Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, Laura M. Bohn ( lbohn@scripps.edu ). The Scripps Research Institute requires that a material transfer agreement (MTA) be signed for the transfer of materials.

Chinese Hamster Ovary (CHO-K1) and Phoenix-AMPHO cells were purchased from ATCC; U2OS-βarrestin-hMOR PathHunter cells were purchased from DiscoveRx and the U2OS-Tango-hOPRL1-bla cells were purchased from ThermoFisher Scientific. The U2OS-βarrestin2-GFP cells were provided by the Addiction Research GPCR Assay Bank. Based on the other reports, all of the parent cell lines are female (query: http://web.expasy.org/cellosaurus/ ). Receptor levels in the CHO-hMOR, hKOR and hDOR cells have been described previously as well as in the current manuscript (). To make the mMOR lines, the HA (haemagglutinin)-N terminus tagged mMOR was packaged into murine stem cell retroviral particles via the phoenix packaging system and then CHO-K1 and U2OS-βarrestin2-GFP cells were transduced with the particles. A BD FACSAria3 flow cytometer was used to select for high expressing cells using an anti-HA AlexaFluor 594 conjugate antibody (1:100). All cells lines were cultured according to standard protocols at 37° in the indicated media with 10% fetal bovine serum (FBS) and 1% pen/strep: DMEM/F12—all CHO-K1 lines; DMEM—Phoenix- AMPHO; MEM—all U2OS cell lines. CHO-hMOR, -hKOR and -hDOR cell lines were grown under geneticin selection (500 μg/μl). U2OS mMOR βarrestin2 cells lines were grown under puromycin selection (500 μg/μl). The U2OS-βarrestin-hMOR PathHunter cell line (in which the MOR retains its natural C-terminal tail, tagged with the enzyme fragment) was cultured according to the manufacturer’s protocol (DiscoveRx), as was the U2OS-Tango-OPRL1-bla cell line (ThermoFisher Scientific).

Male and female C57BL/6J and male MOR-KO mice were purchased from The Jackson Laboratory and propagated by homozygous breeding in-house. Mice were group housed (3-5 mice per cage) and maintained on a 12-hour light/dark cycle with food and water ad libitum. Experiments were performed on naive adult mice between 10-14 weeks of age. Same sex littermates were randomly assigned to experimental groups; males and females were separately tested and their responses are separately reported. Experiments were performed by investigators who were blinded to the treatment assignments. Mice were dosed i.p. at a volume of 10 μl/g mouse, except all 48 mg/kg injections were dosed at a volume of 20 μl/g mouse to adjust for compound solubility. The number of mice used in each assay are indicated in Table S4 . All mice were used in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals with approval by The Scripps Research Institute Animal Care and Use Committee.

Method Details

Synthesis of MOR ligands All reagents and anhydrous solvents were used as obtained from commercial vendors. 1H NMR spectra were recorded at 400 MHz, with chemical shifts are reported in parts per million (ppm) using an internal standard, CHCl 3 (δ 7.26), MeOH (δ 3.34) or DMSO (δ 2.54). Mass spectra were recorded by ESI Ion trap. Analytical HPLC retention times were measured using reverse phase conditions with a Zorbax 5 micron column, model Eclipse-XDB-C18 80Å (155 × 4.6 mm), column temperature = 40°C, flow rate = 3.00 mL/min. The method incorporates a gradient elution, beginning with 98% H 2 O / 2% acetonitrile, each with 0.1% TFA. After 1 min, hydrophobicity was increased to 5% acetonitrile and then linearly in a gradient to 95% acetonitrile over an additional 5 min. Purity assessment (> 95%) was made LC using UV absorbance at multiple wavelengths, typically 215, 254, and 280 nm. Obase et al., 1983 Obase H.

Takai H.

Teranishi M.

Nakamizo N. Synthesis of (1-Substituted Piperidin-4-Yl)-1h-Benzimidazoles and (1-Substituted Piperidin-4-Yl)-3,4-Dihydroquinazolines as Possible Antihypertensive Agents. Budzik et al., 2010 Budzik B.

Garzya V.

Shi D.

Walker G.

Woolley-Roberts M.

Pardoe J.

Lucas A.

Tehan B.

Rivero R.A.

Langmead C.J.

et al. Novel N-Substituted Benzimidazolones as Potent, Selective, CNS-Penetrant, and Orally Active M1 mAChR Agonists. Lindsley et al., 2005 Lindsley C.W.

Zhao Z.

Leister W.H.

Robinson R.G.

Barnett S.F.

Defeo-Jones D.

Jones R.E.

Hartman G.D.

Huff J.R.

Huber H.E.

Duggan M.E. Allosteric Akt (PKB) inhibitors: discovery and SAR of isozyme selective inhibitors. Zhao et al., 2005 Zhao Z.

Leister W.H.

Robinson R.G.

Barnett S.F.

Defeo-Jones D.

Jones R.E.

Hartman G.D.

Huff J.R.

Huber H.E.

Duggan M.E.

Lindsley C.W. Discovery of 2,3,5-trisubstituted pyridine derivatives as potent Akt1 and Akt2 dual inhibitors. Patel et al., 2014 Patel V.

Bhatt N.

Bhatt P.

Joshi H.D. Synthesis and pharmacological evaluation of novel 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one derivatives as potential antimicrobial agents. General synthesis methods: the synthesis of all compounds followed the standard methods depicted in Figure S1 . Nucleophilic aromatic substitution followed by nitro group reduction (), urea formation (), standard Boc deprotection, and finally direct alkylation () or reductive amination of an aldehyde () or ketone () gave the indicated SR compounds. The compounds were isolated and characterized in free base form unless indicated (overall yields 15%–40% for 5 steps) and then the compounds were evaluated in all biological and pharmacological assays as their mono mesylate salts. Representative nucleophilic aromatic substitution procedure, R1 = R2 = Cl: 1,2-Dichloro-4-fluoro-5-nitrobenzene (0.43 mL, 3.3 mmol) was added to a mixture of tert-butyl 4-aminopiperidine-1-carboxylate (0.66 g, 3.3 mmol) and K 2 CO 3 (0.50 g, 3.6 mmol) in DMSO (5 mL). The reaction mixture was stirred overnight at room temperature under argon. Water was added and the organic layer extracted with EtOAc, dried over Na 2 SO 4 , and concentrated under reduced pressure. Purification was achieved by flash column chromatography on silica gel using a gradient of EtOAc:hexanes as the eluent to give an orange solid (0.79 g, 62% yield). 1H NMR (400 MHz, CDCl 3 ) δ 8.29 (s, 1H), 8.01 (d, J = 7.2 Hz, 1H), 6.97 (s, 1H), 4.03 (d, J = 13.6 Hz, 2H), 3.65-3.56 (m, 1H), 3.05 (td, J = 12.4, 2.8 Hz, 2H), 2.05 (dd, J = 13.0, 3.4 Hz, 2H), 1.63-1.49 (m, 2H), 1.47 (s, 9H); MS(m/z): [M + H] calc’d for C 16 H 21 Cl 2 N 3 O 4 is 390.26, found 389.49. Representative nitro group reduction procedure, R1 = R2 = Cl: Tert-butyl 4-((4,5-dichloro-2-nitrophenyl)amino)piperidine-1-carboxylate (0.79 g, 2.0 mmol) was dissolved in EtOH (40 mL) and a 50% aqueous suspension of Raney nickel (5 mL) was added. Hydrazine hydrate (0.98 mL, 20 mmol) was then added dropwise. The mixture was heated to 45°C and maintained at that temperature for 10 min. The mixture was filtered through a pad of Celite® which was washed with MeOH. The filtrate was concentrated under reduced pressure. Purification was achieved by flash column chromatography on silica gel using a gradient of EtOAc: hexanes as the eluent to give the diamine product (0.55 g, 76% yield). 1H NMR (400 MHz, CD 3 OD) δ 6.75 (s, 1H), 6.62 (s, 1H), 4.46 (s, 1H), 4.01 (d, J = 13.2 Hz, 2H), 3.36 (tt, J = 10.0, 4.0 Hz, 1H), 2.95 (t, J = 12.2 Hz, 2H), 2.00 (d, J = 13.6 Hz, 2H), 1.45 (s, 9H), 1.36 (qd, J = 12.0, 4.0 Hz, 2H); MS(m/z): [M + H] calc’d for C 16 H 23 Cl 2 N 3 O 2 is 360.28, found 359.58. Representative urea formation and Boc removal procedure, R1 = R2 = Cl: Tert-butyl 4-((2-amino-4,5-dichloro-phenyl)amino)piperidine-1-carboxylate (0.55 g, 1.5 mmol) was dissolved in THF (15 mL) under argon. CDI (0.35 g, 2.1 mmol) was added and the reaction mixture was stirred at room temperature overnight. Upon completion, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc. This mixture was washed with 1M HCl (aq) followed by brine. The organic layer was dried over Na 2 SO 4 and the solvent was removed under reduced pressure. Purification was achieved by flash column chromatography on silica gel using a gradient of EtOAc: hexanes as the eluent to give tert-butyl 4-(5,6-dichloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-1-carboxylate (0.54 g, 91% yield). 1H NMR (400 MHz, CDCl 3 ) δ 9.78 (s, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 4.44-4.34 (m, 3 H), 2.85 (t, J = 11.2 Hz, 2H), 2.26 (qd, J = 12.6, 4.2 Hz, 2H), 1.82 (d, J = 10.8 Hz, 2H), 1.52 (s, 9H); MS(m/z): [M + H] calc’d for C 17 H 21 Cl 2 N 3 O 3 is 386.27, found 385.33. This product was dissolved in a 33% solution of TFA in CH 2 Cl 2 (4 mL). Upon completion, the solvent was removed under reduced pressure and the residue was dissolved in a minimal amount of water-acetonitrile (1:1). The solution was frozen and was then subjected to lyophilization overnight, giving the amine product as a TFA salt (86% crude yield). 1H NMR (400 MHz, CDCl 3 ) δ 7.36 (s, 1H), 7.17 (s, 1H), 4.36 (tt, J = 12.6, 4.0 Hz, 1H), 3.27 (d, J = 12.0 Hz, 2H), 2.79 (td, J = 12.2, 2.0 Hz, 2H), 2.27 (qd, J = 12.4, 4.0 Hz, 2H), 1.83 (dd, J = 12.0, 2.0 Hz, 2H); MS(m/z): [M + H] calc’d for C 12 H 13 Cl 2 N 3 O is 286.16, found 286.12. Representative reductive amination procedure, R1 = R2 = Cl, R3 = Br: NaBH(OAc) 3 (97 mg, 0.44 mmol) was added to an anhydrous DCE (3 mL) solution of 5,6-Dichloro-1-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (59 mg, 0.15 mmol), and 4-bromobenzaldehyde (85 mg, 0.44 mmol). A few drops of AcOH were added to the solution and the reaction mixture was stirred overnight at room temperature under argon. Upon completion, saturated aq. NaHCO 3 was added to the reaction mixture, which was then diluted with CH 2 Cl 2 . The aqueous layer was extracted with CH 2 Cl 2 and the combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated under reduced pressure. Purification was achieved by flash column chromatography on silica gel using with CH 2 Cl 2 :MeOH as the eluent to give the desired product SR-15099 (36 mg, 55% yield). Methanesulfonic acid (5.2 μL, 0.08 mmol) was added to a suspension of SR-15099 free base in EtOH (1 mL). The mixture was heated to 60°C for 30 min. The solvent was evaporated under reduced pressure and the residue was dissolved in a minimal amount of water-acetonitrile (1:1). The solution was frozen and subjected to lyophilization overnight, giving 5,6-Dichloro-1-(1-(4-bromobenzyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one methanesulfonate as an white powder. Analytical data for this tested compound is given later in this section. Note: for reductive amination reactions of methyl ketones rather than aldehydes, the ketone (3 equiv.) and amine was treated with 10 eq. of Ti(Oi-Pr) 4 , heated to 60°C, then 8.75 equiv. of NaBH 3 CN in ethanol was added. After overnight reaction, the mixture was processed as described above. Added note: the unsubstituted N-benzyl compound SR-20437 was prepared by alkyation rather than by reductive amination. To the amine in minimal DMF was added 1.1 equiv. of benzyl bromide, 1.1 equiv. of K 2 CO 3 , 1.0 equiv. of NaI. Heating at 60°C overnight, standard workup, and salt formation gave the desired material in 53% yield.

Analytical data for final compounds 1H NMR (400 MHz, (CD 3 ) 2 SO) δ 10.80 (s, 1H), 7.34 (t, J = 3.0 Hz, 4H), 7.26-7.19 (m, 2H), 6.99-6.95 (m, 3H), 4.05 (tt, J = 12.6, 4.0 Hz, 1H), 3.53 (q, J = 6.8 Hz, 1H), 3.10 (d, J = 10.0 Hz, 1H), 2.89 (d, J = 9.6 Hz, 1H), 2.45-2.23 (m, 2H), 2.07 (td, J = 10.4, 2.4 Hz, 1H), 1.97 (td, J = 10.8, 2.0 Hz, 1H), 1.66 (d, J = 10.8 Hz, 1H), 1.58 (d, J = 10.4 Hz, 1H), 1.33 (d, J = 6.8 Hz, 3H); MS(m/z): [M + H] calculated for C 20 H 23 N 3 O is 321.42, found 321.96; HPLC t R = 3.52 min. 1H NMR (400 MHz, CDCl 3 ) δ 7.93 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.19 (td, J = 7.6, 1.2 Hz, 1H), 7.09-7.02 (m, 2H), 4.62-4.54 (m, 1H), 4.21-4.18 (m, 1H), 3.89 (d, J = 10.4 Hz, 1H), 3.52 (d, J = 12.8 Hz, 2H), 3.27 (qd, J = 11.6, 4.0 Hz, 1H), 2.78 (q, J = 10.4 Hz, 1H), 2.65 (q, J = 11.2 Hz, 1H), 1.97 (d, J = 6.8 Hz, 3H), 1.93-1.89 (m, 2H); MS(m/z): [M + H] calculated for C 20 H 22 ClN 3 O is 355.87, found 355.92; HPLC t R = 3.76 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) 7.56-7.54 (m, 4H), 7.29-7.26 (m, 1H), 7.10-7.07 (m, 3H), 4.56 (tt, J = 12.4, 4.0 Hz, 1H), 4.39 (s, 2H), 3.66 (dd, J = 10.6, 1.8 Hz, 2H), 3.27-3.23 (m, 2H), 2.80 (qd, J = 13.4, 3.8 Hz, 2H), 2.72 (s, 3H), 2.09 (d, J = 14.8 Hz, 2H); MS(m/z): [M + H] calculated for C 19 H 20 ClN 3 O is 341.84, found 342.02; HPLC t R = 3.67 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.56-7.52 (m, 5H), 7.48 (s, 1H), 7.19 (s, 1H), 4.53 (tt, J = 12.4, 4.0 Hz, 1H), 4.39 (s, 2H), 3.65 (d, J = 12.8 Hz, 2H), 3.28-3.22 (m, 2H), 2.77 (qd, J = 13.4, 4.0 Hz, 2H), 2.72 (s, 3H), 2.08 (d, J = 14.8 Hz, 2H); MS(m/z): [M + H] calculated for C 19 H 19 Cl 2 N 3 O is 376.28, found 375.98; HPLC t R = 3.95 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.58-7.53 (m, 4H), 7.48 (s, 1H), 7.19 (s, 1H), 4.52 (tt, J = 12.4, 3.8 Hz, 1H), 4.38 (s, 2H), 3.65 (d, J = 12.8 Hz, 2H), 3.25 (t, J = 12.2 Hz, 2H), 2.80-2.69 (m, 5H), 2.08 (d, J = 13.6 Hz, 2H); MS(m/z): [M + H] calculated for C 19 H 18 Cl 3 N 3 O is 410.72, found 410.01; HPLC t R = 4.12 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.70 (dt, J = 8.4, 2.1 Hz, 2H), 7.50 (dd, J = 6.6, 2.0 Hz, 3H), 7.19 (s, 1H), 4.53 (tt, J = 12.4, 4.0 Hz, 1H), 4.37 (s, 2H), 3.63 (d, J = 12.8 Hz, 2H), 3.24 (t, J = 12.4 Hz, 2H), 2.79-2.70 (m, 6H), 2.07 (d, J = 12.4 Hz, 2H); MS(m/z): [M + H] calculated for C 19 H 18 BrCl 2 N 3 O is 455.18, found 456.23; HPLC t R = 3.76 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.71 (d, J = 8.4 Hz, 2H), 7.49 (t, J = 8.4 Hz, 3H), 7.19 (s, 1H), 4.57 (q, J = 6.8 Hz, 1H), 4.44 (tt, J = 12.4, 4.0 Hz, 1H), 3.87 (dd, J = 11.0, 1.8 Hz, 1H), 3.49 (dd, J = 11.2, 2.0 Hz, 1H), 3.14 (td, J = 13.0, 2.8 Hz, 1H), 3.04 (td, J = 13.0, 2.8 Hz, 1H), 2.84-2.66 (m, 6H), 2.12-2.02 (m, 2H), 1.80 (d, J = 6.8 Hz, 3H); MS(m/z): [M + H] calculated for C 20 H 20 BrCl 2 N 3 O is 469.20, found 469.89; HPLC t R = 4.00 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.67 (t, J = 7.8 Hz, 1H), 7.48-7.43 (m, 3H), 7.19 (s, 1H), 4.93-4.89 (m, 1H), 4.47 (tt, J = 12.2, 4.0 Hz, 1H), 3.89 (d, J = 12.0 Hz, 1H), 3.60 (d, J = 10.4 Hz, 1H), 3.19 (td, J = 13.0, 2.2 Hz, 1H), 3.10 (td, J = 13.0, 2.2 Hz, 1H), 2.88-2.72 (m, 6H), 2.12-2.04 (m, 2H), 1.83 (d, J = 6.8 Hz, 3H); MS(m/z): [M + H] calculated for C 20 H 19 Cl 3 FN 3 O is 442.74, found 441.87; HPLC t R = 3.94 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.67 (t, J = 8.2 Hz, 1H), 7.50 (s, 1H), 7.43 (qd, J = 7.8, 2.0 Hz, 2H), 7.19 (s, 1H), 4.55 (tt, J = 12.4, 4.0 Hz, 1H), 4.46 (s, 2H), 3.70 (d, J = 12.4 Hz, 2H), 3.36-3.30 (m, 2H), 2.82-2.71 (m, 5H), 2.09 (d, J = 13.6 Hz, 2H); MS(m/z): [M + H] calculated for C 19 H 17 Cl 3 FN 3 O is 428.71, found 427.96; HPLC t R = 4.15 min. 1H NMR of the mesylate salt (400 MHz, CD 3 OD) δ 7.30-7.27 (m, 1H), 7.09-7.06 (m, 4H), 7.01 (dd, J = 8.4, 2.0 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.50-4.41 (m, 2H), 4.28 (s, 4H), 3.84 (dt, J = 12.8, 1.9 Hz, 1H), 3.49 (dt, J = 11.8, 1.4 Hz, 1H), 3.14 (td, J = 13.0, 2.8 Hz, 1H), 3.04 (td, J = 13.0, 2.8 Hz, 1H), 2.92-2.72 (m, 6H), 2.12-2.01 (m2H), 1.77 (d, J = 7.2 Hz, 3H); MS(m/z): [M + H] calculated for C 22 H 25 N 3 O 3 is 379.46, found 379.87; HPLC t R = 4.58 min.

Preparation of drug solutions For the in vitro studies, the reference compounds DAMGO, morphine sulfate and nociceptin were prepared in water as a 10 mM stock and a 10 mM stock of U69,593 was prepared in ethanol. All of the other compounds were prepared in DMSO at concentrations spanning from 32 nM to 10 mM, for dilutions. For all assays, the final DMSO concentration was 1%. For the in vivo studies, compounds were dissolved from powder immediately prior to use. Morphine sulfate and the test compounds were prepared in a vehicle of 1:1:8 DMSO: Tween 80: dH 2 O. Fentanyl citrate was dissolved in 0.9% saline for the studies in C57BL/6J male mice. For studies where only one dose was tested (females and MOR-KO mice) all compounds were made in the same vehicle to facilitate blinding of drug preparation and experimenter handling. Compounds were administered intraperitoneally (i.p.) at a concentration of 10 μL per gram mouse, except for the 48 mg/kg dose of the test compounds. In this case, the drugs were administered at a volume of 20 μL per gram mouse due to limited solubility. Morphine sulfate and fentanyl citrate dosing is based on the salt weight of the drugs, while the SR compounds dosing is based on the free base weight.

Saturation and competition radioligand binding Groer et al., 2011 Groer C.E.

Schmid C.L.

Jaeger A.M.

Bohn L.M. Agonist-directed interactions with specific beta-arrestins determine mu-opioid receptor trafficking, ubiquitination, and dephosphorylation. Schmid et al., 2013 Schmid C.L.

Streicher J.M.

Groer C.E.

Munro T.A.

Zhou L.

Bohn L.M. Functional selectivity of 6′-guanidinonaltrindole (6′-GNTI) at κ-opioid receptors in striatal neurons. 3H-DAMGO; KOR, 3H-U69,593; DOR, 3H-diprenorphine) for 2 hr at 25°C. For competition experiments, the concentration of each of the radioligands was approximately 1 nM (0.96–1.10 nM 3H-DAMGO; 1.06–1.19 nM 3H-U69,593; 0.92–0.98 nM 3H-diprenorphine). Nonspecific binding was determined in the presence of 10 μM DAMGO (MOR), 10 μM U69,593 (KOR) or 10 μM Naloxone (DOR). Reactions were terminated by filtration through GF/B glass fiber filter plates (PerkinElmer), which had been pre-incubated with 0.1% polyethyleneimine, on a Brandel cell harvester. Radioactivity was counted with Microscint on a TopCount NXT Scintillation Counter (PerkinElmer). Saturation binding assays and hyperbolic curve fitting of specific binding was used to determine radioligand binding affinities and receptor numbers for the CHO cell lines (hMOR, 1.02 ± 0.10 nM for 3H-DAMGO and 1.58 ± 0.11 pmol/mg; hDOR, 0.70 ± 0.11 nM [3H]-Diprenorphine and 1.46 ± 0.26 pmol/mg; hKOR, 1.07 ± 0.01 nM [3H]-U69,593 and 0.71 ± 0.12 pmol/mg). Receptor binding assays were performed on CHO-hMOR, CHO-hDOR and CHO-hKOR cell lines as previously described (). Cells were serum-starved for 30 min, cells were collected and membrane pellets were prepared by Teflon-on-glass dounce homogenization in membrane buffer containing (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA), followed by centrifugation at 20,000 x g for 30 min at 4°C. Membranes were resuspended in assay buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl). Binding reactions (200 μL volume) were performed on 10 μg membranes with the appropriate radioligand (MOR,H-DAMGO; KOR,H-U69,593; DOR,H-diprenorphine) for 2 hr at 25°C. For competition experiments, the concentration of each of the radioligands was approximately 1 nM (0.96–1.10 nMH-DAMGO; 1.06–1.19 nMH-U69,593; 0.92–0.98 nMH-diprenorphine). Nonspecific binding was determined in the presence of 10 μM DAMGO (MOR), 10 μM U69,593 (KOR) or 10 μM Naloxone (DOR). Reactions were terminated by filtration through GF/B glass fiber filter plates (PerkinElmer), which had been pre-incubated with 0.1% polyethyleneimine, on a Brandel cell harvester. Radioactivity was counted with Microscint on a TopCount NXT Scintillation Counter (PerkinElmer). Saturation binding assays and hyperbolic curve fitting of specific binding was used to determine radioligand binding affinities and receptor numbers for the CHO cell lines (hMOR, 1.02 ± 0.10 nM forH-DAMGO and 1.58 ± 0.11 pmol/mg; hDOR, 0.70 ± 0.11 nM [H]-Diprenorphine and 1.46 ± 0.26 pmol/mg; hKOR, 1.07 ± 0.01 nM [H]-U69,593 and 0.71 ± 0.12 pmol/mg).

35S-GTPγS binding to membranes 35S-GTPγS binding was determined in membranes prepared from CHO-hMOR and CHO-mMOR cells and brainstems isolated from adult male C57BL/6J and MOR-KO mice as described previously ( Schmid et al., 2013 Schmid C.L.

Streicher J.M.

Groer C.E.

Munro T.A.

Zhou L.

Bohn L.M. Functional selectivity of 6′-guanidinonaltrindole (6′-GNTI) at κ-opioid receptors in striatal neurons. 2 , 1 mM EDTA) with 50 μM guanosine-5”-diphosphate (GDP) and 0.1 nM 35S-GTPγS. Reactions were terminated by filtration through GF/B filter plates and radioactivity was counted as described above. For [35S]-GTPγS binding on brainstems isolated from C57BL/6J and MOR-KO mice, tissues were homogenized by polytronic tissue tearor and membranes were prepared as described above. Binding reactions, containing 2.5 μg protein, 1 mM dithiothreitol (DTT), 20 μM GPD and 0.1 nM 35S-GTPγS, were incubated at room temperature for 2 hrs prior to harvesting. The average vehicle value for the CHO-hMOR membranes was 786 ± 78 cpm and the average fold over vehicle for DAMGO was 4.6 ± 0.26. The average vehicle value for the CHO-mMOR cell membranes was 694 ± 28 cpm and the average fold over vehicle for DAMGO was 5.9 ± 0.57. The average vehicle for the C57BL/6J brainstem membranes was 657 ± 62 cpm and the average fold over vehicle for DAMGO was 1.9 ± 0.03. The average vehicle for the MOR-KO brainstem membranes was 1647 ± 507 cpm. S-GTPγS binding was determined in membranes prepared from CHO-hMOR and CHO-mMOR cells and brainstems isolated from adult male C57BL/6J and MOR-KO mice as described previously (). CHO-hMOR and CHO-mMOR cellular membranes, collected and prepared as described above with in GTPγS binding membrane buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA). Reactions (200 μL volume) were performed for 1 hr at 25°C on 10 μg membranes suspended in assay buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 5 mM MgCl, 1 mM EDTA) with 50 μM guanosine-5”-diphosphate (GDP) and 0.1 nMS-GTPγS. Reactions were terminated by filtration through GF/B filter plates and radioactivity was counted as described above. For [S]-GTPγS binding on brainstems isolated from C57BL/6J and MOR-KO mice, tissues were homogenized by polytronic tissue tearor and membranes were prepared as described above. Binding reactions, containing 2.5 μg protein, 1 mM dithiothreitol (DTT), 20 μM GPD and 0.1 nMS-GTPγS, were incubated at room temperature for 2 hrs prior to harvesting. The average vehicle value for the CHO-hMOR membranes was 786 ± 78 cpm and the average fold over vehicle for DAMGO was 4.6 ± 0.26. The average vehicle value for the CHO-mMOR cell membranes was 694 ± 28 cpm and the average fold over vehicle for DAMGO was 5.9 ± 0.57. The average vehicle for the C57BL/6J brainstem membranes was 657 ± 62 cpm and the average fold over vehicle for DAMGO was 1.9 ± 0.03. The average vehicle for the MOR-KO brainstem membranes was 1647 ± 507 cpm.

cAMP accumumlation assay CHO-hMOR, -hDOR and -hKOR cells were plated at a density of 4,000 cells per well of a 384-well, white-walled, 30 μl-volume microplate (Greiner Bio-One) in Opti-MEM containing 1% FBS 4 hr prior to assaying. Cells were treated with 20 μM forskolin, 25 μM 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro-20-1724) and increasing concentrations of test compounds for 30 min at 25°C. Inhibition of cAMP was then determined using the Homogeneous Time-Resolved Fluorescence resonance energy transfer (FRET) cAMP HiRange assay by Cisbio (Cisbio-62AM6PEC). Fluorescence was measured at 620 and 665 nm using an Envision Multilabel Reader (PerkinElmer). FRET was calculated by the ratio of 665 nm / 620 nm. The average vehicle ratio for CHO-hMOR cells was 3134 ± 99 and the average fold over vehicle for DAMGO was 2.2 ± 0.04. The average vehicle ratio for CHO-hDOR cells was 2962 ± 181 and the average fold over vehicle for SNC80 was 1.6 ± 0.04. The average vehicle ratio for CHO-hKOR cells was 2965 ± 153 and the average fold over vehicle for U69,593 was 1.9 ± 0.12.

βArrestin2 recruitment assays To determine βarrestin2 recruitment to the human MOR a commercial enzyme fragment complementation assay (β-galactosidase) was used. U2OS-βarrestin-hMOR PathHunter cells were plated at a density of 5,000 cells per well of a 384-well, white-walled assay microplate (Greiner Bio-One) in Assay Complete Cell Plating 5 Reagent (DiscoveRx) 16-20 hr prior to measuring the signal. Cells were treated for 90 min with increasing concentrations of test compounds at 37°C and βarrestin2 recruitment was determined using the PathHunter Detection Kit with the β-galactosidase substrate to detect functional β-galactosidas. The resulting increase in luminescence was measured using a SpectraMax M5e Microplate Reader (Molecular Devices). The average vehicle for the PathHunter U2OS OPRM1 βarrestin cells was 446 ± 25 RLU and the average fold over vehicle for DAMGO was 36 ± 1. Zhou et al., 2013 Zhou L.

Lovell K.M.

Frankowski K.J.

Slauson S.R.

Phillips A.M.

Streicher J.M.

Stahl E.

Schmid C.L.

Hodder P.

Madoux F.

et al. Development of functionally selective, small molecule agonists at kappa opioid receptors. To determine βarrestin2 recruitment to the mMOR, an imaging-based assay as was used (). U2OS-βarrestin2-GFP-mMOR cells were plated at a density of 5,000 cells per well of a 384-well, black-walled, clear-bottom optical imaging microplate (Brooks) in normal media 16-20 hr prior to assaying. Cells were serum-starved for 1 hr and then treated with increasing concentrations of test compounds for 20 min at 37°C. Cells were fixed with 4% paraformaldehyde (PFA) containing Hoechst nuclear stain at a dilution of 1:1000. βArrestin 2 translocation was measured using the 20X objective on a CellInsight CX5 High Content Screening Platform (ThermoFisher Scientific). Punctae (normalized to Hoechst stain) were quantified using the Cellomics’ Spot Detection BioApplication (ThermoFisher Scientific). The average punctae / Hoechst ratio for vehicle treated U2OS-βarrestin2-GFP-mMOR cells was 2.2 ± 0.54 and the average fold over vehicle for DAMGO was 61 ± 13. To determine whether the compounds have activity at NOP, βarrestin2 recruitment to the receptor was determined in the U2OS-Tango-hOPRL1-bla cells. U2OS-Tango-hOPRL1-bla cells were plated at a density of 10,000 cells per well of a 384-well, black-walled, clear-bottom assay plate in 32 μL assay media (DMEM + 10% dialyzed FBS, 0.1 mM NEAA, 25 mM HEPES and 1% pen/strep) 16-20 hr prior to assaying. Cells were treated with increasing concentrations of test compounds for 5 hr at 37°C. NOP activation was determined using the LiveBLAzer FRET-B/G loading kit with Solution D (ThermoFisher Scientific), according to the manufacturer’s protocol. FRET signal (excitation 409 nm, emissions at 460 nm and 530 nm) was measured using a SpectraMax M5e Microplate Reader (Molecular Devices). The average 460/530 ratio vehicle treated U2OS-Tango-hOPRL1-bla cells was 0.31 ± 0.03 and the average fold over vehicle for nociceptin was 7.6 ± 0.68.

Pharmacokinetics and plasma protein binding Brust et al., 2016 Brust T.F.

Morgenweck J.

Kim S.A.

Rose J.H.

Locke J.L.

Schmid C.L.

Zhou L.

Stahl E.L.

Cameron M.D.

Scarry S.M.

et al. Biased agonists of the kappa opioid receptor suppress pain and itch without causing sedation or dysphoria. Kieltyka et al., 2016 Kieltyka K.

McAuliffe B.

Cianci C.

Drexler D.M.

Shou W.

Zhang J. Application of Cassette Ultracentrifugation Using Non-labeled Compounds and Liquid Chromatography-Tandem Mass Spectrometry Analysis for High-Throughput Protein Binding Determination. Pharmacokinetic parameters were determined in the C57BL/6J mice by i.p. dosing. Plasma was generated by standard centrifugation techniques, resulting in ∼10 μL of plasma that was immediately frozen. For brain collection, mice were sacrificed by cervical dislocation and brains were isolated and flash frozen in liquid nitrogen. Drug levels were determined using a LC (Shimadzu)-tandem mass spectrometry (AB Sciex) operated in positive-ion mode using multiple reaction monitoring methods (). Plasma protein binding for fentanyl and morphine was determined using Rapid Equilibrium Dialysis (RED) devices (ThermoFisher). For the SR compounds, plasma samples (0.5 mL at 0.5 μM test compound) were prepared and 900 μL was transferred to a 2 mL polycarbonate ultracentrifuge tube. The sample was centrifuged at 400,000 x g for two hr using a Beckman Coulter Optima Max ultracentrifuge (130,000 RPM max) with a TLA 120.2 rotor held at 25°C. The centrifuged sample separates into three layers. The protein-rich bottom layer contains most of the albumin and is easily visualized. The top layer is not as easily discerned, but contains a high concentration of lipoproteins. The middle layer (1-2 mm below surface using the described conditions) has very low protein concentrations and can be used to determine the amount of unbound drug. The percent unbound compound was determined by LC-MS/MS by comparison of the compound concentration in the middle layer of the centrifuged sample to the concentration of a parallel sample that did not undergo centrifugation ().

Antinociception Bohn et al., 1999 Bohn L.M.

Lefkowitz R.J.

Gainetdinov R.R.

Peppel K.

Caron M.G.

Lin F.T. Enhanced morphine analgesia in mice lacking beta-arrestin 2. Raehal et al., 2011 Raehal K.M.

Schmid C.L.

Groer C.E.

Bohn L.M. Functional selectivity at the μ-opioid receptor: implications for understanding opioid analgesia and tolerance. ∗ 100. Antinociceptive responses to thermal stimuli were determined according to previously published protocols (). Basal nociceptive responses were determined by measuring the amount of time until a mouse rapidly flicked its tail when placed into a 49°C water bath (tail flick test) or until it licked or flicked its fore- or hind-paws when placed on a to a 52°C hot plate (hot plate test; Hotplate Analgesia Meter, Columbus Instruments). Baseline response latencies averaged 2.95 ± 0.07 s (tail flick) and 6.17 ± 0.06 s (hot plate) for C57BL/6J male mice, 2.34 ± 0.18 s (tail flick) and 6.78 ± 0.14 s (hot plate) for C57BL/6J female mice and 2.29 ± 0.12 s (tail flick) and 6.54 ± 0.17 s (hot plate) for MOR-KO male mice. Antinociceptive responses were determined at the indicated time points over the course of 6 hr immediately following injection. To minimize tissue damage, maximum response latencies were limited to 30 and 20 s for tail flick and hot plate assays, respectively. Data are presented as “% maximum possible effect” which was calculated by (response latency – baseline) / (maximal response cutoff latency – baseline)100.