■
General Methods. Unless stated otherwise, chemicals were
obtained from commercial sources and used without further
purification. The MW-assisted synthesis was performed using a
MicroSYNTH microwave labstation. Flash chromatography was
performed on silica gel (230−400 mesh), using mixtures of distilled
solvents. Purities were determined to be ≥95% by analytical RP-HPLC
and combustion analysis. Analytical RP-HPLC was performed on an
ODS column (4.6 μm particle size, 100 Å pore diameter, 250 μm,
DAD 210 nm, from a 9:1 H2O/CH3CN to a 2:8 H2O/CH3CN in 20
min) at a flow rate of 1.0 mL/min, followed by 10 min at the same
composition. Elemental analyses were performer using a Thermo Flash
2000 CHNS/O analyzer. Semipreparative RP-HPLC was performed
on a C18 column (7 μm particle size, 21.2 mm × 150 mm, from 8:2
H2O/CH3CN to 100% CH3CN in 10 min) at a flow rate of 12 mL/
Computations essentially gave one major cluster for each
compound. The representative geometries of 4 and 5 with the
lowest internal energy were selected and analyzed (Figure 2).
These structures are roughly compatible with an inverse type II
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min. RP-HPLC of 7−12 were performed as reported above, with the
medium containing 0.5 mM 3-isobutyl-1-methylxanthine (Sigma−
Aldrich) and exposed for 15 min to 10 μM forskolin without and with
each compound (0.01 nM to 100 μM) at 37 °C. Cells were then lysed
in 0.1 N HCl, scraped off, and centrifuged (2000g, 5 min).
Supernatants were assayed for cAMP concentration by using a
cAMP EIA kit (Cayman Chemical Co., Ann Arbor, MI, U.S.)
according to the manufacturer’s instructions. Each well was
determined individually. The triplicates were averaged, and IC50
values were determined. Activities were expressed as percent inhibition
of forskolin-induced cAMP production.
Conformational Analysis. ROESY cross-peak intensities in 8:2
DMSO-d6/H2O were classified as very strong, strong, medium, and
weak and associated with distances of 2.2, 2.6, 3.0, and 4.2 Å,
respectively. Geminal and obvious correlations were discarded. For the
absence of Hα(i,i+1) cross peaks, the ω bonds were set at 180° (force
constant: of 16 kcal mol−1 Å−2). The restrained MD simulations were
conducted using the AMBER force field in a 30 Å × 30 Å × 30 Å box
of standard TIP3P models of equilibrated water. All water molecules
with atoms that come closer than 2.3 Å to a solute atom were
eliminated. A 50 ps simulation at 1200 K was used for generating 50
random structures that were subsequently subjected to a 20 ps
restrained MD with a 50% scaled force field at the same temperature,
followed by 20 ps with full restraints (distance force constant of 7 kcal
mol−1 Å−2), after which the system was cooled in 5 ps to 50 K. H-
Bond interactions were not included nor were torsion angle restraints.
The resulting structures were minimized with 3000 cycles of the
steepest descent and 3000 cycles of the conjugated gradient
(convergence of 0.01 kcal Å−1 mol−1). The backbones of the
structures were clustered by the rmsd analysis module of HyperChem.
Unrestrained MD simulation in explicit water was performed for 10 ns
at 298 K at constant temperature and pressure (Berendsen scheme,35
bath relaxation constant of 0.2). For 1−4 scale factors, van der Waals
and electrostatic interactions are scaled in AMBER to half their
nominal values. The integration time step was set to 0.1 fs. Box
equilibration was set to 10 ps.
addition of 0.1% TFA in the mobile phase. Mass analysis was done by
1
ESI. H NMR and 13C NMR spectra were recorded at 400 and 100
MHz, respectively, in 5 mm tubes, at rt. Chemical shifts are reported as
δ values relative to the solvent peak. 2D spectra were recorded in the
phase sensitive mode and processed using a 90°-shifted, squared sine-
bell apodization.
Synthesis of 5. HOBt (1.2 mmol) and HBTU (1.2 mmol) were
added to a stirred solution of Boc-PheOH (1.0 mmol) in 4:1 DCM/
DMF (5 mL) at rt under an inert atmosphere. After 5 min, H-
GlyNH2-HCl (1.2 mmol) and DIPEA (2.4 mmol) were added at rt,
and the mixture was stirred under MW irradiation at 150 W. After 10
min the mixture was diluted with DCM, and the solution was washed
with 0.5 M HCl (5 mL) and saturated NaHCO3 (5 mL). The organic
layer was dried over Na2SO4, and solvent was removed at reduced
pressure. Boc deprotection was performed by treatment with 1:3
TFA/DCM (4 mL). After 30 min the mixture was concentrated at
reduced pressure, and the procedure was repeated. The resulting H-
Phe-GlyNH2-TFA was coupled with Ac-D-TrpOH under the same
conditions described above. The procedure was also utilized to obtain
the other peptides. Purification by semipreparative RP-HPLC (general
methods) gave 4−12 as waxy solids (70−85% yield, 95−98% pure by
analytical RP-HPLC).
Receptor Binding to Cloned Human OR. Displacement binding
assays have been performed in HEK-293 cells stably expressing human
MOR, human DOR, or human KOR obtained as previously
reported.14 Cell surface human MOR receptors were measured on
intact cells using [3H]DAMGO (0.1−5.0 nm) as radioligand and
naloxone (30 μM) to determine nonspecific binding. For displacement
binding assays, HEK-293 cells expressing human MOR were incubated
at rt for 2 h with [3H]DAMGO (2.5 nM) in the presence or absence of
compounds at various concentrations (10−12−10−4 M); nonspecific
binding was determined in the presence of naloxone (30 μM). After
incubation with the ligands, cells were washed in PBS (pH 7.4) and
lysed with 0.1 N NaOH. Lysed samples were buffered with an equal
amount of 0.1 N HCl and left in scintillation fluid for 8 h before
counting. Cell membranes from DOR and KOR expressing HEK-293
cells were prepared as previously reported.14 Receptor binding assays
were carried out by using [3H]diprenorphine to label DOR and
[3H]U69,593 to label KOR and by incubating the membrane
preparations at 25 °C for 90 min in buffer containing 100 mM Tris-
HCl and 0.3% BSA. For saturation binding assays, the concentrations
of [3H]diprenorphine and [3H]U69,593 ranged from 40 pM to 3 nM
and from 20 pM to 5 nM, respectively. For competition binding assays,
the concentration of [3H]diprenorphine or [3H]U69,593 was 1 and 2
nM, respectively. Nonspecific binding was determined in the presence
of 10 μM DPDPE (DOR) or 10 μM U50,488 (KOR) and
corresponded to 8−12% and 12−15% of total [3H]diprenorphine
and [3H]U69,593 binding, respectively. Triplicate determinations were
made for each experiment. Reactions were terminated by filtration
through Whatman GF/C filters presoaked with 0.3% polyethyleni-
mine, which were washed three times with 5 mL of ice-cold buffer
containing 50 mM Tris-HCl, pH 7.4. The radioactivity trapped was
determined by liquid scintillation spectrometry. Data from at least
three independent experiments were fitted by nonlinear regression
analysis using GraphPad Prism. Ki values were calculated from the IC50
using the Cheng−Prusoff equation.34 IC50 values represent mean
values from no less than four experiments. IC50 values, relative potency
estimates, and their associated standard errors were determined by
fitting the data to the Hill equation by a computerized nonlinear least-
squares method.
ASSOCIATED CONTENT
* Supporting Information
■
S
ROESY cross-peaks observed for 4 and 5; analytical character-
ization of 4−12. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*For S.S: phone, +39 051 20991851; e-mail, santi.
spampinato@unibo.it. For L.G.: phone, +39 0512099570; fax,
+39 051 2099456; e-mail, luca.gentilucci@unibo.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank MIUR (PRIN 2008), Fondazione Umberto Veronesi,
and the Italian Ministry for Foreign Affairs (bilateral project
Italy−Mexico) for financial support.
ABBREVIATIONS USED
■
MOR, μ-opioid receptor; DOR, δ-opioid receptor; KOR, κ-
opioid receptor; Dmt, 2′,6′-dimethyltyrosine; VT, variable
temperature; MW, microwave; DIPEA, diisopropylethylamine;
HOBt, hydroxybenzotriazole; HBTU, O-benzotriazole-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; RP, re-
versed phase
Determination of Inhibition of cAMP Accumulation. The
agonist activity was determined by measuring the inhibition of
forskolin-stimulated cAMP accumulation in whole HEK-293 cells
stably expressing MOR. Cells were grown at 37 °C and 5% CO2 in
MEM, 2 mM Gln, and 1× nonessential amino acids supplemented
with 10% FBS. Samples in a 75 cm2 flask at 95−100% confluence were
split into 24 wells and incubated overnight. When the confluence
became 85−95%, the medium was removed and the cells were washed
three times with PBS; thereafter, cells were incubated in serum-free
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Journal of Medicinal Chemistry
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