Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at μOR

The mu opioid receptor (μOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in μOR with a supraspinally active analgesic named C6guano. Presently, to improve systemic activity of this ligand, we used structure-based design, identifying a new ligand named RO76 where the flexible alkyl linker and polar guanidine guano group is swapped with a benzyl alcohol, and the sodium site is targeted indirectly through waters. A cryoEM structure of RO76 bound to the μOR-Gi complex confirmed that RO76 interacts with the sodium site residues through a water molecule, unlike C6guano which engages the sodium site directly. Signaling assays coupled with APEX based proximity labeling show binding in the sodium pocket modulates receptor efficacy and trafficking. In mice, RO76 was systemically active in tail withdrawal assays and showed reduced liabilities compared to those of morphine. In summary, we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved.

General synthetic procedure for the reductive amination followed by propionylation (procedure no. 1) N-phenylethylpiperidin-4-one 4-piperidone monohydrate hydrochloride (3.6 g, 23.4 mmol, 1 equiv.)was dissolved in acetonitrile (20 mL) in a 100 mL round bottom flask equipped with a stir bar.To this mixture, cesium carbonate (15.2 g, 46.8 mmol, 2 equiv.)and (2-iodoethyl) benzene (3.05 mL, 4.88 g, 21.06 mmol) were added at room temperature.The reaction mixture was refluxed at 80 o C for 18 h.After 18 h, the mixture was cooled to room temperature, extracted with DCM (15 mL x 3), washed with saturated NaHCO3 (15 mL) and brine (15 mL).The organic layer was dried over Na2SO4 and concentrated in vacuo, purified by flash column chromatography using hexane: ethyl acetate (0 to 100%) 1,2 1 H NMR (400 MHz, chloroform-d): δ 7.33 -7.27 (m, 2 H), 7.22 (d, J = 6.6 Hz, 3 H), 2.88 -2.81 (m, 6 H), 2.76 -2.73 (m, 1 H), 2.73 -2.70 (m, 1 H), 2.48 (t, J = 6.0 Hz, 4 H) General procedure for reductive amination followed by propionylation N-phenylethylpiperidin-4-one (0.983 mmol) was dissolved in 5 mL methanol dried over 4 Å MS in 10 mL microwave vial equipped with a stir bar.To this, amine (0.983 mmol) was added followed by addition of 2-3 drops of acetic acid (cat.)The microwave vial was sealed and heated in microwave reactor at 80 o C for 45 minutes.After 45 minutes of microwave, sodium cyanoborohydride (1.96 mmol) was added slowly to the reaction mixture at ambient temperature and the reaction stirred for 18 h at ambient temperature.The formation of desired product was confirmed by TLC and LCMS analysis.The reaction mixture was filtered through a celite pad and washed with 20% MeOH: DCM.The solution was evaporated in vacuo.The intermediate secondary amine was forwarded to the next step without any purification.Next the intermediate amine was dissolved in dry DCM (5 mL) and to it DIPEA (1.96 mmol) was added followed by propionyl chloride (0.885 mmol).The reaction mixture was stirred at room temperature for 10 to 15 mins and quenched with ice water after completion of the reaction.The reaction mixture was extracted with 10% MeOH: DCM (10 mL x 3), washed with saturated NaHCO3 (10 mL), brine (10 mL) and dried over Na2SO4.The organic layer was evaporated in vacuo and purified by combi flash chromatography (DCM/Methanol -0 to 20%) to obtain RO76 and analogues.These compounds were further purified using reverse phase column chromatography (solvent system -acetonitrile with 0.05%TFA: H2O with 0.05%TFA) with 45 min of runtime.The final compounds were isolated as a TFA salt.
Characterization of compounds synthesized by reductive amination followed by propionylation (procedure no.Yield: 42% dissolved in dry DCM (5 mL) and to it DIPEA (1.17 mmol) was added followed by propionyl chloride (0.528 mmol).
The reaction mixture was stirred at room temperature for 10 to 15 mins and quenched with ice water after completion of reaction.The reaction mixture was extracted with 10% MeOH: DCM (5 mL x 3), washed with saturated NaHCO3 (5 mL), brine (5 mL) and dried over Na2SO4.The organic layer was evaporated in vacuo and purified by combi flash chromatography (DCM/Methanol -0 to 20%) to obtain RO76 analogues.These compounds further purified using reverse phase column chromatography (solvent system -acetonitrile with 0.05%TFA: H2O with 0.05%TFA) afforded colorless sticky compound as a TFA salt.
BRET based assays (TRUPATH and arrestin signaling) [3][4] To measure G proteins dissociation or β-arrestin recruitment, 2.5 million cells were plated in 10 cm dishes and incubated overnight at 37 ºC.The next day, cells were co-transfected, using Lipofectamine 2000 following vendor protocol.Human MOR, Gα-RLuc8, Gβ, Gγ-GFP2 plasmids (2.5 μg per construct) were transfected at a ratio of 1:1:1:1 for G protein dissociation and a 1:5 DNA ratio of Rluc tagged MOR, Venus-tagged N-terminal β-arrestin (1 or 2) was used for β-arrestin recruitment.The following day, transfected cells were plated into a 384-well Poly-D-lysine coated plate with DMEM supplemented with 1% dialyzed FBS at a density of 20,000 cells per well and incubated overnight at 37 ºC.On the day of the assay, the media was removed, and cells were rinsed with 30 μL of assay buffer (1×Hank's balanced salt solution (HBSS), 20 mM HEPES, pH 7.4), followed by the addition of the 30 μL substrate buffer (7.85   μM coelenterazine 400a for G-protein or 7.85 μM coelenterazine h for β-arrestin in assay buffer) for 5 min in the dark at room temperature.After a 5 min equilibration period, the cells were treated with 15 μL of drug (3X drug concentration in assay buffer with 0.3% bovine serum albumin) for an additional 5 min in the dark at room temperature.The plates were then read four times in a BioTek Synergy Neo Alpha plate reader using 395 nm (RLuc8coelenterazine 400a) and 510 nm (GFP2) emission filters and the measurements from the 10 min read were used for G protein analyses.For β-arrestin, the plates were read six times using 480 (RLuc8-coelenterazine h) and 530 nm (YFP2) emission filters and the measurements from the 15 min read were used for β-arrestin recruitment analyses.
The ratio of GFP2/RLuc8 for G protein and eYFP/RLuc for β-arrestin were calculated per well in quadruplicates and plotted as a function of drug concentration, normalized to % control agonist stimulation and analyzed using ''log(agonist) vs. response'' in GraphPad Prism 10.0.

Nb39 assay 5
HEK293T cells were transiently transfected with MOR-Rluc8 and Nb39-Venus in a 1:3 ratio, after 16 hrs cells were plated onto a poly-D-lysine coated 384-well plate in DMEM supplemented with 1% dialyzed FBS at 20,000 cells/well in 40 µl.After a further 24 hrs, cells were washed with 20 µl assay buffer (1X HBSS, 20 mM HEPES) then incubated with 20 µl per well coelenterazine h (5 µM, in assay buffer) for 5 min in the dark.Drugs were diluted to 30 µM in drug buffer (assay buffer + 0.3 g/ml BSA), 10 µl was added onto the plate (final concentration of 10 µM in the well) and incubated for 5 min in the dark.The plate was then immediately read for luminescence at 480 nm and Venus emission at 530 nm on a BioTek Synergy Neo plate reader.The ratio of Venus/Rluc8 signal was calculated, Nb39 recruitment was calculated by subtracting the mean ratio of the wells that received DMSO vehicle control.

Cryo-EM Expression and purification of the μ-opioid receptor (μOR)
The μOR construct was expressed and purified as previously described 6 .Briefly, receptor was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus method (Expression Systems), and media was supplemented with 1 μM Naloxone.Cells expressing μOR were harvested and solubilized from membranes using 20 mM hydroxy-ethylpiperazine ethane sulfonic acid (HEPES), pH 7.4, 100 mM sodium chloride (NaCl), 1% n-dodecylβ-D-maltopyranoside (DDM), 0.1% cholesteryl hemisuccinate (CHS), 2 mM MgCl2, 1 μM Naloxone and protease inhibitors.Membranes were homogenized with a douncer, stirred for one hour at 4 degrees, and the soluble fraction was isolated by centrifugation and applied to a nickel-chelating sepharose resin.The Ni-NTA elution in 0.1%DDM/0.01%CHSwas then incubated with 0.1% lauryl maltose neopentyl glycol (LMNG)/0.01%CHS for 1 hour on ice to exchange the detergents.After detergent exchange, 2 mM CaCl2 was added, and the sample was loaded onto M1 anti-Flag resin and washed with progressively lower concentrations of salt and naloxone.The µOR was then eluted from M1 resin in a buffer consisting of 20 mM Hepes pH 7.5, 100 mM NaCl, 0.003% L-MNG/0.0003%CHS supplemented with 1 uM naloxone, Flag peptide and 5 mM EDTA.The M1 elute was further purified by size exclusion chromatography on the Superdex 200 10/300 gel filtration column (GE Healthcare) in 20 mM HEPES pH 7.5, 100 mM NaCl, 0.003% L-MNG/0.0003%CHS.The monomeric fractions were pooled, concentrated, and flash frozen in liquid nitrogen.

Expression and purification of heteromeric Gi1
Heterotrimeric Gi was expressed and purified as previously described. 7Briefly, heterotrimeric Gi1 was expressed in Trichoplusia ni (T.ni) insect cells using the baculovirus method (Expression Systems).Two viruses were used to infect the insect cells, one encoding the wild-type human Gαi1 subunit and another one encoding the wild-type human β1γ2 subunits.The cells were harvested 48 hours post-transfection, and the pellet was flash frozen with liquid nitrogen and stored at -80°C.Cells lysis was conducted in hypotonic buffer.Membranes were harvested by centrifugation and solubilized using a douncer in 20 mM Hepes pH 7.4, 100 mM NaCl, 1% sodium cholate, 0.05% DDM, 5 mM magnesium chloride, 5 mM bME, 5 mM imidazole, 20 mM GDP and protease inhibitors.The solubilization mixture was stirred for one hour at 4 degrees.After centrifugation, the supernatant was loaded on a Ni-NTA chromatography column, washed in 0.05% DDM buffer to remove the cholate.After elution using 250 mM imidazole, human rhinovirus 3C protease (3C protease) was added and the histidine tag was cleaved overnight at 4 °C during dialysis.
Then the heterotrimeric Gi without tag will be further purified through reverse Ni-NTA chromatography.Gi heterotrimer was separated from excess betagamma using a MonoQ 5/50 GL column (GE Healthcare).The protein was diluted to lower the imidazole concentration and loaded onto the column in 20 mM Hepes pH 7.4, 50 mM NaCl, 1 mM MgCl2, 0.05% DDM/CHS, 100 mM TCEP and 20 mM GDP.The heterotrimer was eluted with a linear gradient of 0-50% with buffer containing 1 M NaCl.Eluted fractions were concentrated to 200-250 uM and after addition of 20% glycerol the protein was flash frozen and stored at -80 °C.

Expression and Purification of scFv16
scFv16 was developed and purified as previously described. 8Basically, scFv with C terminal His tag was expressed in Trichuplusia ni Hi5 insect cells.After infection and expression, the insect cell supernatant was loaded onto Ni-NTA resin and the scFv was eluted in 20 mM HEPES pH 7.5, 500 mM NaCl, and 250 mM imidazole.The eluate was incubated with 3C protease overnight to cleave the C-terminal His tag.After dialysis into the buffer consisting of 20 mM HEPES pH 7.5 and 100 mM NaCl, scFv16 was further purified by reverse Ni-NTA chromatography.The flow-through was collected and applied over a Superdex 200 16/60 column (GE Healthcare).
The scFv16 fractions were pooled, concentrated, and flash frozen.at pH 7, except for D114 2.50 and D164 3.49 , which were protonated (neutral) in all simulations, as these conserved residues are reported to be protonated in the active states of several class A GPCRs. [20][21] Histidine residues were modeled as neutral, with a hydrogen atom bound to the epsilon nitrogen, as we did not find any cases where moving the hydrogen to the delta nitrogen would help optimize the local hydrogen bonding network.Using Dabble, the prepared protein structures were inserted into a pre-equilibrated palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayer, the system was solvated, and sodium and chloride ions were added to neutralize the system and to obtain a final concentration of 150 mM.The final systems comprised approximately 59,000 atoms, and system dimensions were approximately 80 × 80 × 90 Å 3 .

MD simulation and analysis protocols
3] All simulations were performed using the Compute Unified Device Architecture (CUDA) version of particle-mesh Ewald (PME) MD in AMBER20 on graphics processing units (GPUs). 24stems were first minimized using three rounds of minimization, each consisting of 500 cycles of steepest descent followed by 500 cycles of conjugate gradient optimization.Thus, 10.0 and 5.0 kcal mol −1 Å −2 harmonic restraints were applied to protein, lipids and ligand for the first and second rounds of minimization, respectively.Further, 1 kcal mol −1 Å −2 harmonic restraints were applied to protein and ligand for the third round of minimization.Systems were then heated from 0 K to 100 K in the constant number (N), constant-volume (V) and constant-temperature (T) or NVT ensemble over 12.5 ps and then from 100 K to 310 K in the constant number (N), pressure (P) and constanttemperature (T) or NPT ensemble over 125 ps, using 10.0 kcal mol −1 Å −2 harmonic restraints applied to protein-and ligand-heavy atoms.Subsequently, systems were equilibrated at 310 K and 1 bar in the NPT ensemble, with harmonic restraints on the protein and ligand nonhydrogen atoms tapered off by 1.0 kcal mol −1 Å −2 starting at 5.0 kcal mol −1 Å −2 in a step-wise fashion every 2 ns for 10 ns, and then by 0.1 kcal mol −1 Å −2 every 2 ns for 20 ns.Production simulations were performed without restraints at 310 K and 1 bar in the NPT ensemble using the Langevin thermostat and the Monte Carlo barostat, and using a timestep of 4.0 fs with hydrogen mass repartitioning.Bond lengths were constrained using the SHAKE algorithm. 25Nonbonded interactions were cut off at 9.0 Å, and long-range electrostatic interactions were calculated using the PME method with an Ewald coefficient of approximately 0.31 Å, and 4th order B-splines.
The PME grid size was chosen such that the width of a grid cell was approximately 1 Å.The use of a 9.0-Å nonbonded interaction cutoff and a 4-fs timestep with hydrogen mass repartitioning may have affected the structural and kinetic properties of the lipid bilayer, potentially introducing artifacts in our simulations.The AmberTools17 CPPTRAJ package was used to reimage trajectories to 1 ns per frame. 26Simulations were visualized and analyzed using Visual Molecular Dynamics (VMD) and PyMOL (Schrodinger). 27drogen bond contacts were measured using the GetContacts package, which sets the donor-to-acceptor distance as less than 3.5 Å and donor-hydrogen-acceptor angle as greater than 110 degrees. 28Water-mediated bonds were calculated, as defined by GetContacts, as interactions where the two contact regions hydrogen bond to either the same water molecule or to two consecutive water molecules that form an extended water bridge.For this paper, interactions with one water or two waters were considered separately, with plots showing only a single water interaction unless otherwise specified.When the frequencies of both conditions were calculated, frames with both one and two waters forming interactions were categorized as single water mediated interaction frames to avoid overlap.The water mediated interactions described in this paper were calculated between the closer side-chain oxygen of D114 2.50 and either the hydroxyl of RO76 or the carbonyl of RO152.The Y 7.43 -Q 2.60 hydrogen bond was calculated between the tyrosine side chain oxygen and the glutamine side chain nitrogen.Pi stacking interactions were quantified by computing the distance between the centroid of the ligand and tyrosine phenyls.Each centroid was calculated as the center of three alternating carbons on the aromatic rings.A centroid distance below 5 Å indicates the presence of pipi stacking.All images from MD simulation were chosen to reflect representative behavior across all simulation trajectories.
APEX reaction, biotinylated protein enrichment and preparation for mass spectrometry analysis.
HEK293T cells expressing the APEX2 enzyme fused to the human μOR (μOR-APEX) and the spatial reference APEX constructs were incubated with 500 μM biotin-phenol at 37 °C for 30 min.The receptor was activated with 10 μM RO76 over a time course of 30 min.The spatial reference APEX samples were not treated with RO76.APEX labeling was initiated pre-activation (time 0) and after 1, 5, 10 and 30 min of activation by 1:1 mixing of the H2O2 containing media (1 mM H2O2 final) with the biotin-phenol containing media at room temperature.After 45 s of the biotinylation reaction, the cells were washed three times (1 min each) with ice cold quenching buffer (PBS supplemented with 10 mM sodium ascorbate, 10 mM sodium azide, and 5 mM Trolox).Cells were then collected in 8 mL of quench buffer and pelleted by centrifugation at 4 °C for 10 min at 3000 x g.For cell lysis, cells were homogenized using probe sonication in RIPA (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 0.25% SDS, pH 7.4) supplemented with 10 mM sodium ascorbate, 10 mM sodium azide, 5 mM Trolox, 1 mM DTT, and protease inhibitors (Roche Complete).To remove the cell debris, cell lysate was centrifuged at 10,000 x g for 10 min, and the supernatant was taken for streptavidin enrichment of biotinylated proteins.
The enrichment of biotinylated proteins was automated with the KingFisher Flex (Thermo Fisher Scientific).
Supernatants were incubated at 4 °C for 18 hrs with magnetic streptavidin beads (Pierce™ Streptavidin Magnetic Beads, Thermo Fisher Scientific) which were pre-washed twice with RIPA buffer.Following incubation, beads were washed three times with RIPA buffer, one time with 1 M KCl, one time with 0.1 M Na2CO3, one time with 2 M urea in 50 mM Tris-HCl (pH 8) buffer, and two times with 50 mM Tris-HCl (pH 8) buffer.Beads were maintained in 200 uL of 2 M urea in 50 mM Tris-HCl (pH 8) buffer for on-bead digestion of proteins.Samples were reduced with 5 mM TCEP at 37 °C for 30 min, followed by alkylation with 5 mM IAA at room temperature in the dark for another 30 min, which was quenched by addition of DTT (5 mM final).For tryptic digestion, 1 ug of trypsin and LysC was added to beads and incubated with shaking at 37 °C for 4 hrs.To ensure complete digestion another 0.5 ug of trypsin were added the next morning with an incubation at 37 °C with shaking.Supernatants were taken and saved for desalting using NEST C18 MicroSpin columns.
Unbiased mass spectrometric data acquisition and protein quantification for APEX samples.μOR-APEX and spatial APEX reference samples were analyzed on an Orbitrap Exploris 480 mass spectrometry system (Thermo Fisher Scientific) coupled to a Easy nLC 1200 nano-flow ultra high-pressure liquid chromatography (Thermo Fisher Scientific) interfaced via a Nanospray Flex nanoelectrospray source.Samples were reconstituted in 1% formic acid and loaded onto a C18 column (25 cm x 75 μm I.D. packed with ReproSil Pur C18 AQ 1.9 μm particles).Mobile phase A consisted of 0.1% FA, and mobile phase B consisted of 0.1% FA/80% ACN.Peptides were separated at a flow rate of 300 nl/min using a gradient increasing buffer B over 40 min to 16% B, followed by an increase over 26 min to 28% B and 4 min to 44% B. The mass spectrometer acquired data in a data-independent acquisition (DIA) mode, collecting one full scan in the Orbitrap at 120,000 resolution followed by DIA MS/MS scans within a m/z range of 350-1050 with a fragmentation window size set to 20 m/z.The resolution of orbitrap for MS2 scans was set to 15,000 and a normalized collision energy of 30 was used for fragmentation.The DIA data were analyzed with Spectronaut (Biognosys) using direct DIA analysis default parameters for the identification and quantification of proteins.Normalization in Spectronaut was turned off.Data were searched against the Uniprot Human database.Peptide ion intensities from Spectronaut were summarized to protein intensities using the MSstats (version 4.4.1)function dataProcess with default settings except missing-value imputation was disabled and only highquality and informative features were used (MBimpute = FALSE, remove_uninformative_feature_outlier = TRUE, featureSubset = "highQuality").All proteins with only one quantified peptide were left out of further analysis.

Statistical analysis of APEX-MS samples
Each protein's trend over the time course after RO76 treatment was scored by fitting the log2 intensities with a continuous cubic-polynomial curve over time using the R functions lm and poly.To better fit the rapid changes, especially between time 0 and 1 minute, the collected timepoints were encoded by their ranks (1, 2, 3, 4, and 5 for 0, 1, 5, 10, and 30 minutes).The model included an additive term for the batch-a protein's background intensity was expected and allowed to vary between batches.The time-dependent model was compared with a null-model that contained only the batch term using the R function anova to compute an F statistic and p-value.The maximum mean change between time 0 and any single later time, after imputing any missing values using the fitted model, was used as the maximum log2 fold change for that protein.

Figure S4 :
Figure S4: RO76 Demonstrates Lower Y 7.43 -Q 2.60 H-Bond Frequency vs LFT a. Representative frame of LFT interactions in simulation.All relevant residues are shown in licorice representation and colored teal.LFT is colored periwinkle.The typical distance between the side chain hydroxyl on Y7.43  and the side chain nitrogen on Q 2.60 was about 2.5-5 Å. b.Representative frame of RO76 interactions in simulation.All relevant residues are shown in licorice representation and colored teal.RO76 is colored in salmon.The distance between the side chain hydroxyl on Y7.43  and the side chain nitrogen on Q 2.60 varied widely from 4-10 Å. c. Hydrogen bond contact frequency between Y 7.43 -Q2.60  .Black dots are used to represent averages for each simulation replicate, and black bars give the standard error of the mean (s.e.m.) for each condition.G protein biased MP shows no contact between these residues, while arrestin-biased LFT shows hydrogen bonds between these two residues in most frames.RO76 lies in the middle of these two values, with the hydrogen bond occurring about a third of the time.Significance was calculated using the Mann-Whitney U test (p values: * < 0.05, ** < 0.01).