title: Two Affinity Sites of the Cannabinoid Subtype 2 Receptor Identified by a Novel Homogeneous Binding Assay.
abstract: Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-yl]sulfonyl-2-pyridyl]oxy]propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may serve to better characterize agonist binding to CB2R and to identify specific properties of CB2R on living cells.
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.) martinezpinillaeva@gmail.com rfranco@ub.edu julenoyarzabal@unav.es.
authors.forename: Eva
authors.identifier: 0000-0002-0572-0926
authors.initials: E
authors.lastname: Martínez-Pinilla
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: Obdulia
authors.initials: O
authors.lastname: Rabal
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: Irene
authors.initials: I
authors.lastname: Reyes-Resina
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: Marta
authors.initials: M
authors.lastname: Zamarbide
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: Gemma
authors.initials: G
authors.lastname: Navarro
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: Juan A
authors.initials: JA
authors.lastname: Sánchez-Arias
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: Irene
authors.initials: I
authors.lastname: de Miguel
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.).
authors.forename: José L
authors.initials: JL
authors.lastname: Lanciego
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.) martinezpinillaeva@gmail.com rfranco@ub.edu julenoyarzabal@unav.es.
authors.forename: Julen
authors.initials: J
authors.lastname: Oyarzabal
authors.affiliation: Neurosciences Division (E.M.-P., M.Z., J.L.L.) and Small Molecule Discovery Platform, Molecular Therapeutics Program (O.R., J.A.S.-A., I.d.M., J.O.), Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Instituto de Neurociencias del Principado de Asturias, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain (E.M.-P.); Departament de Bioquímica i Biología Molecular, Facultat de Biología,. Universitat de Barcelona, Barcelona, Spain (I.R.-R., G.N., R.F.); Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, Madrid, Spain (I.R.-R., G.N., J.L.L., R.F.); Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain (J.L.L.); and Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain (R.F.) martinezpinillaeva@gmail.com rfranco@ub.edu julenoyarzabal@unav.es.
authors.forename: Rafael
authors.initials: R
authors.lastname: Franco
mesh_terms: D001665:Binding Sites; D001681:Biological Assay; D002911:Chrysenes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D008024:Ligands; D062105:Molecular Docking Simulation; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry / Q000378:metabolism*; D043882:Receptors, Cannabinoid; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D002911:Chrysenes; D005456:Fluorescent Dyes; C524885:GPR55 protein, human; D007211:Indoles; D008024:Ligands; D043885:Receptor, Cannabinoid, CB2; D043882:Receptors, Cannabinoid; D043562:Receptors, G-Protein-Coupled; C094023:iodopravadoline