Advertisement

Development of Functionally Selective, Small Molecule Agonists at Kappa Opioid Receptors*

Open AccessPublished:November 01, 2013DOI:https://doi.org/10.1074/jbc.M113.504381
      The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

      Introduction

      The kappa opioid receptor (KOR)
      The abbreviations used are: KOR
      kappa opioid receptor
      GPCR
      G protein-coupled receptor
      h
      human
      EFC
      enzyme fragment complementation
      GTPγS
      guanosine 5′-3-O-(thio)triphosphate
      p-ERK
      phosphorylated ERK
      MOR
      mu opioid receptor
      DOR
      delta opioid receptor
      DAMGO
      [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin.
      is a seven-transmembrane G protein-coupled receptor (GPCR) (
      • Cox B.M.
      Recent developments in the study of opioid receptors.
      ). The cognate neuropeptides for KOR are endogenous opioids, including the dynorphin peptides. Dynorphins and KOR are widely expressed throughout the central nervous system (
      • Chavkin C.
      • James I.F.
      • Goldstein A.
      Dynorphin is a specific endogenous ligand of the kappa opioid receptor.
      ,
      • Mansour A.
      • Fox C.A.
      • Burke S.
      • Meng F.
      • Thompson R.C.
      • Akil H.
      • Watson S.J.
      Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study.
      ,
      • Minami M.
      • Satoh M.
      Molecular biology of the opioid receptors: structures, functions and distributions.
      ). In a canonical sense, KOR activation is defined by agonist-induced coupling of the Gα subunit of the heterotrimeric G proteins in the pertussis toxin-sensitive Gi/o family (
      • Prather P.L.
      • McGinn T.M.
      • Claude P.A.
      • Liu-Chen L.Y.
      • Loh H.H.
      • Law P.Y.
      Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual Gα subunit and is similar to that of other opioid receptors.
      ), subsequent inhibition of adenylyl cyclase (
      • Lawrence D.M.
      • Bidlack J.M.
      The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein.
      ), activation of inward-rectifying potassium channels (
      • Ma G.H.
      • Miller R.J.
      • Kuznetsov A.
      • Philipson L.H.
      kappa-Opioid receptor activates an inwardly rectifying K+ channel by a G protein-linked mechanism: coexpression in Xenopus oocytes.
      ), and blockade of calcium channels (
      • Macdonald R.L.
      • Werz M.A.
      Dynorphin A decreases voltage-dependent calcium conductance of mouse dorsal root ganglion neurones.
      ).
      There is considerable evidence that selective KOR agonists produce antinociception in animal models (
      • Pasternak G.W.
      Multiple opiate receptors: [3H]ethylketocyclazocine receptor binding and ketocyclazocine analgesia.
      ,
      • Vonvoigtlander P.F.
      • Lahti R.A.
      • Ludens J.H.
      U-50,488: a selective and structurally novel non-Mu (kappa) opioid agonist.
      ,
      • Dykstra L.A.
      • Gmerek D.E.
      • Winger G.
      • Woods J.H.
      Kappa opioids in rhesus monkeys. I. Diuresis, sedation, analgesia and discriminative stimulus effects.
      ,
      • Millan M.J.
      • Czl̸onkowski A.
      • Lipkowski A.
      • Herz A.
      Kappa-opioid receptor-mediated antinociception in the rat. II. Supraspinal in addition to spinal sites of action.
      ), and mice lacking KOR expression are no longer responsive to the antinociceptive effects of a selective KOR agonist U50,488 (
      • Simonin F.
      • Valverde O.
      • Smadja C.
      • Slowe S.
      • Kitchen I.
      • Dierich A.
      • Le Meur M.
      • Roques B.P.
      • Maldonado R.
      • Kieffer B.L.
      Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal.
      ). The G protein-mediated signaling events are believed to contribute to the analgesic properties of KOR agonists (
      • Goicoechea C.
      • Ormazábal M.J.
      • Abalo R.
      • Alfaro M.J.
      • Martín M.I.
      Calcitonin reverts pertussis toxin blockade of the opioid analgesia in mice.
      ,
      • Gullapalli S.
      • Ramarao P.
      Role of L-type Ca2+ channels in pertussis toxin induced antagonism of U50,488H analgesia and hypothermia.
      ). Unlike MOR agonists, KOR agonists do not cause physical dependence nor do they produce respiratory failure; thus, they are attractive as potent analgesics (
      • Raehal K.M.
      • Bohn L.M.
      Mu opioid receptor regulation and opiate responsiveness.
      ). However, KOR activation has also been implicated in producing an array of undesirable side effects, including dysphoria (
      • Pfeiffer A.
      • Brantl V.
      • Herz A.
      • Emrich H.M.
      Psychotomimesis mediated by kappa opiate receptors.
      ,
      • Land B.B.
      • Bruchas M.R.
      • Lemos J.C.
      • Xu M.
      • Melief E.J.
      • Chavkin C.
      The dysphoric component of stress is encoded by activation of the dynorphin kappa-opioid system.
      ), sedation (
      • Vonvoigtlander P.F.
      • Lahti R.A.
      • Ludens J.H.
      U-50,488: a selective and structurally novel non-Mu (kappa) opioid agonist.
      ), diuresis (
      • Dykstra L.A.
      • Gmerek D.E.
      • Winger G.
      • Woods J.H.
      Kappa opioids in rhesus monkeys. I. Diuresis, sedation, analgesia and discriminative stimulus effects.
      ), hallucination (
      • Roth B.L.
      • Baner K.
      • Westkaemper R.
      • Siebert D.
      • Rice K.C.
      • Steinberg S.
      • Ernsberger P.
      • Rothman R.B.
      Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist.
      ), and depression (
      • Knoll A.T.
      • Carlezon Jr., W.A.
      Dynorphin, stress, and depression.
      ). These adverse effects limit the therapeutic potential of KOR agonists as analgesics.
      It is becoming widely evident that activation of GPCRs by chemically distinct agonists can promote receptor coupling to distinct pathways in different tissues (
      • Bohn L.M.
      ,
      • Urban J.D.
      • Clarke W.P.
      • von Zastrow M.
      • Nichols D.E.
      • Kobilka B.
      • Weinstein H.
      • Javitch J.A.
      • Roth B.L.
      • Christopoulos A.
      • Sexton P.M.
      • Miller K.J.
      • Spedding M.
      • Mailman R.B.
      Functional selectivity and classical concepts of quantitative pharmacology.
      ,
      • Reiter E.
      • Ahn S.
      • Shukla A.K.
      • Lefkowitz R.J.
      Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors.
      ). The KOR also has the potential to signal through multiple downstream signaling cascades, and there is increasing evidence that signaling to alternative cascades may promote side effects associated with KOR activation. In addition to G protein coupling, another proximal event following GPCR activation is the agonist-promoted recruitment of βarrestins. As regulatory and scaffolding proteins, βarrestins can lead to desensitization of the receptor by impeding further G protein coupling; in some cases, βarrestins facilitate the assembly of protein scaffolds, thereby acting as signal transducers (
      • Luttrell D.K.
      • Luttrell L.M.
      Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases.
      ,
      • Schmid C.L.
      • Bohn L.M.
      Physiological and pharmacological implications of β-arrestin regulation.
      ,
      • Shenoy S.K.
      • Lefkowitz R.J.
      β-Arrestin-mediated receptor trafficking and signal transduction.
      ). For example, βarrestin2 recruitment has been shown to promote recruitment and activation of MAPKs, including ERK1/2 (
      • Lefkowitz R.J.
      • Shenoy S.K.
      Transduction of receptor signals by β-arrestins.
      ,
      • McLennan G.P.
      • Kiss A.
      • Miyatake M.
      • Belcheva M.M.
      • Chambers K.T.
      • Pozek J.J.
      • Mohabbat Y.
      • Moyer R.A.
      • Bohn L.M.
      • Coscia C.J.
      Kappa opioids promote the proliferation of astrocytes via Gβγ and β-arrestin 2-dependent MAPK-mediated pathways.
      ) and p38 (
      • Bruchas M.R.
      • Macey T.A.
      • Lowe J.D.
      • Chavkin C.
      Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes.
      ). Interestingly, studies in AtT-20 cells and mouse striatal neurons suggest that the KOR may signal via a GPCR kinase 3 (GRK3)- and a βarrestin2-dependent mechanism to activate the stress MAPK p38 and that this signaling cascade may mediate the dysphoric effects of KOR agonists (
      • Bruchas M.R.
      • Macey T.A.
      • Lowe J.D.
      • Chavkin C.
      Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes.
      ,
      • Bruchas M.R.
      • Land B.B.
      • Aita M.
      • Xu M.
      • Barot S.K.
      • Li S.
      • Chavkin C.
      Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria.
      ). Moreover, KOR activation has been shown to lead to ERK1/2 phosphorylation in both neurons and astrocytes through both G protein and βarrestin-dependent mechanisms (
      • McLennan G.P.
      • Kiss A.
      • Miyatake M.
      • Belcheva M.M.
      • Chambers K.T.
      • Pozek J.J.
      • Mohabbat Y.
      • Moyer R.A.
      • Bohn L.M.
      • Coscia C.J.
      Kappa opioids promote the proliferation of astrocytes via Gβγ and β-arrestin 2-dependent MAPK-mediated pathways.
      ,
      • Bruchas M.R.
      • Macey T.A.
      • Lowe J.D.
      • Chavkin C.
      Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes.
      ,
      • Bohn L.M.
      • Belcheva M.M.
      • Coscia C.J.
      Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade.
      ,
      • Belcheva M.M.
      • Clark A.L.
      • Haas P.D.
      • Serna J.S.
      • Hahn J.W.
      • Kiss A.
      • Coscia C.J.
      Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes.
      ). Endogenously elevated dynorphin levels produced by repeated swim stress tests lead to KOR-dependent ERK1/2 phosphorylation in the mouse brain that was not dependent on GRK3 expression (
      • Bruchas M.R.
      • Xu M.
      • Chavkin C.
      Repeated swim stress induces kappa opioid-mediated activation of extracellular signal-regulated kinase 1/2.
      ). Therefore, ERK activation may be a useful indicator of both βarrestin-dependent and G protein-dependent signaling states downstream of KOR (
      • Bruchas M.R.
      • Chavkin C.
      Kinase cascades and ligand-directed signaling at the kappa opioid receptor.
      ).
      Because G protein-mediated signaling is implicated in KOR-induced antinociception and βarrestin2-mediated signaling has been implicated in the aversive and dysphoric properties of KOR agonists, the development of G protein-biased agonists may provide a means to optimally tune KOR therapeutics. In two different screening campaigns, we discovered two new classes of KOR agonists, triazole probe 1 and isoquinolinone probe 2 (Fig. 1). The triazole scaffold was identified under the auspices of the Molecular Libraries Probe Production Centers Network via a high throughput screening campaign. In this work, which was done in collaboration with colleagues at Sanford-Burnham Research Institute and Duke University, the National Institutes of Health Small Molecule Repository was screened to identify KOR ligands based on activation of βarrestin2 recruitment (

      Hedrick, M. P., Gosalia, P., Frankowski, K., Shi, S., Prisinzano, T. E., Schoenen, F., Aube, J., Su, Y., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Ghosh, P., Milan, L., Heynen-Genel, S., Chung, T. D. Y., Dad, S., Caron, M., Bohn, L. M., Barak, L. S., (2010) Probe Reports from the NIH Molecular Libraries Program, Bethesda, MD

      ,
      • Frankowski K.J.
      • Hedrick M.P.
      • Gosalia P.
      • Li K.
      • Shi S.
      • Whipple D.
      • Ghosh P.
      • Prisinzano T.E.
      • Schoenen F.J.
      • Su Y.
      • Vasile S.
      • Sergienko E.
      • Gray W.
      • Hariharan S.
      • Milan L.
      • Heynen-Genel S.
      • Mangravita-Novo A.
      • Vicchiarelli M.
      • Smith L.H.
      • Streicher J.M.
      • Caron M.G.
      • Barak L.S.
      • Bohn L.M.
      • Chung T.D.
      • Aubé J.
      Discovery of small molecule kappa opioid receptor agonist and antagonist chemotypes through a HTS and hit refinement strategy.
      ). The isoquinolinone scaffold arose from a 72-member library prepared by a tandem Diels-Alder acylation reaction that was screened for binding at potential GPCR targets by the NIMH Psychoactive Drug Screening Program (
      • Frankowski K.J.
      • Hirt E.E.
      • Zeng Y.
      • Neuenswander B.
      • Fowler D.
      • Schoenen F.
      • Aubé J.
      Synthesis of N-alkyl-octahydroisoquinolin-1-one-8-carboxamide libraries using a tandem Diels-Alder/acylation sequence.
      ,
      • Frankowski K.J.
      • Ghosh P.
      • Setola V.
      • Tran T.B.
      • Roth B.L.
      • Aubé J.
      N-Alkyl-octahydroisoquinolin-1-one-8-carboxamides: a novel class of selective, nonbasic, nitrogen-containing kappa-opioid receptor ligands.
      ). The lead isoquinolinone was reported as a KOR agonist showing selectivity and high binding affinity for KOR over MOR, DOR, and other screened GPCRs (
      • Frankowski K.J.
      • Hirt E.E.
      • Zeng Y.
      • Neuenswander B.
      • Fowler D.
      • Schoenen F.
      • Aubé J.
      Synthesis of N-alkyl-octahydroisoquinolin-1-one-8-carboxamide libraries using a tandem Diels-Alder/acylation sequence.
      ,
      • Frankowski K.J.
      • Ghosh P.
      • Setola V.
      • Tran T.B.
      • Roth B.L.
      • Aubé J.
      N-Alkyl-octahydroisoquinolin-1-one-8-carboxamides: a novel class of selective, nonbasic, nitrogen-containing kappa-opioid receptor ligands.
      ); moreover, the triazole lead also displayed high selectivity for KOR over MOR and DOR (Fig. 1) (

      Hedrick, M. P., Gosalia, P., Frankowski, K., Shi, S., Prisinzano, T. E., Schoenen, F., Aube, J., Su, Y., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Ghosh, P., Milan, L., Heynen-Genel, S., Chung, T. D. Y., Dad, S., Caron, M., Bohn, L. M., Barak, L. S., (2010) Probe Reports from the NIH Molecular Libraries Program, Bethesda, MD

      ,
      • Frankowski K.J.
      • Hedrick M.P.
      • Gosalia P.
      • Li K.
      • Shi S.
      • Whipple D.
      • Ghosh P.
      • Prisinzano T.E.
      • Schoenen F.J.
      • Su Y.
      • Vasile S.
      • Sergienko E.
      • Gray W.
      • Hariharan S.
      • Milan L.
      • Heynen-Genel S.
      • Mangravita-Novo A.
      • Vicchiarelli M.
      • Smith L.H.
      • Streicher J.M.
      • Caron M.G.
      • Barak L.S.
      • Bohn L.M.
      • Chung T.D.
      • Aubé J.
      Discovery of small molecule kappa opioid receptor agonist and antagonist chemotypes through a HTS and hit refinement strategy.
      ). The isoquinolinone compounds are particularly interesting in that they lack the basic nitrogen center common in small molecule KOR ligands (
      • Vonvoigtlander P.F.
      • Lahti R.A.
      • Ludens J.H.
      U-50,488: a selective and structurally novel non-Mu (kappa) opioid agonist.
      ,
      • Lahti R.A.
      • Mickelson M.M.
      • McCall J.M.
      • Von Voigtlander P.F.
      [3H]U-69593 a highly selective ligand for the opioid kappa receptor.
      ,
      • Jones R.M.
      • Portoghese P.S.
      5′-Guanidinonaltrindole, a highly selective and potent kappa-opioid receptor antagonist.
      ,
      • Thomas J.B.
      • Atkinson R.N.
      • Rothman R.B.
      • Fix S.E.
      • Mascarella S.W.
      • Vinson N.A.
      • Xu H.
      • Dersch C.M.
      • Lu Y.
      • Cantrell B.E.
      • Zimmerman D.M.
      • Carroll F.I.
      Identification of the first trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine derivative to possess highly potent and selective opioid kappa receptor antagonist activity.
      ); the best known ligand lacking this feature is salvinorin A, a natural neoclerdane diterpene found to be a highly selective, potent KOR agonist (
      • Roth B.L.
      • Baner K.
      • Westkaemper R.
      • Siebert D.
      • Rice K.C.
      • Steinberg S.
      • Ernsberger P.
      • Rothman R.B.
      Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist.
      ).
      Figure thumbnail gr1
      FIGURE 1Structure of KOR agonists. A, triazole probes (PubChem compound ID 44601470) were reported to have binding affinity for KOR (Ki = 2.4 nm) over MOR (Ki = 1900 nm) and DOR (Ki = 5351 nm) (
      • Frankowski K.J.
      • Hedrick M.P.
      • Gosalia P.
      • Li K.
      • Shi S.
      • Whipple D.
      • Ghosh P.
      • Prisinzano T.E.
      • Schoenen F.J.
      • Su Y.
      • Vasile S.
      • Sergienko E.
      • Gray W.
      • Hariharan S.
      • Milan L.
      • Heynen-Genel S.
      • Mangravita-Novo A.
      • Vicchiarelli M.
      • Smith L.H.
      • Streicher J.M.
      • Caron M.G.
      • Barak L.S.
      • Bohn L.M.
      • Chung T.D.
      • Aubé J.
      Discovery of small molecule kappa opioid receptor agonist and antagonist chemotypes through a HTS and hit refinement strategy.
      ). Analogues of the triazole probe are shown. B, isoquinolinone probe and analogues were reported to have binding affinity to KOR (Ki = 5 nm) over MOR (Ki = 3550 nm) and DOR (Ki > 10 μm) (
      • Frankowski K.J.
      • Ghosh P.
      • Setola V.
      • Tran T.B.
      • Roth B.L.
      • Aubé J.
      N-Alkyl-octahydroisoquinolin-1-one-8-carboxamides: a novel class of selective, nonbasic, nitrogen-containing kappa-opioid receptor ligands.
      ).
      Following their initial disclosure, these scaffolds were subjected to iterative rounds of medicinal chemistry and structure-activity relationship studies with the goal of developing KOR agonists that are biased toward G protein coupling. Here, we report five triazole analogues and two isoquinolinone analogues (Fig. 1) that activate KOR in a manner that is preferentially biased toward G protein signaling with minimal effects on βarrestin2 recruitment and downstream ERK1/2 activation.

      RESULTS

      KOR-mediated G protein signaling was evaluated using a [35S]GTPγS binding assay in cell membranes from CHO-K1 cells stably expressing the human KOR (CHO-hKOR) (
      • 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 kappa opioid receptors in striatal neurons.
      ). Concentration-response curves were run in parallel with U69,593, a well characterized potent, full agonist at KOR, as a reference agonist for the assay; EC50 values and maximal responses of test compounds were determined by nonlinear regression analysis and are presented in Table 1. Compared with U69,593 (51 nm potency), the triazole and isoquinolinone analogues show similar potencies for stimulating G protein signaling, with EC50 values ranging from ∼30 to 270 nm and with maximal stimulations roughly equivalent to that achieved with U69,593 stimulation (Fig. 2; Table 1). Furthermore, none of these compounds stimulate G protein signaling in CHO cells expressing hMOR when tested at a 10 μm concentration (data not shown) demonstrating selectivity for KOR.
      TABLE 1Signaling parameters for KOR agonists across functional assay platforms
      Entry[35S]GTPγS (mG)βarrestin2 EFCβarrestin2 imaging
      EC50EmaxEC50EmaxEC50Emax
      nm%nm%nm%
      U69,59351.5 ± 4.5100131 ± 22.4100205.3 ± 21.4100
      1.131.0 ± 3.894 ± 14129 ± 74697 ± 53138 ± 40096 ± 10
      1.2101.4 ± 20.593 ± 43210 ± 60865 ± 45991 ± 105579 ± 26
      1.366.0 ± 9.598 ± 12201 ± 267106 ± 203154 ± 84592 ± 10
      1.4250.1 ± 53.692 ± 56257 ± 204868 ± 103993 ± 111577 ± 29
      1.532.7 ± 8.193 ± 18918 ± 284172 ± 20>10,000NC
      2.184.7 ± 12.489 ± 1>10,000NC3784 ± 79783 ± 2
      2.2264.5 ± 43.484 ± 2> 10,000NC>10,000NC
      Figure thumbnail gr2
      FIGURE 2Triazole analogues (A) and isoquinolinone analogues (B) are potent, full agonists in membrane [35S]GTPγS binding assays. CHO-hKOR cell membrane preparations were incubated with increasing concentrations of indicated agonists in the presence of [35S]GTPγS; activity is calculated as percentage of maximal U69,593 stimulation following base-line subtraction. Calculated potencies and efficacies are presented in . Data are presented as the mean ± S.E. (n ≥ 3).
      To determine the relative potencies for recruiting βarrestin2 to the agonist-stimulated KOR, two cell-based assays, as described in the Molecular Libraries Probe Production Centers Network probe report, were used (

      Hedrick, M. P., Gosalia, P., Frankowski, K., Shi, S., Prisinzano, T. E., Schoenen, F., Aube, J., Su, Y., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Ghosh, P., Milan, L., Heynen-Genel, S., Chung, T. D. Y., Dad, S., Caron, M., Bohn, L. M., Barak, L. S., (2010) Probe Reports from the NIH Molecular Libraries Program, Bethesda, MD

      ). The commercial EFC assay consists of U2OS cells expressing human KOR and βarrestin2 (U2OS-hKOR-βarrestin2-EFC), each tagged with a fragment of β-galactosidase. The degree of βarrestin2 recruitment is measured as increases in luminescence intensities triggered by enzyme complementation occurring upon receptor and βarrestin2 engagement (
      • Bohn L.M.
      • McDonald P.H.
      Seeking ligand bias: Assessing GPCR coupling to beta-arrestins for drug discovery.
      ). The second assay utilizes U2OS cells expressing human KOR and βarrestin2 tagged with green fluorescent protein (U2OS-hKOR-βarrestin2-GFP) (
      • Barak L.S.
      • Ferguson S.S.
      • Zhang J.
      • Caron M.G.
      A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation.
      ,
      • Barak L.S.
      • Warabi K.
      • Feng X.
      • Caron M.G.
      • Kwatra M.M.
      Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and β-arrestin 2 during homologous desensitization of the substance P receptor.
      ). The translocation of βarrestin2-GFP is measured by automated high content imaging analysis that detects a difference in diffuse cellular GFP distribution to the agonist-induced formation of fluorescent punctae (
      • Barak L.S.
      • Ferguson S.S.
      • Zhang J.
      • Caron M.G.
      A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation.
      ). In both cases, U69,593 is used as the full agonist reference ligand.
      While maintaining potent agonism in the G protein signaling assays, the triazole and isoquinolinone compounds only weakly recruit βarrestin2, with potencies exceeding 2 μm in both assays, whereas the potency of U69,593 is ∼130 nm in the βarrestin2 EFC assay and ∼205 nm in the imaging assay (Fig. 3; Table 1). Examination of the concentration-response curves in both assay platforms (Fig. 3, A and C) reveals that each of the triazole compounds shows minimal βarrestin2 recruitment at lower concentrations (<1 μm). However, stimulation dramatically increases at higher concentrations (>1 μm); this is also true for compound 2.1 of the isoquinolinones (Fig. 3, B and D). This phenomenon was verified for representative compounds using confocal microscopy to visualize βarrestin2-GFP translocation in the U2OS cells (data not shown) that confirmed the results obtained in the high content imaging platform (Fig. 3, C and D).
      Figure thumbnail gr3
      FIGURE 3Compared with U69,593, the triazole and isoquinolinone analogues are weak agonists for βarrestin2 recruitment to KOR. Concentration-response curves of triazole analogues (A) and isoquinolinone analogues (B) in the commercial EFC assay or the high content imaging assay (C, triazoles and D, isoquinolinones) reveal that these compounds lead to βarrestin2 recruitment at low potencies compared with U69,593. Calculated potencies and efficacies are presented in . Data are presented as the mean ± S.E. E, representative images of CHO-hKOR cells expressing βarrestin2-YFP treated with U69,593, 1.1, or 2.1. U69,593 robustly recruits βarrestin2 at 100 nm and 1 and 10 μm, inducing green fluorescent punctae formation. 1.1 does not induce green fluorescent punctae formation at 100 nm and 1 μm, although βarrestin2 recruitment becomes apparent at 10 μm. 2.1 does not recruit βarrestin2-YFP even at 10 μm dose. Insets: 4× magnifications showing the βarrestin2-YFP punctae (arrows). Scale bars, 20 μm.
      G protein signaling and βarrestin2 recruitment were, by design, performed in different cellular backgrounds (CHO-hKOR and U2OS-hKOR-βarrestin2-EFC or U2OS-hKOR-βarrestin2-GFP). The U2OS cell line was used for the commercial enzyme fragment complementation assay because it gave a larger assay window than that observed for the CHO-hKOR-βarrestin2-EFC commercial assay that is also available from the manufacturer. This was preferred to bias the system toward detecting even weak βarrestin2-KOR interactions. Furthermore, even though CHO-hKOR-βarrestin2-EFC cells could be used, it is important to realize that the KOR and βarrestin2 are linked to fragments of β-galactosidase and therefore, although the parental cell line is of the same origin, the stably transfected cell lines are not identical.
      To determine whether the cell line (U2OS versus CHO-K1) contributed to the apparent bias, we also evaluated βarrestin2 (tagged with YFP) recruitment in the CHO-hKOR cell line by confocal microscopy for select agonists of each class. In the confocal microscopy studies of CHO-hKOR cells, 1.1 does not promote βarrestin2 recruitment at low concentrations (100 nm and 1 μm) but induces punctae formation at 10 μm (Fig. 3E), consistent with the studies in U2OS cell imaging assays and the EFC βarrestin2 assay. The ability to visualize the agonist-induced βarrestin2 recruitment also suggests that the positive signal at high concentrations is not an artifact of the assay. In contrast to the observations in the other βarrestin2 assay platforms, 2.1 revealed no visible recruitment of βarrestin2-YFP even at 10 μm, in the CHO-hKOR cells (Fig. 3E). The lack of response to 2.1 in the CHO-hKOR cells transfected with βarrestin2-YFP is likely due to a low detection window with the low degree of stimulation falling below detectable thresholds. Finally, when tested in the MOR EFC assay, no stimulation is seen at any dose (1, 10, and 100 μm), which also argues against the effect being an artifact of the assay (data not shown).
      The studies presented in Tables 1 and in FIGURE 2, FIGURE 3 suggest that the triazole and isoquinolinone analogues presented here bias KOR function toward G protein signaling over the recruitment of βarrestin2. To compare the relative differences within and between assays, curves were fit to the operational model (
      • Black J.W.
      • Leff P.
      Operational models of pharmacological agonism.
      ) to derive transduction coefficients (log(τ/KA)) for the reference ligand (U69,593) and the “test” ligands using GraphPad Prism Version 6.01, as described previously (
      • 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 kappa opioid receptors in striatal neurons.
      ,
      • Kenakin T.
      • Christopoulos A.
      Signalling bias in new drug discovery: detection, quantification and therapeutic impact.
      ,
      • Kenakin T.
      • Watson C.
      • Muniz-Medina V.
      • Christopoulos A.
      • Novick S.
      A simple method for quantifying functional selectivity and agonist bias.
      ). The transduction coefficient represents the relative propensity of the ligand to generate a signal proportional to the agonist's calculated relative affinity for engaging the receptor based on its performance in the assay. Subtracting the log(τ/KA) for U69,593 from the values obtained for the test ligands generates a “normalized” transduction coefficient for the test ligand within the assay, or the Δlog(τ/KA). To determine preference between assays, bias factors were calculated as described under “Experimental Procedures” (Table 2) (
      • Kenakin T.
      • Watson C.
      • Muniz-Medina V.
      • Christopoulos A.
      • Novick S.
      A simple method for quantifying functional selectivity and agonist bias.
      ). Although such modeling may be imperfect for extreme cases of bias, we have presented the analysis here for qualitative comparison; each of the compounds displays bias for activation of KOR toward G protein signaling over βarrestin2 recruitment.
      TABLE 2Analysis of bias comparing G protein signaling and βarrestin2 recruitment in reference to U69,593 activity
      Entry[35S]GTPγS (mG), log(τ/KA)βArrestin2 EFC, log(τ/KA)βArrestin2 imaging log(τ/KA)Bias factors
      mG/βarr2 EFCmG/βarr2 Imaging
      U69,5937.36 ± 0.017.09 ± 0.046.79 ± 0.051.01.0
      1.17.47 ± 0.035.42 ± 0.10*5.61 ± 0.1161.220.0
      1.26.95 ± 0.035.38 ± 0.16*5.37 ± 0.1519.910.4
      1.37.40 ± 0.045.66 ± 0.09*5.68 ± 0.1130.114.4
      1.46.67 ± 0.064.92 ± 0.135.47 ± 0.1230.84.3
      1.57.52 ± 0.045.21 ± 0.155.31 ± 0.65111.744.3
      2.17.01 ± 0.055.25 ± 0.185.59 ± 0.5831.47.2
      2.26.48 ± 0.054.54 ± 0.813.91 ± 0.30*46.7100.0
      Because the [35S]GTPγS binding assay was performed in membrane preparations whereas the βarrestin2 recruitment studies were performed in whole cells, we considered that these contextual differences could confound our interpretations of bias. Therefore, we selected a representative triazole and isoquinolinone and performed two additional G protein signaling assays in whole cell preparations (Fig. 4). Using a low concentration of saponin to permeabilize the CHO-hKOR cells allowing uptake of the radionucleotide, [35S]GTPγS binding was performed in whole cells plated on 96-well plates (Fig. 4A). U69,593, 1.1, and 2.1 potently stimulate G protein signaling in this whole cell assay. Furthermore, in the absence of the brief permeabilization step, U69,593 does not induce [35S]GTPγS binding due to the lack of available radionucleotide inside the cell.
      Figure thumbnail gr4
      FIGURE 4Triazole and isoquinolinone KOR agonists are potent, full agonists in whole cell G protein signaling assays. A, whole cell G coupling assay. CHO-hKOR cells were permeabilized prior to performing agonist-stimulated [35S]GTPγS binding assays directly on plated cells in 96-well plates. Activity is calculated as percentage of maximal U69,593 stimulation following base-line subtraction. U69,593 induces no stimulation in the absence of permeabilization (−saponin). B, cellular impedance assay. Changes in cellular impedance in CHO-hKOR cells were recorded for 35 min after treatment of increasing doses of U69,593, 1.1 and 2.1. Maximal changes in impedance are calculated as percentage of maximal U69,593 (U69) stimulation. C, example traces comparing vehicle (Veh) and the maximum dose of 1 μm of each compound are shown (bottom). Calculated potencies and efficacies for both assays are presented in . Data are presented as the mean ± S.E. (n ≥ 5).
      A label-free, cellular impedance assay was also utilized to assess relative potencies in the living CHO-hKOR cells (Fig. 4B). This assay measures changes in impedance resulting from cytoskeletal re-organization leading to changes in cell shape (
      • McGuinness R.
      Impedance-based cellular assay technologies: recent advances, future promise.
      ). GPCRs promote actin reassembly patterns reflective of the coupling of particular Gα proteins. In our analysis, the cellular response signature resembles that of a typical Gαi-coupled GPCR (Fig. 4C) (
      • Peters M.F.
      • Scott C.W.
      Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.
      ). Importantly, no response was observed in the parental CHO cell line (data not shown). In this live cell assay format, 1.1 and 2.1 are fully efficacious and nearly as potent as U69,593 in promoting KOR-dependent responses indicative of G protein signaling (Table 3).
      TABLE 3Representative triazole and isoquinolinone compounds in whole cell G protein signaling assays
      Table thumbnail fx2
      Further analysis was undertaken to compare all G protein signaling assays to all βarrestin2 recruitment assays. Data from TABLE 2, TABLE 3 were used to determine bias factors that are presented graphically in Fig. 5. In summary, regardless of the assay format and the cell type, the triazole and isoquinolinone maintain bias toward KOR-induced G protein signaling over βarrestin2 recruitment.
      Figure thumbnail gr5
      FIGURE 5Triazole and isoquinolinone agonists bias KOR toward G protein signaling pathways. Transduction efficiencies presented in TABLE 2, TABLE 3 for G protein signaling assays and βarrestin2 recruitment assays were used to calculate bias factors. Bias factors are presented in TABLE 2, TABLE 3 and are plotted here on a logarithmic scale (base 10). As the reference agonist, the bias of U69,593 conforms to unity in all assays. The pathways represented are as follows: membrane [35S]GTPγS binding (mG protein); βarr2 EFC, βarr2 imaging, cellular impedance, and whole cell [35S]GTPγS binding (wcG protein). Independent of the platform used to assess G protein signaling or βarrestin2 recruitment, both 1.1 and 2.1 display bias for G protein signaling.
      To further characterize the compounds at a downstream signaling pathway, ERK1/2 phosphorylation was investigated. GPCRs can utilize both G protein-dependent and βarrestin-mediated signaling pathways to activate ERK1/2 MAPKs (
      • Bruchas M.R.
      • Chavkin C.
      Kinase cascades and ligand-directed signaling at the kappa opioid receptor.
      ). Interestingly, both classes of compounds activate ERK with potencies between 300 and 6000 nm as compared with ∼5 nm for U69,593. Although this appears to correlate with their relative potency for recruiting βarrestin2 (Fig. 6, A and B, and Table 4), further experiments must be undertaken to further define these pathways.
      Figure thumbnail gr6
      FIGURE 6Ligands induced ERK1/2 phosphorylation in CHO-hKOR cells. A, CHO-hKOR cells were treated with increasing doses of triazole (A) and isoquinolinone analogues (B) for 10 min, and phosphorylated (p-ERK) and total ERK (t-ERK) were detected by fluorescence intensity in 96-well plate “In-cell Western” assays. The ratio of p-ERK1/2 to total ERK1/2 was calculated and reported normalized to the percentage of maximal response induced by U69,593. C and D, Western blot analysis confirms that the p42 and p44 bands increase with drug treatment when examined with the p-ERK1/2 antibody. The high degree of ERK1/2 phosphorylation in CHO-hKOR cells induced by the triazole (C) or isoquinolinone (D) stimulation exceeds that observed for U69,593 (U69) (10 μm, 10 min), confirming the observations made in the In-cell Western format. Ratios of p-ERK1/2 over total ERK1/2 were normalized to the maximal U69,593 stimulation observed (n ≥ 3; *, p < 0.05; **, p < 0.01, Student's t test); molecular masses are indicated in kDa. Veh, vehicle.
      TABLE 4Signaling parameters for KOR agonists in ERK1/2 phosphorylation studies
      EntryERK1/2 phosphorylation
      EC50Imax
      nm%
      U69,5935.4 ± 0.78100
      1.1329 ± 84109 ± 7
      1.22282 ± 426139 ± 11
      1.3432 ± 152167 ± 14
      1.42778 ± 605173 ± 9
      1.55642 ± 2215241 ± 49
      2.12812 ± 1116149 ± 20
      2.2553 ± 23893 ± 10
      It is noteworthy that although these agonists are not very potent in this assay, their efficacy for activating ERK exceeds that of the reference compound U69,593. Because the assay utilized here involves an immunohistochemistry approach, it is possible that the increase in fluorescence could be due to the induction of other kinases that may be recognized by the phospho-specific ERK1/2 antibodies used. Therefore, Western blot analysis was performed to determine whether the increase in phosphorylation detected could be attributed to the 42- and 44-kDa bands, p-ERK1 and p-ERK2, respectively. Western blot analysis confirmed that the intensity of 42- and 44-kDa bands are elevated over that observed for U69,593-induced phosphorylation suggesting that this effect is not an artifact of the 384-well plate immunohistochemistry approach (Fig. 6C). Furthermore, none of the compounds activate ERK1/2 in the CHO-K1 parental cell line when tested at 1, 10, or 20 μm suggesting that these effects are KOR-dependent (data not shown). Because the efficacy of the test compounds exceeded that of the reference compound in this assay, U69,593 was insufficient to define the maximum potential of the system, and therefore, these data were not fit to the operational model. Additional studies, investigating differences in ERK1/2 populations (cytosol and nuclear) as well as the temporal regulation of ERK1/2 phosphorylation, will be needed to make accurate comparisons between the agonists.
      It is possible that, given the different receptor expression levels and cellular context, the ligands may not have comparable opportunities to interact with the receptor. To address this question, radioligand competition binding assays were used to determine whether the test ligands maintained comparable affinity and ability to displace [3H]U69,593 binding to membranes from the CHO-hKOR and U2OS-hKOR-βarrestin2-EFC cell lines. Saturation binding of [3H]U69,593 was performed initially to determine equilibrium dissociation constants (KD values) and receptor numbers (Bmax values) in each cell line (with unlabeled U69,593 at 10 μm defining the nonspecific binding). The CHO-hKOR cell line expresses 2.07 ± 0.48 pmol/mg membrane protein with a [3H]U69,593 binding affinity of 0.27 ± 0.11 nm (Table 5). The commercial U2OS-hKOR-βarrestin2-EFC cell line expresses nearly 6-fold more receptors (11.45 ± 2.26 pmol/mg of membrane protein), and the affinity for [3H]U69,593 binding is 0.36 ± 0.10 nm. When the test compounds were compared in each of the cell lines, the triazole and isoquinolinone compounds all fully displace [3H]U69,593 binding and all do so with high affinity (0.25–1.79 nm), confirming comparable opportunity to bind to the KOR (Table 5). Further studies were performed to determine selectivity profiles of binding to MOR and DOR expressed in CHO-K1 cells. The triazole and isoquinolinone derivatives, like the initial probes, display high selectivity for KOR binding over mu and delta opioid receptors (Table 5) (

      Hedrick, M. P., Gosalia, P., Frankowski, K., Shi, S., Prisinzano, T. E., Schoenen, F., Aube, J., Su, Y., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Ghosh, P., Milan, L., Heynen-Genel, S., Chung, T. D. Y., Dad, S., Caron, M., Bohn, L. M., Barak, L. S., (2010) Probe Reports from the NIH Molecular Libraries Program, Bethesda, MD

      ,
      • Frankowski K.J.
      • Hedrick M.P.
      • Gosalia P.
      • Li K.
      • Shi S.
      • Whipple D.
      • Ghosh P.
      • Prisinzano T.E.
      • Schoenen F.J.
      • Su Y.
      • Vasile S.
      • Sergienko E.
      • Gray W.
      • Hariharan S.
      • Milan L.
      • Heynen-Genel S.
      • Mangravita-Novo A.
      • Vicchiarelli M.
      • Smith L.H.
      • Streicher J.M.
      • Caron M.G.
      • Barak L.S.
      • Bohn L.M.
      • Chung T.D.
      • Aubé J.
      Discovery of small molecule kappa opioid receptor agonist and antagonist chemotypes through a HTS and hit refinement strategy.
      ,
      • Frankowski K.J.
      • Ghosh P.
      • Setola V.
      • Tran T.B.
      • Roth B.L.
      • Aubé J.
      N-Alkyl-octahydroisoquinolin-1-one-8-carboxamides: a novel class of selective, nonbasic, nitrogen-containing kappa-opioid receptor ligands.
      ).
      TABLE 5Binding affinities for KOR agonists in the U2OS-hKOR-βarrestin2-EFC, CHO-hKOR, CHO-hDOR, and CHO-hMOR cell lines
      EntryEFC-KOR, Ki or KDCHO-hKOR, Ki or KDCHO-hDORCHO-hMORKi ratios in CHO lines
      Ki% at 10 μmKi% at 10 μmDOR/KORMOR/KOR
      nmnmnmnm
      U690.36 ± 0.100.27 ± 0.11>10,00048 ± 121638 ± 54862 ± 12>36,9466052
      1.10.33 ± 0.050.25 ± 0.022225 ± 57158 ± 10>10,00031 ± 118905>40,011
      1.20.55 ± 0.050.54 ± 0.09>10,00048 ± 8712 ± 48277 ± 13>18,7031332
      1.30.47 ± 0.070.25 ± 0.062681 ± 19260 ± 41687 ± 25168 ± 10102996636
      1.41.79 ± 0.181.16 ± 0.142061 ± 18167 ± 82720 ± 71460 ± 1517792348
      1.50.67 ± 0.130.47 ± 0.11>10,00031 ± 16>10,00033 ± 11>21,216>21,216
      2.10.28 ± 0.050.35 ± 0.13>10,00033 ± 10368 ± 13680 ± 8>28,5421051
      2.21.45 ± 0.101.31 ± 0.33>10,0009 ± 11>10,00020 ± 11>7,649>7,649
      Ultimately, these compounds, of which there is a growing collection, may become important tools for ascertaining the relative contributions of G protein-dependent, βarrestin2-independent signaling at KOR in vivo. As an early step in characterization, the pharmacokinetic properties of 1.1 and 2.1 were assessed in mice. Brain and plasma compound contents were determined following a single systemic, intraperitoneal administration (10 mg/kg, intraperitoneal), and both compounds were detected at low micromolar concentrations in brain at 30 and 60 min (Table 6). Pharmacokinetic distribution and clearance rates were determined following a 2 mg/kg (i.v.) tail vein injection and timed blood collections. Plasma protein binding and nonspecific brain binding were also determined for these compounds. These parameters, along with the determined “logP” values are presented in Table 6 and indicate good brain exposure and reasonable pharmacokinetic properties. Together these findings suggest that these scaffolds may represent favorable leads for development of biased KOR agonists with CNS penetration.
      TABLE 6Triazole (1.1) and isoquinolinone (2.1) brain and blood distribution, pharmacokinetics, and logP values
      Assay1.12.1
      [Brain] (μm)
      30 min1.91 ± 0.831.09 ± 0.04
      60 min1.31 ± 0.151.21 ± 0.11
      [Plasma] (μm)
      30 min0.43 ± 0.150.79 ± 0.06
      60 min0.22 ± 0.020.82 ± 0.02
      Brain (% bound)
      Rat>99.599.20
      Plasma (% bound)
      Human>99.5>99.5
      Mouse>99.596.90
      Rat>99.599.20
      Pharmacokinetics
      t½2.80 h2.03 h
      tmax0.25 h0.14 h
      Cmax4.94 μm4.16 μm
      AUClast4.62 μm·h6.83 μm·h
      Cl_obs31.53 ml/min/kg26.82 ml/min/kg
      Vss_obs4.20 liter/kg3.43 liter/kg
      Determined logP3.684.16
      Because G protein-mediated KOR signaling has been proposed as the mechanism underlying KOR-mediated antinociception, the antinociceptive effects of 1.1 and 2.1 were then assessed using the warm water tail-flick assay (Fig. 6) (
      • Vonvoigtlander P.F.
      • Lahti R.A.
      • Ludens J.H.
      U-50,488: a selective and structurally novel non-Mu (kappa) opioid agonist.
      ,
      • McLaughlin J.P.
      • Myers L.C.
      • Zarek P.E.
      • Caron M.G.
      • Lefkowitz R.J.
      • Czyzyk T.A.
      • Pintar J.E.
      • Chavkin C.
      Prolonged kappa opioid receptor phosphorylation mediated by G-protein receptor kinase underlies sustained analgesic tolerance.
      ). Mice were injected with 30 mg/kg, intraperitoneal doses of either 1.1, 2.1, or the selective KOR agonist U50,488H, as this dose of U50,488H has been shown to induce antinociception in mice in this assay (
      • Mogil J.S.
      • Wilson S.G.
      • Chesler E.J.
      • Rankin A.L.
      • Nemmani K.V.
      • Lariviere W.R.
      • Groce M.K.
      • Wallace M.R.
      • Kaplan L.
      • Staud R.
      • Ness T.J.
      • Glover T.L.
      • Stankova M.
      • Mayorov A.
      • Hruby V.J.
      • Grisel J.E.
      • Fillingim R.B.
      The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans.
      ). The triazole and isoquinolinone compounds significantly increased the latency to tail withdrawal similar to that seen with U50,488H, with the effects peaking at 20 min post drug treatment (Fig. 7) demonstrating that these agonists are capable of inducing antinociception in mice.
      Figure thumbnail gr7
      FIGURE 7Compounds 1.1 and 2.1 induce antinociceptive effects in mice. C57BL/6J male mice were treated with U50,488H, 1.1, or 2.1 at 30 mg/kg, and intraperitoneal and tail withdrawal response latencies were recorded in response to exposure of the tail to warm water (49 °C) at 10, 20, 30, 45, 60, and 90 min post-treatment. Data are presented as the mean ± S.E. U50,488H and both test compounds induced similar time-dependent antinociceptive responses (one-way ANOVA, for U50,488H, F(6,49) = 5.58, p < 0.0001; for 1.1, F(6,49) = 10.55, p < 0.0001; for 2.1, F(6,49) = 13.42, p < 0.0001; Bonferroni's post test, basal versus treatment time within each drug treatment: U50,488H (10, 20 min, p < 0.001; 30, 45 min, p < 0.01; 60 min, p < 0.05) and 1.1 (10, 20 min, p < 0.001; 30, 45 min, p < 0.05), 2.1 (20, 30 min, p < 0.001, 10; 45 min, p < 0.01) n = 8–10 mice per drug treatment).

      DISCUSSION

      The triazole and isoquinolinone analogues described herein are highly selective KOR agonists that induce receptor signaling biased toward G protein signaling over βarrestin2 recruitment. When tested in vivo, 1.1 and 2.1, a triazole and an isoquinolinone, respectively, prove to be brain-penetrant. Subsequently, 1.1 and 2.1 produce antinociception in the mouse tail flick test, which correlates with the ability of these compounds to potently activate G protein signaling in cell-based assays.
      Prior studies suggest that KOR agonists that do not engage βarrestin2-mediated signaling pathways may prove to have less aversive properties such as dysphoria (
      • Bruchas M.R.
      • Land B.B.
      • Aita M.
      • Xu M.
      • Barot S.K.
      • Li S.
      • Chavkin C.
      Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria.
      ,
      • Chavkin C.
      The therapeutic potential of kappa-opioids for treatment of pain and addiction.
      ,
      • Rives M.L.
      • Rossillo M.
      • Liu-Chen L.Y.
      • Javitch J.A.
      6′-Guanidinonaltrindole (6′-GNTI) is a G protein-biased kappa-opioid receptor agonist that inhibits arrestin recruitment.
      ) (Fig. 8). It is hopeful that the generation of compounds, such as those in the series described here, will provide pharmacological tools that will aid in the elucidation of KOR-mediated signaling cascades in cellular systems and, importantly, in vivo. The fact that these series possess very high affinity and selectivity for KOR over other opioid receptors, are brain penetrant, and have favorable pharmacokinetic parameters will certainly aid in these ventures.
      Figure thumbnail gr8
      FIGURE 8Schematic of isoquinolinone and triazole compound signaling at KOR compared with U69,593. Activation at KOR mediates multiple signaling cascades leading to G protein coupling, βarrestin recruitment, and ERK kinase phosphorylation. The isoquinolinones and triazoles act as biased agonists, preferentially inducing G protein coupling over βarrestin2 recruitment. This lack of preference for βarrestin2 recruitment appears to correlate with a loss in potency for ERK1/2 activation. Because KOR-mediated antinociception has been attributed to G protein signaling mechanisms and because KOR-induced interactions with βarrestin2 are proposed to induce dysphoria, biasing KOR activation toward G protein signaling and away from βarrestin2 pathways may be the key to induce antinociception and avoid dysphoria (
      • Bruchas M.R.
      • Land B.B.
      • Aita M.
      • Xu M.
      • Barot S.K.
      • Li S.
      • Chavkin C.
      Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria.
      ,
      • Barak L.S.
      • Warabi K.
      • Feng X.
      • Caron M.G.
      • Kwatra M.M.
      Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and β-arrestin 2 during homologous desensitization of the substance P receptor.
      ,
      • Peters M.F.
      • Scott C.W.
      Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.
      ).
      Recently, Rives et al. (
      • Rives M.L.
      • Rossillo M.
      • Liu-Chen L.Y.
      • Javitch J.A.
      6′-Guanidinonaltrindole (6′-GNTI) is a G protein-biased kappa-opioid receptor agonist that inhibits arrestin recruitment.
      ) found the naltrindole-derived ligand 6′-GNTI to be a potent partial agonist for KOR-stimulated G protein signaling yet an antagonist for recruiting βarrestins. From our laboratory, Schmid et al. (
      • 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 kappa opioid receptors in striatal neurons.
      ) recently confirmed the delineation of this bias in vitro and further explored it in cultured striatal neurons. Although 6′-GNTI potently activates ERK1/2 in CHO-hKOR cells, in neurons 6′-GNTI does not activate ERK1/2 although U69,593 does so in a βarrestin2-dependent manner, suggesting that KOR utilizes βarrestin2 to activate ERK in the endogenous setting (
      • 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 kappa opioid receptors in striatal neurons.
      ). The triazole and isoquinolinone compounds investigated herein differ from 6′-GNTI in that they are full agonists in G protein signaling and they are able to activate βarrestin2 recruitment in the cell-based assays, although at a greatly diminished potency. Furthermore, the triazoles and isoquinolinones do not potently activate ERK1/2, further distinguishing them from 6′-GNTI. The increasing number of compounds identified with diverse signaling profiles downstream of KOR activation may be useful for docking studies to the continually refined crystal structure of these GPCRs (
      • Wu H.
      • Wacker D.
      • Mileni M.
      • Katritch V.
      • Han G.W.
      • Vardy E.
      • Liu W.
      • Thompson A.A.
      • Huang X.P.
      • Carroll F.I.
      • Mascarella S.W.
      • Westkaemper R.B.
      • Mosier P.D.
      • Roth B.L.
      • Cherezov V.
      • Stevens R.C.
      Structure of the human kappa-opioid receptor in complex with JDTic.
      ), which may provide insight into the chemical signatures driving specific receptor confirmations and signaling events.
      The triazoles and isoquinolinones consistently activate ERK1/2 with less potency than that observed for U69,593. Therefore, it is attractive to speculate that the triazoles and isoquinolinones are biased toward G protein signaling over ERK1/2 phosphorylation. We hesitate, however, to make this claim based on two confounding variables. In this particular pathway, U69,593 does not adequately serve as a reference compound. The second issue, and perhaps the most important, is that ERK1/2 activation is a downstream effector that represents the consolidation of multiple upstream cascades (potentially originating from affecting different G proteins, βarrestins, calcium influx, etc.). Although our negative controls demonstrate that the activation of ERK by the test compounds is due to KOR (no response in parental CHO-K1 cells), it is difficult to interpret what component of cellular signaling is participating more (overshoot in efficacy) or less (rightward shift in potency) in generating the response profile elicited by these compounds downstream of KOR. Regardless, the compounds consistently reveal more potency in the G protein signaling assay than in the ERK1/2 and βarrestin assays. Overall, these results are intriguing and represent an area for further explorations into the compartmental (nuclear versus cytoplasmic (
      • Ahn S.
      • Shenoy S.K.
      • Wei H.
      • Lefkowitz R.J.
      Differential kinetic and spatial patterns of β-arrestin and G protein-mediated ERK activation by the angiotensin II receptor.
      )) and temporal (transient or sustained (
      • McLennan G.P.
      • Kiss A.
      • Miyatake M.
      • Belcheva M.M.
      • Chambers K.T.
      • Pozek J.J.
      • Mohabbat Y.
      • Moyer R.A.
      • Bohn L.M.
      • Coscia C.J.
      Kappa opioids promote the proliferation of astrocytes via Gβγ and β-arrestin 2-dependent MAPK-mediated pathways.
      ,
      • Ahn S.
      • Shenoy S.K.
      • Wei H.
      • Lefkowitz R.J.
      Differential kinetic and spatial patterns of β-arrestin and G protein-mediated ERK activation by the angiotensin II receptor.
      )) aspects of GPCR-stimulated ERK activation in respect to βarrestin and G protein-dependent mechanisms.
      It will be of great interest to utilize these compounds in additional behavioral and physiological assays to determine the contribution of βarrestin2 to KOR signaling in vivo. However, it will first be important to determine that the signaling bias observed across the various cell-based assay platforms is maintained in vivo and whether these pathways are associated with behaviors. If indeed these newly discovered KOR-biased agonists, which are brain penetrant following systemic injections, prove to maintain signaling bias in the endogenous setting, these compounds may serve as important tools for investigating the contributions of ERK activation and/or βarrestin2 recruitment to KOR-mediated effects in vivo, and it is hopeful that these new pharmacological tools will serve the community for exploring relevant KOR biology in an endogenous setting.

      Acknowledgments

      We thank Ben Neuenswander and Patrick Porubsky (Kansas University) for HPLC-MS analysis and Larry Barak (Duke University) for the U2OS-hKOR-βarrestin2-GFP cell line. We are grateful to Arthur Christopoulos (Monash University, Australia) for helpful discussions on determining bias factors.

      REFERENCES

        • Cox B.M.
        Recent developments in the study of opioid receptors.
        Mol. Pharmacol. 2013; 83: 723-728
        • Chavkin C.
        • James I.F.
        • Goldstein A.
        Dynorphin is a specific endogenous ligand of the kappa opioid receptor.
        Science. 1982; 215: 413-415
        • Mansour A.
        • Fox C.A.
        • Burke S.
        • Meng F.
        • Thompson R.C.
        • Akil H.
        • Watson S.J.
        Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study.
        J. Comp. Neurol. 1994; 350: 412-438
        • Minami M.
        • Satoh M.
        Molecular biology of the opioid receptors: structures, functions and distributions.
        Neurosci. Res. 1995; 23: 121-145
        • Prather P.L.
        • McGinn T.M.
        • Claude P.A.
        • Liu-Chen L.Y.
        • Loh H.H.
        • Law P.Y.
        Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual Gα subunit and is similar to that of other opioid receptors.
        Brain Res. Mol. Brain Res. 1995; 29: 336-346
        • Lawrence D.M.
        • Bidlack J.M.
        The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein.
        J. Pharmacol. Exp. Ther. 1993; 266: 1678-1683
        • Ma G.H.
        • Miller R.J.
        • Kuznetsov A.
        • Philipson L.H.
        kappa-Opioid receptor activates an inwardly rectifying K+ channel by a G protein-linked mechanism: coexpression in Xenopus oocytes.
        Mol. Pharmacol. 1995; 47: 1035-1040
        • Macdonald R.L.
        • Werz M.A.
        Dynorphin A decreases voltage-dependent calcium conductance of mouse dorsal root ganglion neurones.
        J. Physiol. 1986; 377: 237-249
        • Pasternak G.W.
        Multiple opiate receptors: [3H]ethylketocyclazocine receptor binding and ketocyclazocine analgesia.
        Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 3691-3694
        • Vonvoigtlander P.F.
        • Lahti R.A.
        • Ludens J.H.
        U-50,488: a selective and structurally novel non-Mu (kappa) opioid agonist.
        J. Pharmacol. Exp. Ther. 1983; 224: 7-12
        • Dykstra L.A.
        • Gmerek D.E.
        • Winger G.
        • Woods J.H.
        Kappa opioids in rhesus monkeys. I. Diuresis, sedation, analgesia and discriminative stimulus effects.
        J. Pharmacol. Exp. Ther. 1987; 242: 413-420
        • Millan M.J.
        • Czl̸onkowski A.
        • Lipkowski A.
        • Herz A.
        Kappa-opioid receptor-mediated antinociception in the rat. II. Supraspinal in addition to spinal sites of action.
        J. Pharmacol. Exp. Ther. 1989; 251: 342-350
        • Simonin F.
        • Valverde O.
        • Smadja C.
        • Slowe S.
        • Kitchen I.
        • Dierich A.
        • Le Meur M.
        • Roques B.P.
        • Maldonado R.
        • Kieffer B.L.
        Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal.
        EMBO J. 1998; 17: 886-897
        • Goicoechea C.
        • Ormazábal M.J.
        • Abalo R.
        • Alfaro M.J.
        • Martín M.I.
        Calcitonin reverts pertussis toxin blockade of the opioid analgesia in mice.
        Neurosci. Lett. 1999; 273: 175-178
        • Gullapalli S.
        • Ramarao P.
        Role of L-type Ca2+ channels in pertussis toxin induced antagonism of U50,488H analgesia and hypothermia.
        Brain Res. 2002; 946: 191-197
        • Raehal K.M.
        • Bohn L.M.
        Mu opioid receptor regulation and opiate responsiveness.
        AAPS J. 2005; 7: E587-E591
        • Pfeiffer A.
        • Brantl V.
        • Herz A.
        • Emrich H.M.
        Psychotomimesis mediated by kappa opiate receptors.
        Science. 1986; 233: 774-776
        • Land B.B.
        • Bruchas M.R.
        • Lemos J.C.
        • Xu M.
        • Melief E.J.
        • Chavkin C.
        The dysphoric component of stress is encoded by activation of the dynorphin kappa-opioid system.
        J. Neurosci. 2008; 28: 407-414
        • Roth B.L.
        • Baner K.
        • Westkaemper R.
        • Siebert D.
        • Rice K.C.
        • Steinberg S.
        • Ernsberger P.
        • Rothman R.B.
        Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11934-11939
        • Knoll A.T.
        • Carlezon Jr., W.A.
        Dynorphin, stress, and depression.
        Brain Res. 2010; 1314: 56-73
        • Bohn L.M.
        Neve K.A. 1st Ed. Functional Selectivity of G Protein-coupled Receptor Ligands. Humana Press Inc., Totowa, NJ2009: 71-85
        • Urban J.D.
        • Clarke W.P.
        • von Zastrow M.
        • Nichols D.E.
        • Kobilka B.
        • Weinstein H.
        • Javitch J.A.
        • Roth B.L.
        • Christopoulos A.
        • Sexton P.M.
        • Miller K.J.
        • Spedding M.
        • Mailman R.B.
        Functional selectivity and classical concepts of quantitative pharmacology.
        J. Pharmacol. Exp. Ther. 2007; 320: 1-13
        • Reiter E.
        • Ahn S.
        • Shukla A.K.
        • Lefkowitz R.J.
        Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors.
        Annu. Rev. Pharmacol. Toxicol. 2012; 52: 179-197
        • Luttrell D.K.
        • Luttrell L.M.
        Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases.
        Assay Drug Dev. Technol. 2003; 1: 327-338
        • Schmid C.L.
        • Bohn L.M.
        Physiological and pharmacological implications of β-arrestin regulation.
        Pharmacol. Ther. 2009; 121: 285-293
        • Shenoy S.K.
        • Lefkowitz R.J.
        β-Arrestin-mediated receptor trafficking and signal transduction.
        Trends Pharmacol. Sci. 2011; 32: 521-533
        • Lefkowitz R.J.
        • Shenoy S.K.
        Transduction of receptor signals by β-arrestins.
        Science. 2005; 308: 512-517
        • McLennan G.P.
        • Kiss A.
        • Miyatake M.
        • Belcheva M.M.
        • Chambers K.T.
        • Pozek J.J.
        • Mohabbat Y.
        • Moyer R.A.
        • Bohn L.M.
        • Coscia C.J.
        Kappa opioids promote the proliferation of astrocytes via Gβγ and β-arrestin 2-dependent MAPK-mediated pathways.
        J. Neurochem. 2008; 107: 1753-1765
        • Bruchas M.R.
        • Macey T.A.
        • Lowe J.D.
        • Chavkin C.
        Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes.
        J. Biol. Chem. 2006; 281: 18081-18089
        • Bruchas M.R.
        • Land B.B.
        • Aita M.
        • Xu M.
        • Barot S.K.
        • Li S.
        • Chavkin C.
        Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria.
        J. Neurosci. 2007; 27: 11614-11623
        • Bohn L.M.
        • Belcheva M.M.
        • Coscia C.J.
        Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade.
        J. Neurochem. 2000; 74: 564-573
        • Belcheva M.M.
        • Clark A.L.
        • Haas P.D.
        • Serna J.S.
        • Hahn J.W.
        • Kiss A.
        • Coscia C.J.
        Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes.
        J. Biol. Chem. 2005; 280: 27662-27669
        • Bruchas M.R.
        • Xu M.
        • Chavkin C.
        Repeated swim stress induces kappa opioid-mediated activation of extracellular signal-regulated kinase 1/2.
        Neuroreport. 2008; 19: 1417-1422
        • Bruchas M.R.
        • Chavkin C.
        Kinase cascades and ligand-directed signaling at the kappa opioid receptor.
        Psychopharmacology. 2010; 210: 137-147
      1. Hedrick, M. P., Gosalia, P., Frankowski, K., Shi, S., Prisinzano, T. E., Schoenen, F., Aube, J., Su, Y., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Ghosh, P., Milan, L., Heynen-Genel, S., Chung, T. D. Y., Dad, S., Caron, M., Bohn, L. M., Barak, L. S., (2010) Probe Reports from the NIH Molecular Libraries Program, Bethesda, MD

        • Frankowski K.J.
        • Hedrick M.P.
        • Gosalia P.
        • Li K.
        • Shi S.
        • Whipple D.
        • Ghosh P.
        • Prisinzano T.E.
        • Schoenen F.J.
        • Su Y.
        • Vasile S.
        • Sergienko E.
        • Gray W.
        • Hariharan S.
        • Milan L.
        • Heynen-Genel S.
        • Mangravita-Novo A.
        • Vicchiarelli M.
        • Smith L.H.
        • Streicher J.M.
        • Caron M.G.
        • Barak L.S.
        • Bohn L.M.
        • Chung T.D.
        • Aubé J.
        Discovery of small molecule kappa opioid receptor agonist and antagonist chemotypes through a HTS and hit refinement strategy.
        ACS Chem. Neurosci. 2012; 3: 221-236
        • Frankowski K.J.
        • Hirt E.E.
        • Zeng Y.
        • Neuenswander B.
        • Fowler D.
        • Schoenen F.
        • Aubé J.
        Synthesis of N-alkyl-octahydroisoquinolin-1-one-8-carboxamide libraries using a tandem Diels-Alder/acylation sequence.
        J. Comb. Chem. 2007; 9: 1188-1192
        • Frankowski K.J.
        • Ghosh P.
        • Setola V.
        • Tran T.B.
        • Roth B.L.
        • Aubé J.
        N-Alkyl-octahydroisoquinolin-1-one-8-carboxamides: a novel class of selective, nonbasic, nitrogen-containing kappa-opioid receptor ligands.
        ACS Med. Chem. Lett. 2010; 1: 189-193
        • Lahti R.A.
        • Mickelson M.M.
        • McCall J.M.
        • Von Voigtlander P.F.
        [3H]U-69593 a highly selective ligand for the opioid kappa receptor.
        Eur. J. Pharmacol. 1985; 109: 281-284
        • Jones R.M.
        • Portoghese P.S.
        5′-Guanidinonaltrindole, a highly selective and potent kappa-opioid receptor antagonist.
        Eur. J. Pharmacol. 2000; 396: 49-52
        • Thomas J.B.
        • Atkinson R.N.
        • Rothman R.B.
        • Fix S.E.
        • Mascarella S.W.
        • Vinson N.A.
        • Xu H.
        • Dersch C.M.
        • Lu Y.
        • Cantrell B.E.
        • Zimmerman D.M.
        • Carroll F.I.
        Identification of the first trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine derivative to possess highly potent and selective opioid kappa receptor antagonist activity.
        J. Med. Chem. 2001; 44: 2687-2690
        • 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 kappa opioid receptors in striatal neurons.
        J. Biol. Chem. 2013; 288: 22387-22398
        • Ananthan S.
        • Saini S.K.
        • Dersch C.M.
        • Xu H.
        • McGlinchey N.
        • Giuvelis D.
        • Bilsky E.J.
        • Rothman R.B.
        14-Alkoxy- and 14-acyloxypyridomorphinans: mu agonist/delta antagonist opioid analgesics with diminished tolerance and dependence side effects.
        J. Med. Chem. 2012; 55: 8350-8363
        • Breivogel C.S.
        • Walker J.M.
        • Huang S.M.
        • Roy M.B.
        • Childers S.R.
        Cannabinoid signaling in rat cerebellar granule cells: G-protein activation, inhibition of glutamate release and endogenous cannabinoids.
        Neuropharmacology. 2004; 47: 81-91
        • McGuinness R.
        Impedance-based cellular assay technologies: recent advances, future promise.
        Curr. Opin. Pharmacol. 2007; 7: 535-540
        • Robinson J.
        • Smith A.
        • Sturchler E.
        • Tabrizifard S.
        • Kamenecka T.
        • McDonald P.
        Development of a high-throughput screening-compatible cell-based functional assay to identify small molecule probes of the galanin 3 receptor (GalR3).
        Assay Drug Dev. Technol. 2013;
        • Bohn L.M.
        • Gainetdinov R.R.
        • Lin F.T.
        • Lefkowitz R.J.
        • Caron M.G.
        Mu-opioid receptor desensitization by β-arrestin-2 determines morphine tolerance but not dependence.
        Nature. 2000; 408: 720-723
        • Valko K.
        • My Du C.
        • Bevan C.
        • Reynolds D.P.
        • Abraham M.H.
        Rapid method for the estimation of octanol/water partition coefficient (log P(oct)) from gradient RP-HPLC retention and a hydrogen bond acidity term (ζα(2)(H)).
        Curr. Med. Chem. 2001; 8: 1137-1146
        • Bohn L.M.
        • Lefkowitz R.J.
        • Gainetdinov R.R.
        • Peppel K.
        • Caron M.G.
        • Lin F.T.
        Enhanced morphine analgesia in mice lacking β-arrestin 2.
        Science. 1999; 286: 2495-2498
        • Raehal K.M.
        • Schmid C.L.
        • Medvedev I.O.
        • Gainetdinov R.R.
        • Premont R.T.
        • Bohn L.M.
        Morphine-induced physiological and behavioral responses in mice lacking G protein-coupled receptor kinase 6.
        Drug Alcohol Depend. 2009; 104: 187-196
        • Black J.W.
        • Leff P.
        Operational models of pharmacological agonism.
        Proc. R. Soc. Lond B Biol. Sci. 1983; 220: 141-162
        • Kenakin T.
        • Christopoulos A.
        Signalling bias in new drug discovery: detection, quantification and therapeutic impact.
        Nat. Rev. Drug Discov. 2013; 12: 205-216
        • Evans B.A.
        • Broxton N.
        • Merlin J.
        • Sato M.
        • Hutchinson D.S.
        • Christopoulos A.
        • Summers R.J.
        Quantification of functional selectivity at the human α(1A)-adrenoceptor.
        Mol. Pharmacol. 2011; 79: 298-307
        • Kenakin T.
        • Watson C.
        • Muniz-Medina V.
        • Christopoulos A.
        • Novick S.
        A simple method for quantifying functional selectivity and agonist bias.
        ACS Chem. Neurosci. 2012; 3: 193-203
        • Bohn L.M.
        • McDonald P.H.
        Seeking ligand bias: Assessing GPCR coupling to beta-arrestins for drug discovery.
        Drug Discov. Today Technol. 2010; 7: e37-e42
        • Barak L.S.
        • Ferguson S.S.
        • Zhang J.
        • Caron M.G.
        A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation.
        J. Biol. Chem. 1997; 272: 27497-27500
        • Barak L.S.
        • Warabi K.
        • Feng X.
        • Caron M.G.
        • Kwatra M.M.
        Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and β-arrestin 2 during homologous desensitization of the substance P receptor.
        J. Biol. Chem. 1999; 274: 7565-7569
        • Peters M.F.
        • Scott C.W.
        Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.
        J. Biomol. Screen. 2009; 14: 246-255
        • McLaughlin J.P.
        • Myers L.C.
        • Zarek P.E.
        • Caron M.G.
        • Lefkowitz R.J.
        • Czyzyk T.A.
        • Pintar J.E.
        • Chavkin C.
        Prolonged kappa opioid receptor phosphorylation mediated by G-protein receptor kinase underlies sustained analgesic tolerance.
        J. Biol. Chem. 2004; 279: 1810-1818
        • Mogil J.S.
        • Wilson S.G.
        • Chesler E.J.
        • Rankin A.L.
        • Nemmani K.V.
        • Lariviere W.R.
        • Groce M.K.
        • Wallace M.R.
        • Kaplan L.
        • Staud R.
        • Ness T.J.
        • Glover T.L.
        • Stankova M.
        • Mayorov A.
        • Hruby V.J.
        • Grisel J.E.
        • Fillingim R.B.
        The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 4867-4872
        • Chavkin C.
        The therapeutic potential of kappa-opioids for treatment of pain and addiction.
        Neuropsychopharmacology. 2011; 36: 369-370
        • Rives M.L.
        • Rossillo M.
        • Liu-Chen L.Y.
        • Javitch J.A.
        6′-Guanidinonaltrindole (6′-GNTI) is a G protein-biased kappa-opioid receptor agonist that inhibits arrestin recruitment.
        J. Biol. Chem. 2012; 287: 27050-27054
        • Wu H.
        • Wacker D.
        • Mileni M.
        • Katritch V.
        • Han G.W.
        • Vardy E.
        • Liu W.
        • Thompson A.A.
        • Huang X.P.
        • Carroll F.I.
        • Mascarella S.W.
        • Westkaemper R.B.
        • Mosier P.D.
        • Roth B.L.
        • Cherezov V.
        • Stevens R.C.
        Structure of the human kappa-opioid receptor in complex with JDTic.
        Nature. 2012; 485: 327-332
        • Ahn S.
        • Shenoy S.K.
        • Wei H.
        • Lefkowitz R.J.
        Differential kinetic and spatial patterns of β-arrestin and G protein-mediated ERK activation by the angiotensin II receptor.
        J. Biol. Chem. 2004; 279: 35518-35525