Journal of Medicinal Chemistry
Article
the filtrate to promote precipitation of the peptide products that were
finally isolated by centrifugation. Reverse-phase purification of crude
peptides was carried out on a Waters Prep 600 high-performance liquid
chromatography (HPLC) system with a Jupiter column C18 (250 × 30
mm, 300 Å, 15 μm spherical particle size) using a gradient, programmed
time by time, of acetonitrile/water [with 0.1% trifluoroacetic acid
(TFA)] at a flow rate of 20 mL/min. Nonpeptide derivatives were
purified through flash column chromatography using a Biotage System
Isolera One. Analytical HPLC was performed with a Beckman 116
liquid chromatograph furnished of a UV detector. The purity of
peptides in Table 1 was assessed with a Symmetry C18 column (4.6 ×
75 mm, 3.5 μm particle size, SYSTEM GOLD) at a flow rate of 0.5 mL/
min using a linear gradient from 100% of A (water + 0.1% TFA) to
100% of B (acetonitrile + 0.1% TFA) over a period of 25 min. The
purity of peptides in Tables 2 and 3 was assessed with an Agilent Zorbax
C18 column (4.6 × 150 mm, 3.5 μm particle size, KARAT32) at a flow
rate of 0.7 mL/min using a linear gradient from 100% of A (water +
0.1% TFA) to 100% of B (acetonitrile + 0.1% TFA) over a period of 25
min. All final compounds were monitored at 220 nm showing ≥95%
purity, and their molecular weights were confirmed using an ESI
Micromass ZQ, Waters (HPLC chromatograms and ESI mass spectra
of the final peptide derivatives have been reported in the Supporting
Information). 1H and 13C NMR spectra were recorded for nonpeptide
derivatives on a Varian 400 MHz instrument, and all experiments were
performed in deuterated DMSO using its residual shifts as reference (s:
singolet, d: doublet, dd: double doublet, t: triplet, m: multiplet).
In Vitro Pharmacological Studies. Drugs and Reagents. [D-
Pen2,D-Pen5]enkephalin (DPDPE) and naltrexone were purchased
from Tocris Bioscience (Bristol, UK). Concentrated solutions (1 mM)
were made in bidistilled water and kept at −20 °C until use. The
medium and reagents for cell culture were from Euroclone (Milan,
Italy). Fluo-4 AM and pluronic acid were from Invitrogen/Thermo-
Fisher Scientific (Waltham, USA). N-(2-Hydroxyethyl)piperazine-N′-
ethanesulfonic acid (HEPES), probenecid, brilliant black, and bovine
serum albumin (BSA) fraction V were from Sigma-Aldrich (St. Louis,
USA).
Calcium Mobilization Assay. CHO cells stably coexpressing the
human NOP or kappa or the mu receptor and the C-terminally
modified Gαqi5 and CHO cells coexpressing the delta receptor and the
GαqG66Di5 protein were generated and cultured as described
previously.45,46 Cells were maintained in Dulbecco’s modified Eagle’s
medium/nutrient mixture F-12 (DEMEM/F-12) supplemented with
10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 100 μg/
mL hygromicin B, and 200 μg/mL G418 and cultured at 37 °C in 5%
CO2 humidified air. Cells were seeded at a density of 50,000 cells/well
into 96-well black, clear-bottom plates. The following day, the cells were
incubated with Hanks’ balanced salt solution (HBSS) supplemented
with 2.5 mM probenecid, 3 μM of the calcium-sensitive fluorescent dye
Fluo-4 AM, and 0.01% pluronic acid for 30 min at 37 °C. After that
time, the loading solution was aspirated and 100 μL/well of HBSS
supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 μM
brilliant black was added. Serial dilutions were carried out in HBSS/
HEPES (20 mM) buffer (containing 0.02% BSA fraction V). After
placing both plates (cell culture and master plate) into the fluorometric
imaging plate reader FlexStation II (Molecular Devices, Sunnyvale,
CA), fluorescence changes were measured. On-line additions were
carried out in a volume of 50 μL/well. To facilitate drug diffusion into
the wells, the present studies were performed at 37 °C. Maximum
change in fluorescence, expressed as percent over the baseline
fluorescence, was used to determine agonist response.
and cultured for 20 h to obtain confluent monolayers. Cells were
starved in the assay buffer (HBSS with 20 mM HEPES, 0.01% BSA
fraction V) for 90 min before the test. Serial dilutions were made in the
assay buffer. After reading the baseline, compounds were added in a
volume of 10 μL; then, DMR changes were recorded for 60 min.
Responses were described as picometer (pm) shifts over time (sec)
following subtraction of values from vehicle-treated wells. Maximum
picometer (pm) modification (peak) was used to generate concen-
tration response curves. All the experiments were carried out at 37 °C.
Data Analysis and Terminology. The pharmacological terminology
adopted in this paper is consistent with IUPHAR recommendations.59
All data are expressed as the mean standard error of the mean (SEM)
of at least three experiments performed in duplicate. For potency values,
95% confidence limits (CL95%) were indicated. Agonist potencies are
given as pEC50, that is, the negative logarithm to base 10 of the molar
concentration of an agonist that produces 50% of the maximal effect of
that agonist. Concentration-response curves to agonists were fitted to
the classical four-parameter logistic nonlinear regression model:
E ff e c t
= B a s e l i n e + ( E m a x − B a s e l i n e ) / ( 1 +
10(LogEC − Log[compound]) × Hillslope). Curve fitting was performed using
PRISM 6.0 (GraphPad Software Inc., San Diego).
50
Molecular Dynamics. The setup of an in silico model of the non-
natural peptides [Dmt1,5] N/OFQ(1-9)-NH2 and N/OFQ(1-9)-NH2
in complex with the human mu receptor has been described in the
receptor-peptide complexes were performed and compared with an
MD simulation of the experimental system DAMGO-mu receptor-Gi
protein complex as derived by the PDB file 6DDF.30 The GROMACS
2018.3 package60 was used under the AMBER parm99sb force field61 at
the full atomistic level using a TIP3P water solvent and an explicit pre-
equilibrated phospholipid bilayer of 128 POPC (1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine) molecules obtained by the Prof. Tiele-
performed in a water−membrane system prepared as previously
described.31,32 The receptor-peptide-membrane systems were solvated
in a triclinic water box (having basis vector lengths of 7, 7.4, and 9.3 nm)
under periodic boundary conditions for a total number of about 45,000
atoms (6400 solvent molecules). The total charge of the system was
neutralized by randomly substituting water molecules with Na+ ions
and Cl− ions to obtain neutrality with a 0.15M salt concentration.
Following a steepest descent minimization algorithm, the system was
equilibrated under canonical ensemble (NVT) conditions for 300 ps
using a V-rescale, modified Berendsen thermostat with position
restrains for both the receptor-peptide complex and the lipids and
thereafter in a isothermal−isobaric ensemble (NPT) for 500 ps,
applying position restraints to the heavy atoms of the protein-peptide
complex, and using a Nose−Hoover thermostat and a Parrinello-
Rahman barostat at 1 atm with a relaxation time of 2.0 ps. The MD
simulation of the mu receptor-DAMGO-Gi protein was carried out on
the whole ternary complex without positional restraints. On the other
hand, in order to reduce the computational time, in the two mu
receptor-peptide complexes, the Gi protein was not included in the
system, but all residues within 5 Å of the Gi protein interface were
restrained to the initial structure of the activated receptor using 5.0 kcal
mol−1 Å−2 harmonic restraints applied to non-hydrogen atoms. Using
such restraints ensures that the receptor maintains an active
conformation throughout the simulation. MD runs were performed
under NPT conditions at 300 K with a T-coupling constant of 1 ps. van
der Waals interactions were modeled using a 6−12 Lennard-Jones
potential with a 1.2 nm cutoff. Long-range electrostatic interactions
were calculated, with a cutoff for the real space term of 1.2 nm. All
covalent bonds were constrained using the LINCS algorithm. The time
step employed was 2 fs, and the coordinates were saved every 5 ps for
analysis.
DMR Assay. CHO cells stably expressing the human NOP and mu
receptors were kindly provided by D.G. Lambert (University of
Leicester, UK). Cells were cultured in DMEM/F-12 medium
supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL
streptomycin, and 2 mmol/L L-glutamine. The medium was
supplemented with 400 μg/mL G418 to maintain expression. Cells
were cultured at 37 °C in 5% CO2 humidified air. For DMR
measurements, the label-free EnSight Multimode Plate Reader (Perkin
Elmer, MA, US) was used. Cells were seeded 15,000 cells/well in a
volume of 30 μL onto fibronectin-coated 384-well DMR microplates
The MD analysis of the DAMGO-mu receptor-Gi protein complex
(Figure S1) shows an overall stability of the starting configuration
(corresponding to the crystal structure) with some motion of the
phenolic head toward the intracellular side of the receptor, still
conserving the water bridge contact with H297. A non-negligible
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J. Med. Chem. 2021, 64, 6656−6669