title: Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation.
abstract: The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.
authors.affiliation: Center for Drug Discovery, Department of Chemistry and Chemical Biology, College of Science, Northeastern University , Boston, Massachusetts 02115-5000, United States.
authors.forename: Dennis W
authors.initials: DW
authors.lastname: Szymanski
authors.forename: Malvina
authors.initials: M
authors.lastname: Papanastasiou
authors.forename: Katja
authors.initials: K
authors.lastname: Melchior
authors.forename: Nikolai
authors.initials: N
authors.lastname: Zvonok
authors.forename: Richard W
authors.initials: RW
authors.lastname: Mercier
authors.forename: David R
authors.initials: DR
authors.lastname: Janero
authors.forename: Ganesh A
authors.initials: GA
authors.lastname: Thakur
authors.forename: Sangwon
authors.initials: S
authors.lastname: Cha
authors.forename: Billy
authors.initials: B
authors.lastname: Wu
authors.forename: Barry
authors.initials: B
authors.lastname: Karger
authors.forename: Alexandros
authors.initials: A
authors.lastname: Makriyannis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry*; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera
chemical_list: C507046:7'-Isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol; D000939:Epitopes; D008024:Ligands; D010455:Peptides; D043885:Receptor, Cannabinoid, CB2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D013759:Dronabinol; D003545:Cysteine