Identification of a Μ-Δ Opioid Receptor Heteromer-Biased Agonist With

Corrections

MEDICAL SCIENCES Correction for “Myc and mTOR converge on a common node Proc Natl Acad Sci USA (110:11988–11993; ﬁrst published June in protein synthesis control that confers synthetic lethality in 26, 2013; 10.1073/pnas.1310230110). Myc-driven cancers,” by Michael Pourdehnad, Morgan L. Truitt, The authors note that Fig. 5 appeared incorrectly. The cor- Imran N. Siddiqi, Gregory S. Ducker, Kevan M. Shokat, and rected ﬁgure and its legend appear below. Davide Ruggero, which appeared in issue 29, July 16, 2013, of

AB C

Fig. 5. Clinical relevance of mTOR-dependent 4EBP1 phosphorylation in Myc-driven human lymphomas. (A) Analysis of apoptosis in the human Raji Burkitt’s lymphoma cell line upon 4EBP1m expression or MLN0128 treatment for 24 h. Graph represents mean ± SD. (B) Representative H&E, Myc staining, and phospho-4EBP1 staining in human diffuse large B-cell lymphoma (DLBCL). (C) Box and whisker plot of IHC intensity for total 4EBP1 and phospho-4EBP1 from a human DLBCL tissue microarray (TMA) consisting of 77 patients.

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NEUROSCIENCE NEUROSCIENCE Correction for “Identification of a μ-δ opioid receptor heteromer- Correction for “Transient, afferent input-dependent, postnatal biased agonist with antinociceptive activity,” by Ivone Gomes, niche for neural progenitor cells in the cochlear nucleus,” Wakako Fujita, Achla Gupta, Adrian S. Saldanha, Ana Negri, by Stefan Volkenstein, Kazuo Oshima, Saku T. Sinkkonen, Christine E. Pinello, Edward Roberts, Marta Filizola, Peter Hodder, C. Eduardo Corrales, Sam P. Most, Renjie Chai, Taha A. Jan, and Lakshmi A. Devi, which appeared in issue 29, July 16, 2013, Alan G. Cheng, and Stefan Heller, which appeared in issue 35, of Proc Natl Acad Sci USA (110:12072–12077; first published July 1, August 27, 2013, of Proc Natl Acad Sci USA (110:14456–14461; 2013; 10.1073/pnas.1222044110). first published August 12, 2013; 10.1073/pnas.1307376110). The authors note that Christina Eberhart should be added to The authors note that Renée van Amerongen should be added the author list between Christine E. Pinello and Edward Roberts. to the author list between Taha A. Jan and Alan G. Cheng. Renée Christina Eberhart should be credited with having performed van Amerongen should be credited with having performed re- research and analyzed data. search and having contributed new reagents/analytic tools. The The authors also note that the author name Adrian S. Saldanha corrected author line, affiliation line, and author contributions should instead appear as S. Adrian Saldanha. appear below. The online version has been corrected. The corrected author line, affiliation line, and author contributions appear below. The online version has been corrected. Stefan Volkensteina,b, Kazuo Oshimaa,b, Saku T. Sinkkonena,b, C. Eduardo Corralesa, Sam P. Mosta, a a a Ivone Gomes , Wakako Fujita , Achla Gupta , Renjie Chaia, Taha A. Jana, Renée van Amerongenc, b c b S. Adrian Saldanha , Ana Negri , Christine E. Pinello , Alan G. Chenga, and Stefan Hellera,b Christina Eberhartb, Edward Robertsd, Marta Filizolac, Peter Hodderb, and Lakshmi A. Devia Departments of aOtolaryngology–Head and Neck Surgery and bMolecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305; and cDivision of Molecular Oncology, Netherlands Cancer Departments of aPharmacology and Systems Therapeutics and cStructural Institute, 1066 CX, Amsterdam, The Netherlands and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bScripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, Jupiter, FL 33458; and dDepartment of Chemistry, The Scripps Research Institute, La Jolla, Author contributions: S.V., K.O., T.A.J., and S.H. designed research; S.V., K.O., CA 92037 S.T.S., C.E.C., S.P.M., R.C., T.A.J., and R.v.A. performed research; R.v.A. contributed new reagents/analytic tools; S.V., K.O., S.T.S., C.E.C., S.P.M., T.A.J., A.G.C., and S.H. analyzed data; and S.V., K.O., S.P.M., and S.H. wrote the paper. Author contributions: P.H. and L.A.D. designed research; I.G., W.F., A.G., S.A.S., A.N., C.E.P., C.E., and E.R. performed research; E.R. and M.F. contributed www.pnas.org/cgi/doi/10.1073/pnas.1317787110 new reagents/analytic tools; I.G., W.F., S.A.S., C.E.P., C.E., M.F., P.H., and L.A.D. analyzed data; and I.G. and L.A.D. wrote the paper.

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17160–17161 | PNAS | October 15, 2013 | vol. 110 | no. 42 www.pnas.org Downloaded by guest on September 28, 2021 NEUROSCIENCE Correction for “The role of long-range connections on the speci- ficity of the macaque interareal cortical network,” by Nikola T. Markov, Maria Ercsey-Ravasz, Camille Lamy, Ana Rita Ribeiro Gomes, Loïc Magrou, Pierre Misery, Pascale Giroud, Pascal Barone, Colette Dehay, Zoltán Toroczkai, Kenneth Knoblauch, David C. Van Essen, and Henry Kennedy, which appeared in issue 13, March 26, 2013, of Proc Natl Acad Sci USA (110:5187– 5192; first published March 11, 2013; 10.1073/pnas.1218972110). The authors note that Fig. 4 appeared incorrectly. The correct figure and its legend appear below. Additionally, on page 5190, right column, first full paragraph, lines 21–22, “These values contrast with the interregion graph, in which the density is 50%” should instead appear as “These values contrast with the interregion graph, in which the density is 61%.”

A intra target regions inter target regions 25 Experimental values 25 Randomized values 20 20 15 15 10 10 5 5 0 0

Number of common areas Front Occ Par Pref Temp Front Occ Par Pref Temp

B C Absent 1.0 Present 100

0.8 80 0.6 60 0.4 Density 40 0.2 Density in %

Number of areas 20 0 Front ParOcc Pref Temp Inter G29x29

0 200 400 600 800 1000 0 D

p < 0.001 (0,10] 0.4 (10,20] (20,30] (30,40] (40,50] (50,60] Distance intervals (mm) 0.2

0.0 Similarity score -0.2 0 102030405060 Distance (mm)

Fig. 4. Inﬂuence of distance on connectivity. (A) Number of intraregion (Left) and interregion (Right) common-source areas and effects of randomization of connections with preservation of target in-degree. Error bars, 5–95% quantiles after 2 × 104 permutation tests. (B) Density of the edge-complete graphs for intra- and interregions. (C) Histogram showing the number of connected and nonconnected areas at given distance intervals from injected target areas. Black bars, connected source areas; white bars, nonconnected areas. In red, connec- tion density percentage (proportion of connected with respect to unconnected areas) of connectivity with distance. (D) Binary similarity index as a function of distance between target pairs. Abbreviations are the same as in Fig. 2.

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PNAS | October 15, 2013 | vol. 110 | no. 42 | 17161 Downloaded by guest on September 28, 2021 Identiﬁcation of a μ-δ opioid receptor heteromer- biased agonist with antinociceptive activity

Ivone Gomesa, Wakako Fujitaa, Achla Guptaa, Adrian S. Saldanhab, Ana Negric, Christine E. Pinellob, Edward Robertsd, Marta Filizolac, Peter Hodderb, and Lakshmi A. Devia,1

Departments of aPharmacology and Systems Therapeutics and cStructural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bScripps Research Institute Molecular Screening Center, Lead Identiﬁcation Division, Translational Research Institute, Jupiter, FL 33458; and dDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037

Edited* by Susan G. Amara, National Institute of Mental Health, Bethesda, MD, and approved June 4, 2013 (received for review December 19, 2012) G protein-coupled receptors play a pivotal role in many physiological or β-arrestin or possible disruption of μOR-δOR heteromers signaling pathways. Mounting evidence suggests that G protein- leads to an enhancement of morphine-mediated antinociception coupled receptors, including opioid receptors, form dimers, and and attenuation in the development of tolerance (8–10). Nota- dimerization is necessary for receptor maturation, signaling, and bly, we observed that a δOR antagonist, H-Tyr-Tic[CH2NH]- trafficking. However, the physiological role of dimerization in Phe-Phe-OH (TIPPψ), can potentiate morphine-mediated an- vivo has not been well-explored because of the lack of tools to algesia (4), and studies using bivalent ligands targeting μOR- study these dimers in endogenous systems. To address this prob- δOR heteromers showed that these ligands induce anti- lem, we previously generated antibodies to μ-δ opioid receptor nociception with attenuated development of tolerance as well as (μOR-δOR) dimers and used them to study the pharmacology and conditioned place preference (11, 12). Taken together, these signaling by this heteromer. We also showed that the heteromer data suggest that occupancy of δOR by an antagonist could exhibits restricted distribution in the brain and that its abundance dissociate the antinociceptive effects of μOR agonists from the is increased in response to chronic morphine administration. development of tolerance and addiction. Therefore, there is Thus, the μOR-δOR heteromer represents a potentially unique a need for ligands that selectively interact with μOR-δOR het- target for the development of therapeutics to treat pain. Here, we eromers to understand their role in antinociception and de- report the identification of compounds targeting μOR-δOR hetero- velopment of tolerance to morphine. NEUROSCIENCE mers through high-throughput screening of a small-molecule li- In an attempt to identify μOR-δOR heteromer-selective ago- brary. These compounds exhibit activity in μOR-δOR cells but not nists, we used a β-arrestin recruitment assay and screened small μOR or δOR cells alone. Among them, CYM51010 was found to molecules available through the Molecular Libraries Probe Pro- be a μOR-δOR–biased ligand, because its activity is blocked by duction Centers Network. This screen identified 94 compounds the μOR-δOR heteromer antibody. Notably, systemic administra- that were biased to μOR-δOR heteromers compared with μOR, tion of CYM51010 induced antinociceptive activity similar to δOR, or serotonin 5HT5A receptors. Among a dozen compounds morphine, and chronic administration of CYM51010 resulted in that were repurchased and tested using secondary screens, one, lesser antinociceptive tolerance compared with morphine. Taken which we named CYM51010 [PubChem compound identifier together, these results suggest that CYM51010, a μOR-δOR–biased (CID)23723457; Probe Report ID ML335], exhibited a strong ligand, could serve as a scaffold for the development of a unique μOR-δOR–biased activity that was blocked by μOR-δOR het- type (heteromer-biased) of drug that is more potent and without the eromer-selective mAb (μ-δ mAb). Furthermore, systemic ad- severe side effects associated with conventional clinical opioids. ministration of CYM51010 led to antinociceptive activity similar to morphine but with a lower antinociceptive tolerance on tudies with mice lacking opioid receptors show that the chronic administration. Notably, although the intrathecal (i.t.) Santinociceptive actions of clinically administered opioids, antinociceptive activity of CYM51010 could be significantly such as morphine or fentanyl, involve the activation of μ-opioid blocked by i.t. administration of μ-δ mAb, the i.t. antinociceptive receptors (μORs) (1). However, continued opioid use leads to activity of morphine was not. These results suggest that CYM51010 undesired side effects, including respiratory depression, con- could serve as a scaffold for the development of unique thera- stipation, immunosuppression, and development of tolerance peutics acting at the μOR-δOR heteromer for the effective man- and addiction (2). In an effort to identify novel compounds that agement of pain. are as effective as morphine in the treatment of chronic pain but Results and Discussion without the associated side effects, our group, among others, has μ δ investigated the modulation of μOR function by receptor het- To screen for OR- OR heteromer-biased ligands, we used a β eromerization. We found that μOR can form interacting com- -arrestin recruitment assay that is based on an enzyme frag- fi plexes with δ-opioid receptors (δORs), that both receptors are in ment complementation technology. Speci cally, receptor acti- β close proximity to interact in live cells, and that, in heterologous vation-mediated -arrestin recruitment leads to reconstitution δ of β-gal activity (Fig. S1). This strategy was used to engineer systems, low nonsignaling doses of some OR ligands can po- βgal βgal βgal μ – cell lines stably expressing μ OR-δOR, δ OR, or μ OR tentiate the binding and signaling of OR agonists (3 5). The fi recently reported crystal structure of μOR (6), in which receptors (DiscoverX). Based on the nding that these cells bind were crystallized as parallel dimers, is consistent with the idea that μOR can associate in complexes. We also generated mAbs selective to μOR-δOR heteromers; Author contributions: P.H. and L.A.D. designed research; I.G., W.F., A.G., A.S.S., A.N., C.E.P., and E.R. performed research; E.R. and M.F. contributed new reagents/analytic we showed that the latter can be detected in the brains of WT tools; I.G., W.F., A.S.S., C.E.P., M.F., P.H., and L.A.D. analyzed data; and I.G. and L.A.D. but not KO mice and that heteromer levels are increased in brain wrote the paper. regions involved in pain processing after chronic morphine ad- The authors declare no conflict of interest. ministration under a paradigm that leads to the development of *This Direct Submission article had a prearranged editor. tolerance (7). The idea that μOR-δOR heteromers may play 1To whom correspondence should be addressed. E-mail: [email protected]. a role in the development of tolerance to morphine is further This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. supported by studies showing that genetic deletion of either δOR 1073/pnas.1222044110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1222044110 PNAS Early Edition | 1of6 radiolabeled μOR or δOR ligands with nanomolar affinity and in β-arrestin recruitment (a phenomenon similar to the phe- exhibit heteromer-mediated increases in binding (potentiation nomenon reported previously) (13) and that these effects are β β β of radiolabeled μOR binding by δOR antagonist and vice versa) seen only in μ galOR-δOR and not μ galOR or δ galOR cells β and agonist-mediated increases in G-protein activity (Fig. S1), we suggests that the μ galOR-δOR cell line would be suitable for proceeded to characterize their suitability to screen for μOR-δOR screening μOR-δOR heteromer-selective ligands. heteromer-biased ligands. To directly test the extent of involvement of μOR-δOR het- We found that treatment with a δOR-selective agonist del- eromers in Delt II-mediated β-arrestin recruitment, we used a μ- torphin II (Delt II) leads to a dose-dependent increase in δ mAb that was previously shown to selectively block μOR-δOR β β-arrestin recruitment to μ galOR-δOR with nanomolar affinity. heteromer activity (7). Treatment with the μ-δ mAb leads to β Similar results were obtained with Flag μOR-δ galOR cells (Fig. a substantial decrease in Delt II-mediated recruitment of β β 1 A and B and Table S1). The Delt II-mediated β-arrestin re- β-arrestin in μ galOR-δOR cells as well as Flag μOR-δ galOR cruitment exhibits a time-dependent increase that plateaus by cells (Fig. 1C and Fig. S3D). Notably, μ-δ mAb-mediated about 40 min; this increase in β-arrestin recruitment is reduced decreases in recruitment are not seen with mAbs directed at μ by the μOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr- other heteromers, such as CB1R-AT1R (CB1-AT1 mAb), OR- μ δ δ C NH2 (CTOP). This effect of CTOP is selective for μOR-δOR CB1R( -CB1 mAb), or OR-CB1R( -CB1 mAb) (Fig. 1 and βgal D δβgal E heteromers, because it is not observed in cells expressing δ OR ), or in cells expressing OR-CB1R(Fig. S3 ), suggesting (Figs. S2 and S3 A–C and Table S1). Reciprocally, the μOR that this effect is selective for μOR-δOR heteromers. These agonist, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), studies gave us confidence to use the β-arrestin recruitment assay increases β-arrestin recruitment, which is reduced by the δOR for high-throughput screening aimed at the identification of antagonist TIPPψ (Fig. S2 and Table S2). These results were small molecules targeting the μOR-δOR heteromer. surprising, because we had previously found that μOR-δOR The set of ∼335,461 small molecules available through the heteromers constitutively recruit β-arrestin (13) and that acti- Molecular Libraries Probe Production Centers Network was βgal βgal βgal vation of the heteromer causes a dissociation of associated screened using cells expressing μ OR-δOR, μ OR, δ OR, βgal β or 5HT5A receptors. Primary screening carried out at a single -arrestin (13). The fact that, in this study, we observe a time- β β concentration (9.3 μM) in cells expressing either μ galOR-δOR dependent increase in -arrestin recruitment suggests that the β βgal gal fi modified receptors and β-arrestin in μ OR-δOR cells behave or 5HT5A receptors led to the identi cation of 993 hits with μβgal δ βgal A differently from the native receptor system. Nonetheless, the OR- OR and 2,039 hits with 5HT5A cells (Fig. 2 ). observations that a combination of δOR agonist and μOR an- Comparison of the hits between the two cell lines showed that μβgal δ tagonist (or μOR agonist and δOR antagonist) causes a decrease 885 of 993 hits were unique to OR- OR. Secondary screening (triplicate at a single concentration of 9.3 μM) led to the β identification of 375 hits for μ galOR-δOR, among which 346 β hits were unique to μ galOR-δOR (Fig. 2B). Tertiary screening A βgal _ B β (10-point dilution series in triplicate) of 229 of these compounds μ OR δOR Flag μOR_δ galOR βgal βgal βgal 1000 in cells expressing μ OR-δOR, 5HT5A receptors, μ OR, βgal 800 or δ OR led to the identification of 125 hits (Fig. 2C). We – 600 compared the dose response curves obtained with cells μβgal δ μβgal δβgal fi 400 expressing OR- OR, OR, or OR and identi ed a number of potential μOR-δOR–biased ligands based on the 200 ≤ μ μβgal RLU (% basal) criteria that they exhibit EC50 values of 10 M with OR- 0 β -11 -9 -7 -9 gal 0 10 -11 10 -7 δOR cells, a fivefold difference in EC50 between μ OR-δOR 10 10 0 10 10 β β Delt II [M] Delt II [M] and either μ galOR or δ galOR cells. These criteria led to the β β μ δ – C μ galOR_δOR μ galOR_δOR selection of 94 compounds as potential OR- OR biased 200 -Ab fi μ δ – -Ab ligands (Table S3). For validation of the identi ed OR- OR 300 +CB -AT mAb 150 *** 1 1 biased ligands, we selected 14 compounds based on their po- +μ-δ mAb tency, efficacy, and uniqueness of structure compared with other 200 100 +μ-δ mAb opioid ligands. Novelty of the chemical scaffolds with respect to 100 50 9,934 annotated opioid receptor ligands in the ChEMBL data-

RLU (% basal) 0 0 base (https://www.ebi.ac.uk/chembl/) was evaluated by way of -9 -5 0 10-11 10 10-7 10 Basal 30 nM Delt II Tanimoto coefficients (Tcs) to the nearest neighbors, excluding Delt II [M] the aforementioned 94 compounds. Of 14 compounds selected β D Flag μOR_δ galOR ≤ 600 -Ab for validation, 13 compounds exhibited Tc 0.3 to the closest +CB1-AT1 mAb annotated opioid ligand (Table S4); 14 compounds were μ 400 + -CB mAb repurchased and retested for their ability to recruit β-arrestin in δ 1 β β β + -CB1 mAb cells expressing μ galOR, δ galOR, or μ galOR-δOR. Among the β 200 + μ-δ mAb compounds tested, six exhibited higher efficacy in μ galOR-δOR β β cells compared with μ galOR or δ galOR cells alone (Fig. 2 D–I RLU (% basal) 0 -9 -8 -7 -6 and Table S4). Among these six compounds, PubChem 0 10 10 10 10 β DAMGO [M] CID23723457 (CYM51010) exhibited a robust -arrestin recruitment (Emax = 1,197 ± 31% basal) that was at least twofold β β Fig. 1. Recruitment of β-arrestin by the δOR agonist Delt II. Cells expressing higher than the activity obtained with μ galOR or δ galOR alone β β (A) μ galOR-δOR or (B) Flag μOR-δ galOR (20,000 cells/well) were subjected to (Fig. 2E and Table S4); this compound was selected for a β-arrestin recruitment assay with the δOR agonist Delt II (0–1 μM) as de- β additional characterization. scribed in Materials and Methods.(C) Cells expressing μ galOR-δOR were β fi – μ μ δ CYM51010-mediated -arrestin recruitment could be signi - treated with Delt II (0 10 M) in the absence or presence of - or CB1-AT1 μ δ μ δ mAb (1 μg/well), and β-arrestin recruitment was measured. (D) Cells ex- cantly reduced by - mAb and to a lesser extent, OR- or OR- μ δβgal – μ selective Ab but not anti-Flag Ab (Fig. 3A). We find that pressing Flag OR- OR were treated with DAMGO (0 1 M) in the ab- 35 sence or presence of μ-δ, δ-CB , μ-CB ,orCB-AT mAb (1 μg/well), and CYM51010 causes a robust increase in [ S]GTPγS binding 1 1 1 1 βgal βgal β-arrestin recruitment was measured. Results are mean ± SE (n = 3–6). in μ OR-δOR cell membranes compared with μ OR or βgal RLU, relative luminescence unit. δ OR cell membranes (Fig. 3B and Table 1). Moreover,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1222044110 Gomes et al. A D E μβgal _δ μβgal _δ β 2600 OR OR OR OR βgal _ HT gal β μ OR δOR 5A 2100 μ galOR 900 335,461 tested 885 108 1931 335,461 tested β 1600 δ galOR 993 hits 2039 hits 1100 500 δβgal β B 600 OR μ galOR RLU (% basal) 100 100 βgal _ βgal μ OR δOR HT5A CID16288533 [M] CID23723457 [M] 834 tested 834 tested (CYM51010) 346 29 36 F G 375 hits 65 hits 400 βgal _ μ OR δOR 900 300 β 700 β μ galOR μ galOR_δOR C 500 β 200 β β δ galOR μ galOR μ galOR_δOR 300 19 229 tested RLU (% basal) 229 tested 94 0 δβgal 100 δβgal 23 hits 100 OR OR 125 hits 4 CID5310710 [M] CID5412959 [M] 8 0 H βgal _ I 300 β 140 μ OR δOR μ galOR_δOR 0 250 μβgalOR 120 μβgal μβgal 229 tested OR 200 OR 12 hits 100 150 β δβgal δ galOR 100 OR RLU (% basal) 80 -9 -7 -5 0 10 10 10 0 10-9 10-7 10-5 CID3799497 [M] CID5464086 [M]

βgal βgal Fig. 2. Screening for μOR-δOR–biased agonists. (A) Cells expressing μ OR-δOR or 5HT5A receptors (1,000 cells/well) were treated with different compounds at a single concentration of 9.3 μM in singlicate, and β-arrestin recruitment was measured as described in Materials and Methods.(B) Compounds (834) βgal βgal βgal identified in A as unique for μ OR-δOR were tested in triplicate at a single concentration (9.3 μM) in cells expressing μ OR-δOR or 5HT5A receptors. (C) Compounds (229) identified in B as unique for μβgalOR-δOR were tested in a 10-point dilution series in triplicate (0–92.6 μM) in cells expressing μβgalOR-δOR, NEUROSCIENCE βgal βgal βgal βgal 5HT5A receptors, μ OR, or δ OR. No hits were observed with cells expressing 5HT5A receptors. (D–I) Validation of hits identified in C carried out with β β β commercially available compounds (Tables S3 and S4) using cells expressing μ galOR, δ galOR, or μ galOR-δOR (5,000 cells/well). Data with six compounds that β exhibited higher efficacy or potency at μ galOR-δOR cells are shown. pretreatment with a μOR antagonist, naltrexone (NTX), but not but not the antinociception mediated by morphine (Fig. S4 D–H) with a δOR antagonist, naltriben (NTB), significantly, albeit par- or CID24891919 [area under the curve (AUC) [percentage of tially, decreases CYM51010-mediated increases in [35S]GTPγS maximum possible effect (%MPE) × time] is 5,177 ± 844 in the binding (Fig. 3C). We also examined CYM51010-mediated absence and 4,998 ± 261 in the presence of μ-δ mAb], a non- increases in G-protein activity using spinal cord membranes selective opioid agonist (Table S3). Together, these results sup- from WT animals and compared them with membranes from port the notion that CYM51010-mediated antinociception is, at mice lacking μOR or δOR. CYM51010 induces a robust dose- least in part, mediated by μOR-δOR heteromers. Finally, we also dependent increase in [35S]GTPγS binding in WT membranes examined the development of tolerance and physical dependence (Fig. 3D and Table 1). In addition, pretreatment with a μOR to CYM51010 and how it compared with morphine. We find antagonist NTX partially decreases this CYM51010-mediated that the development of antinociceptive tolerance to increase in [35S]GTPγS binding (Fig. 3E). Interestingly, a combi- CYM51010(10mg/kg)islessthanwhatisobservedwith nation of NTX with μ-δ mAb, but not CB1-δ mAb, completely morphine (Fig. 4G and Fig. S4 I–K). This observation is more blocks CYM51010-mediated increases in [35S]GTPγS binding apparent when a dose that induces ∼70% maximal anti- (Fig. S3F), consistent with the idea that CYM51010 behaves as nociception(6mg/kg)isused(Fig. S4 L–O). In naloxone pre- a μOR-δOR heteromer-biased ligand. cipitated withdrawal assay, chronic CYM51010 administration Next, we examined if CYM51010 exhibits μOR-δOR hetero- shows less severe signs of withdrawal for diarrhea and body mer-mediated activity in vivo using the tail-flick assay to measure weight loss compared with morphine (Fig. S5). Taken together, antinociception, because previous studies have implied a role for these results indicate that a major portion of the antinociception μOR-δOR heteromer in spinal analgesia (4, 10–12); s.c. admin- observed after i.t. administration of CYM51010 is mediated by istration of CYM51010 leads to dose-dependent antinociception, μOR-δOR heteromers and that the chronic administration of which is similar to antinociception of morphine (Fig. 4 A and B the compound leads to less tolerance compared with morphine. and Fig. S4A). Notably, although the antinociceptive effects A major finding of this study is the identification of CYM51010 of morphine were completely blocked by NTX, the effects of as a biased μOR-δOR agonist. Several recent reports have pro- CYM51010 were only partially blocked by the same dose of NTX posed classic μOR or δOR agonists to have μOR-δOR heteromer (Fig. 4C and Fig. S4 B and C); NTB had no effect on either selectivity. For example, morphine and DAMGO have recently morphine- or CYM51010-mediated antinociception (Fig. 4C and been reported to be more potent and efficacious at inducing + Fig. S4 B and C). These results suggest that a component of the Ca 2 mobilization in cells expressing μOR-δOR heteromers CYM51010-mediated antinociception is through μOR; the fact and more potent at eliciting [35S]GTPγS binding in these cells that only a portion of CYM51010-mediated antinociception was compared with cells expressing μOR (13, 14). Also, 4-[(R)- mediated by μOR suggests that CYM51010 is mediating its ef- [(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl) fects by engaging the μOR-δOR heteromer. methyl]-N,N-diethylbenzamide (SNC80), a δOR-selective ago- The involvement of the μOR-δOR heteromer in CYM51010- nist, was reported as a potent and efficacious μOR-δOR hetero- + mediated antinociception was tested by using μ-δ mAb. We mer-selective agonist based on a Ca 2 mobilization assay and find that coadministration of μ-δ mAb significantly blocks i.t. a decrease in antinociceptive activity in mice lacking μOR or δOR CYM51010-mediated antinociception (Fig. 4 D–F and Fig. S4F) (15). These studies suggest that the ability of a compound to elicit

Gomes et al. PNAS Early Edition | 3of6 currently under phase II clinical studies for the treatment of irri- A Basal + 51010 B μ δ 200 μβgal _δ table bowel syndrome (17, 18). Given that OR- OR heteromers 600 ** OR OR *** 180 represent potential targets for the development of unique ther- 160 βgal 400 *** μ OR apeutics to treat pain, the identiﬁcation of CYM51010 as an 140 μ δ – S (% basal) 120 antinociceptive OR- OR biased agonist is exciting, because it γ 200 100 β δ galOR provides a chemical scaffold for the development of therapeutics RLU (% basal) 0 80 with reduced side effects commonly associated with chronic

_ _ _ _ S]GTP -11 -9 -7 -5 _ + _ _ _ + μ Ab 0 10 10 10 10

______δ 35 morphine use. In addition, compounds developed based on mod- + + Ab [ 51010 [M] _ _ _ + _ _ _ _ + _ μ-δ mAb iﬁcations of the CYM51010 structure (both agonists and antag- _ _ _ _ + _ _ _ _ + Flag Ab onists) will help in the elucidation of the physiological role of β μ galOR_δOR μOR-δOR heteromers in vivo. C D160 Spinal cord 150 *** 140 WT Materials and Methods 100 Cell Culture and Transfections. μβgal – δβgal – μβgal δ – 120 δ k/o OR , OR ,or OR- OR expressing μβgal μ S (% basal) μ k/o UO5S cells were a gift from DiscoverX. OR cells [expressing OR tagged γ 50 100 with a ProLink/β-gal donor (PK) fragment at the C-terminal region and β β 0 _ _ _ + + + 51010 80 -11 -9 -7 -5 -arrestin tagged with a complementary -gal activator (EA) fragment] and S]GTP 0 10 10 10 10 βgal _ + _ _ + _ NTX δ OR cells (expressing δOR tagged with the PK fragment at the C-terminal 35

[ _ _ _ _ 51010 [M] + + NTB region and β-arrestin tagged with the EA fragment) were grown in MEMα E WT δ k/o containing 10% (vol/vol) FBS, streptomycin-penicillin, 500 μg/mL geneticin, μ μβgal δ δ μ 140 and 250 g/mL hygromycin. OR- OR cells expressing WT OR, OR tag- * ged with the PK fragment at the C-terminal region, and β-arrestin tagged α 120 with the EA fragment were grown in MEM containing 10% (vol/vol) FBS, μ μ * streptomycin-penicillin, 500 g/mL geneticin, 250 g/mL hygromycin, and

S (% basal) μ γ 100 0.25 g/mL puromycin. β δ galOR cells were transfected with either Flag-tagged μOR or myc-tagged 80 ’ S]GTP CB R using Fugene 6 (Roche) as described in the manufacturer s protocol. _ + + + _ + + + 51010 1 35 [ _ _ + _ _ _ + _ NTX _ _ _ + _ _ _ + NTB Radioligand Binding Studies. Saturation binding assays were carried out in β β β cells (2 × 105) expressing μ galOR, δ galOR, or μ galOR-δOR using either the Fig. 3. Validation of CYM51010 as a μOR-δOR–biased agonist. (A) Cells radiolabeled δOR agonist ([3H]Delt II) or the radiolabeled μOR agonist ([3H] βgal expressing μ OR-δOR (5,000 cells/well) were treated with CYM51010 DAMGO; 0–10 nM final concentration) in 50 mM Tris·Cl buffer (pH 7.4) (51010; 10 μM) in the absence or presence of μ-δ mAb, μ, δ, or Flag Ab, and containing 0.32 M sucrose and protease inhibitor mixture (Sigma) as de- β-arrestin recruitment was measured. (B) Membranes (20 μg) from cells scribed in refs. 5 and 19. [3H]Delt II exhibits a K = 12.2 ± 3.8 nM and a B = β β β d max expressing μ galOR, δ galOR, or μ galOR-δOR were subjected to a [35S]GTPγS βgal 3,723 ± 748 fmol/mg protein with cells expressing δ OR and a Kd = 12.7 ± binding assay with CYM51010 (0–10 μM final concentration). (C) Membranes = ± β 3.6 nM and a Bmax 6,023 1,099 fmol/mg protein with cells expressing (20 μg) from cells expressing μ galOR-δOR were subjected to a [35S]GTPγS βgal 3 μ OR-δOR (Fig. S1). [ H]DAMGO exhibits a Kd = 8.4 ± 2.3 nM and a Bmax = binding assay with CYM51010 (1 μM) in the absence or presence of the μOR βgal 670 ± 107 fmol/mg protein with cells expressing μ OR and a Kd = 2.5 ± 0.6 μ δ μ β antagonist NTX (10 M) or the OR antagonist NTB (10 M). (D) Spinal cord nM and B = 370 ± 32 fmol/mg protein with cells expressing μ galOR-δOR μ μ μ δ δ max membranes (20 g) from WT mice or mice lacking OR ( k/o) or OR ( k/o) (Fig. S1). 35 γ – μ fi were subjected to a [ S]GTP S binding assay with CYM51010 (0 10 M nal Potentiation of [3H]Delt II binding by μOR antagonist CTOP (10 nM final μ δ concentration). (E) Spinal cord membranes (20 g) from WT or k/o mice 3 δ ψ 35 concentration) or [ H]DAMGO binding by the OR antagonist TIPP (10 nM were subjected to a [ S]GTPγS binding assay with CYM51010 (1 μM) in the 5 βgal final concentration) was examined in cells (2 × 10 ) expressing μ OR, absence or presence of the μOR antagonist NTX (10 μM) or the δOR antag- β β δ galOR, or μ galOR-δOR in 50 mM Tris·Cl buffer (pH 7.4) containing 0.32 M onist NTB (10 μM). Results represent mean ± SE (n = 3–6). sucrose and protease inhibitor mixture as described in refs. 4 and 5.

35 β [ S]GTPγS Binding. Membranes were prepared from cells expressing μ galOR, μ δ β β a signaling response through OR- OR heteromers may depend δ galOR, or μ galOR-δOR and spinal cords of WT, μOR, or δOR KO mice as on the signaling pathway being examined. However, these studies described previously (19). Membranes (20 μg) were subjected to a [35S]GTPγS did not critically evaluate whether the μOR-δOR heteromer was binding assay using Delt II, DAMGO, or CYM51010 (0–10 μM final concen- the actual target of these agonists by either disrupting the het- tration) as described in ref. 4. In studies examining the effects of Ab, eromer or blocking the heteromer with selective Ab. In this membranes were incubated with 1 μg indicated Ab for 30 min before the study, we used the heteromer-selective mAb to infer that addition of agonists. In studies examining the effects of antagonists, mem- CYM51010 is a μOR-δOR heteromer-biased agonist. It is in- branes were incubated with 10 μM μOR antagonist NTX or δOR antagonist teresting to note that CYM51010 exhibits higher potency at NTB for 30 min before addition of agonists. μβgal δ μβgal δβgal OR- OR compared with OR and OR alone for β-Arrestin Recruitment. μβgal δβgal μβgal δ activating G proteins (∼50 vs. ∼300 nM) and lower potency for Cells expressing OR, OR, or OR- OR were plated in each well (20,000 cells) of a 96-well white clear bottom plate in β-arrestin recruitment (∼8 vs. 3 μM) (Table 1). Improving the G- μ μ δ 100 L media. The next day, cells were treated with either Delt II or DAMGO protein signaling bias in addition to increasing its OR- OR (0–1 μM final concentration) for 60 min at 37 °C, and β-arrestin recruitment selectivity are likely to make CYM51010 an ideal tool to explore was measured using the PathHunter Chemiluminescence Detection Kit as theinvitroandinvivopharmacologyofμOR-δOR heteromers. described in the manufacturer’s protocol (DiscoverX). In studies examining Early studies showing that morphine-mediated antinociception the effects of μOR antagonist CTOP or δOR antagonist TIPPψ, cells were pre- could be potentiated by δOR antagonists (4, 16) suggested a func- incubated without or with the antagonists (10 μM final concentration) for tional interaction between μOR and δOR. More recent studies 30 min before the addition of agonists. In studies examining the time course using bivalent ligands targeting the μOR-δOR heteromer (μOR of β-arrestin, recruitment cells were treated with either 1 μMDeltII(±100 nM μ ± ψ agonist linked to δOR antagonist by a spacer) showed that they CTOP) or 1 MDAMGO( 100 nM TIPP ) for the indicated time periods (30 s and 1, 3, 5, 10, 30, and 60 min). In studies examining the effects of Ab, cells exhibit antinociceptive activity without the development of toler- βgal βgal βgal expressing μ OR-δOR, Flag μOR-δ OR, or δ OR-myc CB1R were incu- ance (11, 12). Recently, a compound named MuDelta, exhibiting bated with 1 μg/well μ-δ,CB cannabinoid-AT angiotensin receptor (CB -AT ), μ δ 1 1 1 1 OR agonistic activity and OR antagonistic activity, was identi- μ-CB1,orδ-CB1 heteromer-selective mAbs for 30 min before the addition of fied using structure–activity relationship studies with the hetero- Delt II (0–1 μM). Validation studies were carried out using 5,000 cells/well and cyclic core of opioid receptor agonists (17, 18). This compound is commercially available compounds identified as μOR-δOR–biased agonists in

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1222044110 Gomes et al. A B C ** 12000 ** 120 51010 * ** 10000 Mor 51010; 10mg/kg 8000 * n.s. 80 Mor; 10mg/kg 6000 51010; 6mg/kg 4000 51010; 3mg/kg % MPE 40 51010; 1mg/kg 2000 vehicle 0 0 1 1 1 1 mg/kg 0 -2000 24 6810 Mor 0.1 0.1 30 60 90 0.01 0.01 120 Drug (mg/kg) 51010 Time (min) AUC (%MPE x time) +NTX +NTB +NTX +NTB D E F G 4000 5000 * ** 100 51010; 30nmol 12000 3000 4000 * Mor * 51010 3000 8000 ** 60 2000 51010; 10nmol 2000 20 1000 4000 % MPE 51010; 3nmol 1000 vehicle 0 0 δ 3 - 0 -20 30 60 90 10 30 nmol AUC (%MPE x time) +μ 120 +IgG d1 d2 d3 d4 d5 d8 Time (min) mAb vehicle 51010 51010

Fig. 4. Antinociceptive activity of CYM51010. (A and B) Mice were s.c. administered with CYM51010 (51050) or morphine (Mor; 1, 3, 6, or 10 mg/kg body weight). (C) Mice were i.p. administered with vehicle, NTX, or NTB at the indicated doses 30 min before Mor (10 mg/kg s.c.) or CYM51010 (10 mg/kg s.c.) administration. (D) Mice were i.t. administered with CYM51010 (3, 10, or 30 nmol). E represents AUC calculated from data in D.(F) Mice were i.t. administered with vehicle, control IgG (anti-Flag IgG; 1 μg), or μ-δ mAb (1 μg) 30 min before CYM51010 (30 nmol i.t.) administration. (G) Mice were administered with either Mor or CYM51010 (10 mg/kg s.c.) one time daily for 8 d, and antinociception was measured daily. (A–G) Antinociceptive activity was measured as described in ± = – < < Materials and Methods. Results are mean SE (n 3 18 mice per group). *P 0.05; **P 0.01 as determined by ANOVA followed by multiple comparison test NEUROSCIENCE (Student Newman–Keuls test) or unpaired t test. n.s., not significant. the high-throughput screen (0–100 μM final concentration). Differences ob- jCompoundMapper (20) after converting linear text SMILES formats for each served in data obtained with the validated compounds and data from the molecule into corresponding SDF files with Corina (21). A Tc = 0 indicates max- high-throughput screening (described below) could be because of the dif- imally dissimilar compounds, whereas a Tc = 1 indicates maximally similar ones ferences in assay conditions. (22), with values below 0.40 suggesting reasonably unique compounds (23).

High-Throughput Screening for μOR-δOR–Biased Agonists. Cells expressing Animals. Male C57BL/6 mice (25–35 g; 6–12 wk) were obtained from Jackson βgal βgal βgal βgal μ OR, δ OR, μ OR-δOR, or 5HT5A receptors were plated in each well Laboratories. All mice were maintained on a 12-h light/dark cycle with ro- (1,000 cells) of a 1,536-well white plate. Cells were incubated for 3 h at 37 °C dent chow and water available ad libitum, and they were housed in groups with different compounds followed by 1 h of incubation with the Path- of five until testing. Animal studies were carried out according to protocols Hunter Chemiluminescence Detection Kit. In the primary screening, com- approved by the Mount Sinai School of Medicine Animal Care and pounds were screened at a single concentration (9.3 μM) in singlicate in cells Use Committee. βgal βgal expressing either μ OR-δOR or 5HT5A receptors. In secondary screening β assays, 834 compounds identified in primary screens as unique for μ galOR- Drug Administration. Morphine sulfate (Sigma) and NTX (Tocris) were dis- δOR were tested in triplicate at a single concentration (9.3 μM) in the same solved in saline. NTB (Tocris) was dissolved in 2% DMSO in saline. CYM51010 β cell lines. This screen led to the identification of 375 hits with μ galOR-δOR (ChemBridge) and CID24891919 (Life Chemicals) were dissolved in 6% DMSO βgal and 5% Tween80 in saline. μ-δ mAb (7) and control IgG (anti-Flag Ab; 1 μg) and 65 hits with 5HT5A cells. In tertiary assays, 229 compounds identified β in secondary screens as unique for μ galOR-δOR were tested in a 10-point were diluted in saline. Corresponding vehicle was used for the control β dilution series in triplicate (0–92.6 μM) in cells expressing μ galOR-δOR, group. Mice were administered drugs s.c. or i.p. for systemic treatment. For βgal βgal βgal i.t. administration, the direct lumbar puncture method (24) was applied in 5HT5A receptors, μ OR, or δ OR. Detailed screening assay protocols as awake, conscious mice. Mice were covered with a soft cloth over the head well as all screening results can be found in PubChem, a publically available and upper body and gripped firmly by the pelvic girdle (iliac crest); 5 μLdrug database (pubchem.ncbi.nlm.nih.gov; BioAssay AID 504355) Hits identified was administered with a 50-μL Hamilton syringe (Hamilton Co) attached to a 30- as μOR-δOR–selective are summarized in Table S3. gauge, 1-in sterile disposable needle, which was inserted into the i.t. space at the cauda equine region according to the method described in ref. 24. Puncture Similarity Analysis. Tc values were calculated with an in-house script using ex- of the dura was indicated by a flick of the tail. For measuring development of tended connectivity fingerprint maximum distance 4. They were generated with tolerance, mice were s.c. administered with 10 mg/kg morphine or CYM51010 one time per day for 8 d or 6 mg/kg daily for 14 d, and antinociception was measured daily from 0 to 120 min. Naloxone-precipitated withdrawal symp- Table 1. EC50 and Emax for CYM51010 toms were measured in mice after administration of 10 mg/kg morphine or β-Arrestin recruitment [35S]GTPγS CYM51010 s.c. one time per day for 9 d. Withdrawal was induced by i.p. administration of naloxone (5 mg/kg) 2 h after the last drug administration, and

EC50 (M) Emax (% basal) EC50 (M) Emax (% basal) withdrawal signs were recorded for 30 min as described (25). Body weight was measured before and 30 min after last naloxone injection. μβgal OR 1.8E-6 557 ± 11 2.1E-7 138 ± 6 βgal δ OR 2.7E-6 423 ± 49 3.0E-7 113 ± 1 Analgesia Assays. Drug-induced antinociception was evaluated by using the β μ galOR-δOR 8.3E-6 1,197 ± 31 5.4E-8 168 ± 3 tail-flick test (26). Using a tail-flick apparatus (IITC Life Science), the intensity WT spinal cord n.d n.d 7.1E-7 141 ± 2 of the heat source was set at 10, which resulted in the basal tail-flick latency μ k/o spinal cord n.d n.d 7.9E-6 106 ± 4 occurring between 5 and 7 s for most of the animals. The tail-flick latency – δ k/o spinal cord n.d n.d 1.8 E-7 117 ± 3 was recorded at the indicated time period (0 120 min) after drug administration; %MPE was calculated for each mouse at each time point according n.d., not determined. to the following formula: %MPE = [(latency after drug − baseline latency)/

Gomes et al. PNAS Early Edition | 5of6 (20 − baseline latency)] × 100. Cutoff latency was selected at 20 s to mini- ACKNOWLEDGMENTS. We thank Dr. John Pintar for the gift of brains from β β β mize tissue damage. The area under the %MPE vs. time curves (AUCs) for mice lacking μOR or δOR, DiscoverX for the μ galOR, δ galOR, and μ galOR-δOR each treatment condition is shown in Fig. 4 and Fig. S4. cells, and PathHunter Assay kits and Pierre Baillargeon, Lina DeLuca, and Louis Scampavia for compound management and analysis. These studies Statistical Analyses. The data were expressed as means ± SEMs. One-way were supported by National Institutes of Health Molecular Library Probe ANOVA and multiple comparison tests (Student Newman–Keuls tests) were Production Center Grant U54 MH084512 (to E.R. and P.H.) and National used to analyze the data. Tolerance and withdrawal data were analyzed by Institutes of Health Grants DA034049 (to M.F.), DA026434 (to M.F.), unpaired t tests. A difference was considered to be signiﬁcant at P < 0.05. DA008863 (to L.A.D.), and DA019521 (to L.A.D.).

1. Matthes HW, et al. (1996) Loss of morphine-induced analgesia, reward effect and 14. Yekkirala AS, Kalyuzhny AE, Portoghese PS (2010) Standard opioid agonists activate withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383 heteromeric opioid receptors: Evidence for morphine and [d-Ala(2)-MePhe(4)-Glyol (6603):819–823. (5)]enkephalin as selective μ-δ agonists. ACS Chem Neurosci 1(2):146–154. δ 2. Waldhoer M, Bartlett SE, Whistler JL (2004) Opioid receptors. Annu Rev Biochem 73: 15. Metcalf MD, et al. (2012) The opioid receptor agonist SNC80 selectively activates μ δ – 953–990. heteromeric - opioid receptors. ACS Chem Neurosci 3(7):505 509. 3. Gomes I, et al. (2000) Heterodimerization of mu and delta opioid receptors: A role in 16. Abul-Husn NS, Sutak M, Milne B, Jhamandas K (2007) Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu- and delta-opioid opiate synergy. J Neurosci 20(22):RC110. receptor antagonists. Br J Pharmacol 151(6):877–887. 4. Gomes I, et al. (2004) A role for heterodimerization of mu and delta opiate receptors 17. Wade PR, et al. (2012) Modulation of gastrointestinal function by MuDelta, a mixed in enhancing morphine analgesia. Proc Natl Acad Sci USA 101(14):5135–5139. μ opioid receptor agonist/ μ opioid receptor antagonist. Br J Pharmacol 167(5): 5. Gomes I, Ijzerman AP, Ye K, Maillet EL, Devi LA (2011) G protein-coupled receptor 1111–1125. heteromerization: A role in allosteric modulation of ligand binding. Mol Pharmacol 18. Breslin HJ, et al. (2012) Identification of a dual δ OR antagonist/μ OR agonist as – 79(6):1044 1052. a potential therapeutic for diarrhea-predominant Irritable Bowel Syndrome (IBS-d). μ 6. Manglik A, et al. (2012) Crystal structure of the -opioid receptor bound to a mor- Bioorg Med Chem Lett 22(14):4869–4872. phinan antagonist. Nature 485(7398):321–326. 19. Gomes I, Filipovska J, Devi LA (2003) Opioid receptor oligomerization. Detection and 7. Gupta A, et al. (2010) Increased abundance of opioid receptor heteromers after functional characterization of interacting receptors. Methods Mol Med 84:157–183. chronic morphine administration. Sci Signal 3(131):ra54. 20. Hinselmann G, Rosenbaum L, Jahn A, Fechner N, Zell A (2011) jCompoundMapper: An 8. Zhu Y, et al. (1999) Retention of supraspinal delta-like analgesia and loss of morphine open source Java library and command-line tool for chemical fingerprints. J Chem- tolerance in delta opioid receptor knockout mice. Neuron 24(1):243–252. inform 3(1):3. 9. Bohn LM, et al. (1999) Enhanced morphine analgesia in mice lacking beta-arrestin 2. 21. Gasteiger JR, Rudolph C, Sadowski J (1990) Automatic generation of 3D-atomic co- Science 286(5449):2495–2498. ordinates for organic molecules. Tetrahedron Comp Method 3(6):537–547. 10. He SQ, et al. (2011) Facilitation of μ-opioid receptor activity by preventing δ-opioid 22. Rogers D, Brown RD, Hahn M (2005) Using extended-connectivity fingerprints with fi receptor-mediated codegradation. Neuron 69(1):120–131. Laplacian-modi ed Bayesian analysis in high-throughput screening follow-up. J Bio- – 11. Daniels DJ, et al. (2005) Opioid-induced tolerance and dependence in mice is modu- mol Screen 10(7):682 686. lated by the distance between pharmacophores in a bivalent ligand series. Proc Natl 23. Wawer M, Bajorath J (2010) Similarity-potency trees: A method to search for SAR information in compound data sets and derive SAR rules. J Chem Inf Model 50(8): Acad Sci USA 102(52):19208–19213. 1395–1409. 12. Lenard NR, Daniels DJ, Portoghese PS, Roerig SC (2007) Absence of conditioned place 24. Hylden JL, Wilcox GL (1980) Intrathecal morphine in mice: A new technique. Eur J preference or reinstatement with bivalent ligands containing mu-opioid receptor Pharmacol 67(2–3):313–316. agonist and delta-opioid receptor antagonist pharmacophores. Eur J Pharmacol 25. Abul-Husn NS, et al. (2011) Chronic morphine alters the presynaptic protein profile: – – 566(1 3):75 82. Identification of novel molecular targets using proteomics and network analysis. PLoS 13. Rozenfeld R, Devi LA (2007) Receptor heterodimerization leads to a switch in sig- One 6(10):e25535. naling: Beta-arrestin2-mediated ERK activation by mu-delta opioid receptor hetero- 26. D’Amour FE, Smith DL (1941) A method for determining loss of pain sensation. J dimers. FASEB J 21(10):2455–2465. Pharmacol Exp Ther 72(1):74–79.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1222044110 Gomes et al.