title: Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.
abstract: Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [3H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [3H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Han
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Aneetha
authors.initials: A
authors.lastname: Halikhedkar
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Pusheng
authors.initials: P
authors.lastname: Fan
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: David R
authors.identifier: 0000-0002-4633-1038
authors.initials: DR
authors.lastname: Janero
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Ganesh A
authors.identifier: 0000-0002-7468-8819
authors.initials: GA
authors.lastname: Thakur
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Mercier
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Ma
authors.affiliation: Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Makriyannis
mesh_terms: D001665:Binding Sites; D063386:Cannabinoid Receptor Agonists / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists* / Q000737:chemistry*
chemical_list: C588309:CNR2 protein, human; D063386:Cannabinoid Receptor Agonists; D008024:Ligands; D043885:Receptor, Cannabinoid, CB2; D003545:Cysteine
keywords: Binding motif; G-protein coupled receptor; cannabinoid receptor 2; covalent chemical probe; cysteine; isothiocyanate; ligand-binding domain; molecular probe; signal transduction; transmembrane helix