title: Molecular-interaction and signaling profiles of AM3677, a novel covalent agonist selective for the cannabinoid 1 receptor.
abstract: The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.
authors.affiliation: †Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Janero
authors.affiliation: †Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Suma
authors.initials: S
authors.lastname: Yaddanapudi
authors.affiliation: †Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Zvonok
authors.affiliation: †Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Kumar V
authors.initials: KV
authors.lastname: Subramanian
authors.affiliation: †Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Vidyanand G
authors.initials: VG
authors.lastname: Shukla
authors.affiliation: ‡Departments of Molecular Therapeutics and Neuroscience, Scripps Research Institute, Jupiter, Florida 33458, United States.
authors.forename: Edward
authors.initials: E
authors.lastname: Stahl
authors.affiliation: ‡Departments of Molecular Therapeutics and Neuroscience, Scripps Research Institute, Jupiter, Florida 33458, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.affiliation: §Center for Drug Discovery, University of North Carolina at Greensboro, Greensboro, North Carolina 27402, United States.
authors.forename: Dow
authors.initials: D
authors.lastname: Hurst
authors.affiliation: ∥Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana 47405, United States.
authors.forename: James
authors.initials: J
authors.lastname: Wager-Miller
authors.affiliation: ‡Departments of Molecular Therapeutics and Neuroscience, Scripps Research Institute, Jupiter, Florida 33458, United States.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Bohn
authors.affiliation: §Center for Drug Discovery, University of North Carolina at Greensboro, Greensboro, North Carolina 27402, United States.
authors.forename: Patricia H
authors.initials: PH
authors.lastname: Reggio
authors.affiliation: ∥Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana 47405, United States.
authors.forename: Ken
authors.initials: K
authors.lastname: Mackie
authors.affiliation: †Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Makriyannis
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000494:pharmacology*; D063386:Cannabinoid Receptor Agonists / Q000737:chemistry / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004705:Endocytosis / Q000187:drug effects; D057809:HEK293 Cells; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D017879:Isothiocyanates / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D009154:Mutation; D011869:Radioligand Assay; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C505563:20-isothiocyanatoeicosa-5,8,11,14-tetraenoic acid cyclopropylamide; D001095:Arachidonic Acids; C571130:CNR1 protein, human; D063386:Cannabinoid Receptor Agonists; D017879:Isothiocyanates; D043884:Receptor, Cannabinoid, CB1; D005576:Colforsin; D000242:Cyclic AMP
keywords: 7-transmembrane receptor; Amino acid; G protein-coupled receptor; binding domain; central nervous system; chemical probe; cysteine; homology modeling; isothiocyanate; ligand-binding motif; receptor activation; signal transduction