Method
Homology modeling
The homo sapiens active μ opioid receptor was built by taking the X-ray
structure of musculus active μ opioid receptor (PDB code: 5C1M) as a
template by using the homology modeling module of Discovery Studio 3.5
software(15 ). All water molecules
in the X-ray structure were retained. Ramachandran plot was employed to
evaluate the validity of the homology models in Figure S1 .
Residues were numbered according to the generalized numbering scheme
proposed by Ballesteros and
Weinstein(19 ).
Molecular docking
The morphine, fentanyl and sufentanil were docked into the 3D structure
of homo sapiens active μ opioid receptor. We used the Glide Docking
module in Maestro 3.5 program to dock these compounds into the binding
site of the active μ opioid receptor. These three systems were subjected
to Monte Carlo Multiple Minimum conformational searches using the
OPLS_2005 force field. The minimized conformations were selected for
the next molecular dynamics simulations.
Molecular dynamic
simulations
Eight systems were built for molecular dynamics simulations: (1) μ
opioid receptor without ligand (Apo); (2) morphine-bound active MOR; (3)
sufentanil-bound active MOR; and (4) fentanyl-bound active MOR; (5)
mutant μ opioid receptor without ligand (Apo-W6.48L); (6) morphine-bound
active mutant MOR (W6.48L); (7) sentinel-bound active mutant MOR
(W6.48L); and (8) fentanyl-bound mutant MOR (W6.48L). MD simulations
were performed using the Gromacs5.1.2 package. All systems were embedded
in a hydrated POPC lipid bilayer. Water molecules were used to solvate
the protein. After that, Na+ and Cl-ions were added in the water to neutralize the system using 0.15 mol/L
NaCl. The steepest descent followed by conjugate gradient methods were
used for the energy minimization for these systems. Then, we gradually
heated the systems from 0 K to 310 K. The systems were subjected to
equilibrate at constant pressure and temperature for 1 ns (310 K, 1
atm). Finally, the production MD simulations of eight systems were
performed for 100 ns. All analyses of MD trajectories were performed
using the tools implemented in the Gromacs5.1.2 package.
Plasmid construction and site-directed mutagenesis
To make wild-type and mutant (WT & mutant) MOR plasmids, full-length
hOPRM1 cDNA was subcloned into the Tag-lite® pT8-SNAP vector (Cisbio,
France). The Vazyme® Fast Mutagenesis Kit V2 (Vazyme, China)was used to
introduce the D3.32A, W6.48L, H6.52L site mutation to the MOR
recombinant plasmid, the nucleotide sequences of mutant MOR were
confirmed by DNA sequencing and sequence alignment. Primers designed to
amplify MOR plasmid containing mutant site are listed here:
D3.32A
F 5’-TCCATAGC TTACTATAACATGTTCACCAGC-3’
R 5’-ATAGTAAG CTATGGAGATCACTATCTTGCA-3’
W6.48L
F 5’-GTCTGCTT GACTCCCATTCACATTTACGTC-3’
R 5’-GGGAGTCA AGCAGACGATGAACACAGCCAC-3’
H6.52L
F 5’-CCCATTCT CATTTACGTCATCATTAAAGCC-3’
R 5’-GTAAATGA GAATGGGAGTCCAGCAGACGAT-3’
Cell lines
Chinese Hamster Ovary (CHO-K1) cells were purchased from ATCC and
maintained in F12 medium containing 10% fetal bovine serum
(FBS).
To obtain CHO stably expressing MOR (WT & mutant) cell lines, wild-type
and D3.32A, W6.48L, H6.52L mutant MOR plasmids were respectively
transfected into CHO-K1 cells using lipofectamine® LTX
regent. The transfected cell mixtures were grown under the selection
pressure of 200 μg/ml hygromycin for 10 days, then cells were seeded in
96-well plates at an approximate density of 1 cell per well to isolate
clones. An appropriate clone was selected for further experimental
studies. The MOR expression of CHO-MOR cell lines was confirmed by
western-blot analysis (Figure S3A ). To verify the site mutation
of each CHO-MOR cell line, we amplified the target MOR fragments from
total genomic DNA (Figure S3B ). Then DNA sequencing analysis of
the PCR products verified the correct site mutation of MOR in CHO-MOR
cell lines. Membrane MOR expression of the CHO-MOR cell lines was
measured by immunofluorescence analysis (Figure S3D ). The
expression level of wild-type and D3.32A MOR on membranes was confirmed
to be similar, and the W6.48L and H6.52L mutant MOR is approximately
63% of wild-type. CHO-MOR (WT & mutant) cells were maintained in F12
medium supplemented with 10% FBS and 40 μg/ml hygromycin.
To obtain CHO-stably expressing both MOR (WT & mutant) and
β-arrestin2-EGFP cell lines, we transfected the β-arrestin2-EGFP plasmid
into each CHO-MOR (WT & mutant) cell line. After growing under the
selection pressure of 6 μg/ml puromycin for 10 days, cell mixtures were
seeded in 96-well plates at an approximate density of 1 cell per well to
isolate clones. An appropriate clone was selected for further
experiments. The expression of β-arrestin2 was verified by western-blot
analysis and the expression level of each cell line was confirmed to be
similar (Figure S3C ). CHO-MOR (WT & mutant)-β-arrestin2-EGFP
cells were maintained in F12 medium supplemented with 10% FBS and 40
μg/ml hygromycin and 2 μg/ml puromycin.
All cells were cultured according to the standard protocol in 37 ℃
incubator with 5% CO2.
Western-blot
Adherent CHO cells expressing MOR (WT & mutant) or β-arrestin2 were
lysed in RIPA lysis buffer (50 mM pH 7.4 Tris, 150 mM NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease
inhibitor cocktail and phosphatase inhibitor. The lysate was incubated
at 37 ℃ for 10 min, then separated by SDS-PAGE of 8% polyacrylamide
gels and transferred to PVDF membranes. Membranes were immunoblotted
with anti-MOR antibody (Millipore, AB1580-Ⅰ) to verify the expression of
MOR or immunoblotted with anti-β-arrestin 2 antibody (Cell signaling
technology, 3857) to verify the expression of β-arrestin 2. To ensure
equal loading into each lane, blots were also incubated with GAPDH
antibody.
Immunofluorescence
Membrane MOR can be labelled with SNAP-Lumi4-Tb, and MOR-Tb level could
be quantified by Envision Multilabel Reader at 615 nm. CHO-MOR (WT &
mutant) cells were seeded on 35 mm dishes and grown to 80% confluence.
Cells were incubated with SNAP-Lumi4-Tb solution (Cisbio, France) for 1
h. Then cells were digested and dispensed into 384-well plate at a
density of 20000 cells per well, and the total test volume is 20 μl.
Read the plate on Envision Multilabel Reader (Perkin Elmer) at 615 nm.
The response represents the expression level of MOR.
Genomic DNA PCR
CHO-MOR (WT & mutant) cells were seeded on 35 mm dishes and grown to
80% confluence. Genomic DNA was extracted from cells using the Gentra
pure gene cell kit (Qiagen, Germany). MOR DNA fragments were amplified
from genomic DNA by PCR primers F: 5’-CTCGCCGTGAAAGAGTGGCT-3’, R:
5’-GGGCAACGGAGCAGTTTCT-3’. Then the PCR products were sequenced to
verify the mutant site.
Radioligand saturation binding
assay
Radioligand saturation binding assay was performed to determine the
affinity of fentanyl in binding with MOR. Membrane proteins were
extracted from CHO cells expressing MOR (WT & mutant), and 20 μg
proteins were used for each reaction. In specific binding reaction,
membranes were incubated with a series of concentrations of
[3H]Fentanyl (ranging from 1.5625 nM to 50 nM) for
30 min at 37 ℃ in Tris-HCl buffer (50 mM Tris, pH 7.4). In nonspecific
binding reaction, membranes were incubated with 5 μM naloxone and
[3H]Fentanyl (ranging from 1.5625 nM to 50 nM) for
30 min at 37 ℃ in Tris-HCl buffer. The reaction mixtures were filtered
over GF/C filters, and then the filters were washed three times by cold
Tris-HCl buffer. Radioactivity was assayed by liquid scintillation
counting overnight.
HTRF competitive binding
assay
HTRF competitive binding assay was performed according to the Tag-lite®
binding assay recommended protocol (Cisbio, France). This assay is based
on the competition between the Tag-lite® fluorescent ligand and test
compounds. CHO-MOR (WT & mutant) cells were labeled in batch with
SNAP-Lumi4-Tb and suspended in Tag-lite® labeling buffer (TLB), then
cells dispensed into 384 well small volume white plate
(Thermo Scientific Nunc, USA) at
a density of 5000 cells per well. To determine saturation binding
constant Kd of the fluorescent ligand, cells were incubated with a
series of concentrations (ranging from 0.1 nM to 100 nM) of the
fluorescent ligand in TLB. Non-specific binding signal wells were
incubated with 100 nM Naltrindole. For competition binding experiments,
cells were incubated with varying concentrations (ranging from 10-4 M to
10-14 M) of test compounds in the presence of 8 nM fluorescent ligand.
The total binding reaction volume is 20 μl, and the concentration
mentioned above means final concentration. Read the plate on Envision
Multilabel Reader (Perkin Elmer) at 665 nm and 615 nm after 3 h
incubation. The HTFR ratio was calculated as the equation:
\begin{equation}
\begin{matrix}HTRF\ Ratio=\frac{Signal\ 665\ nm}{Signal\ 615\ nm}\times 10^{4}\#Equation\ 1\\
\end{matrix}\nonumber \\
\end{equation}cAMP assay
The cAMP assay was performed according to the cAMP-Gi kit protocol
(Cisbio, France). CHO-MOR (WT & mutant) cells were harvested and
suspended in stimulation buffer supplemented with 0.5 mM IBMX. Then
cells were dispensed into 384-well small volume white plate (Thermo
Scientific Nunc, USA) at a density of 5000 cells per well. Add a series
of concentrations (ranging from 10-12 M to
10-6 M) of test compounds and 3 μM forskolin into
wells (non-stimulated control wells add stimulation buffer instead of
test compounds at this step), seal the plate and incubate at room
temperature (RT) for 15 min. Add cAMP-Cryptate and anti-cAMP-d2 working
solution into wells, seal the plate and incubate at RT for 1 h. The
total binding reaction volume is 20 μl, and the concentration mentioned
above means final concentration. Read the plate on Envision Multilabel
Reader (Perkin Elmer) at 665 nm and 615 nm after 3 h incubation. The
response was calculated as Equation 1 .
β-arrestin2 recruitment
assay
CHO-MOR (WT & mutant)-βarrestin2 cells were seeded into 96-well,
black-walled, clear-bottom assay plate (Costar, USA) at an appropriate
density to ensure they will be 50%~70% confluent next
day. For the assay, cells were serum-starved for 1 h, then treated with
a series of concentrations (ranging from 10-12 M to
10-5 M) of test compounds for 5 min at 37 ℃. Cells
were fixed and dyed using 4% paraformaldehyde (PFA) with Hoechst
nuclear stain (1:10000) for 30 min. β-arrestin2 translocation images
were captured with a 20× objective on an ArrayScan™ XTI High Content
Analysis (HCA) Reader (Thermo Scientific, USA). The spots/nuclear ratio
per well was quantified using Cellomics Spot® Detection BioApplication
(Thermo Scientific, USA).
Data analysis and statistical
procedures
GraphPad Prism (V7.0) software was used for curve fitting and data
analysis. All data are shown as mean ± SEM of at least three independent
experiments run in duplicate or triplicate.
For binding assays, concentration-response curves were fit to one-site
binding models provided in Prism software to determine
Kd, Bmax, and Ki.
For functional assays, data are shown by subtracting basal values and
presented as percentage of DAMGO, then were fit to a non-linear
regression (three-parameter) model to determine EC50 and
Emax. As indicated, the difference between all the
fitting parameters of wild-type MOR versus W6.48L mutant MOR was
acquired via unpaired, two-tailed t-test.
We used the classic operational model analysis method to calculate
ligand bias. The operational model (Black and Leff,
1983)(20 ) was applied to calculate
bias as described by van der Westhuizen et al. (2014)
(21 )and Laura M. Bohn et al.
(2017)(22 ). The transduction ratio
(τ/KA) was determined based on the equation:
\begin{equation}
\begin{matrix}E=\frac{E_{\max}}{1+\left(\frac{1+\frac{A}{10^{\log\left(K_{A}\right)}}}{A\times 10^{\log\left(\frac{\tau}{K_{A}}\right)}}\right)^{n}}\#Equation\ 2\\
\end{matrix}\nonumber \\
\end{equation}Where E is the effect of ligand, Emax is the maximal
response, A is the molar concentration of the ligand, KAis the equilibrium dissociation constant, and the
log(τ/KA) is the transduction
coefficient(21 ,23 ). For each assay, the
Emax is constrained to be a shared value, the basal is
constrained to be the value of zero and n is constrained to the value of
1.
DAMGO was chosen as the reference ligand. To eliminate system deviation,
the Δ log (τ/KA) of test ligand and SEM were calculated
by Equation 3 and Equation 4 :
\begin{equation}
\begin{matrix}\log\left(\frac{\tau}{K_{A}}\right)={\log\left(\frac{\tau}{K_{A}}\right)}_{\text{test}}-{\log\left(\frac{\tau}{K_{A}}\right)}_{\text{DAMGO}}\#\text{Equation\ }3\\
\end{matrix}\nonumber \\
\end{equation}\begin{equation}
\begin{matrix}\text{SE}_{\left(\text{Δ\ log}\left(\frac{\tau}{K_{A}}\right)\right)}=\sqrt{\left(\text{SE}_{\left(\log\left(\frac{\tau}{K_{A}}\right)\right)_{\text{test}}}\right)^{2}+\left(\text{SE}_{\left(\log\left(\frac{\tau}{K_{A}}\right)\right)_{\text{DAMGO}}}\right)^{2}}\#Equation\ 4\\
\end{matrix}\nonumber \\
\end{equation}The bias parameter ΔΔ log (τ/KA) and SEM were calculated
by Equation 5 and Equation 6 :
\begin{equation}
\begin{matrix}\ {\log\left(\frac{\tau}{K_{A}}\right)}_{\frac{\text{cAMP}}{\beta arr2}}={\operatorname{log}\left(\frac{\tau}{K_{A}}\right)}_{\text{cAMP}}-{\ \log\left(\frac{\tau}{K_{A}}\right)}_{\beta arr2}\#\text{Equation\ }5\\
\end{matrix}\nonumber \\
\end{equation}\begin{equation}
\begin{matrix}\text{SE}_{\left(\text{ΔΔ\ log}\left(\frac{\tau}{K_{A}}\right)\right)}=\sqrt{\left(\text{SE}_{\left(\text{Δ\ log}\left(\frac{\tau}{K_{A}}\right)\right)_{test:cAMP}}\right)^{2}+\left(\text{SE}_{\left(\text{Δ\ log}\left(\frac{\tau}{K_{A}}\right)\right)_{test:\beta arr2}}\right)^{2}}\#\text{Equation\ }6\\
\end{matrix}\nonumber \\
\end{equation}Materials
[3H]Fentanyl was purchased from Perkin Elmer
(USA). DAMGO and Naltrindole were purchased from Tocris (UK). Forskolin
and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich
(USA). Morphine was purchased from QINHAI pharmaceutical factory
(China). Fentanyl derivatives were synthesized by our institute. Cell
culture reagents were purchased from Gibco (USA).