title: Chimeric, mutant orexin receptors show key interactions between orexin receptors, peptides and antagonists.
abstract: Orexin receptor antagonists are being investigated as therapeutic agents for insomnia and addictive disorders. In this study the interactions between the orexin receptors (orexin 1 receptor and orexin 2 receptor), orexin peptides, and small molecule orexin antagonists were explored. To study these phenomena, a variety of mutant orexin receptors was made and tested using receptor binding and functional assays. Domains of the two orexin receptors were exchanged to show the critical ligand binding domains for orexin peptides and representative selective orexin receptor antagonists. Results from domain exchanges between the orexin receptors suggest that transmembrane domain 3 is crucially important for receptor interactions with small molecule antagonists. These data also suggest that the orexin peptides occupy a larger footprint, interacting with transmembrane domain 1, the amino terminus and transmembrane domain 5 as well as transmembrane domain 3. Transmembrane domain 3 has been shown to be an important part of the small molecule binding pocket common to rhodopsin and β2-adrenergic receptors. Additional orexin receptor 2 point mutations were made based on the common arrangement of receptor transmembrane domains shown in the G-protein coupled receptor crystal structure literature and the impact of orexin 2 receptor residue threonine 135 on the ligand selectivity of the 2 orexin receptors. These data support a model of the orexin receptor binding pocket in which transmembrane domains 3 and 5 are prominent contributors to ligand binding and functional activity. The data also illustrate key contact points for ligand interactions in the consensus small molecule pocket of these receptors.
authors.affiliation: Neuroscience Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, USA. DTran2@its.jnj.com
authors.forename: Da-Thao
authors.initials: DT
authors.lastname: Tran
authors.forename: Pascal
authors.initials: P
authors.lastname: Bonaventure
authors.forename: Michael
authors.initials: M
authors.lastname: Hack
authors.forename: Taraneh
authors.initials: T
authors.lastname: Mirzadegan
authors.forename: Curt
authors.initials: C
authors.lastname: Dvorak
authors.forename: Michael
authors.initials: M
authors.lastname: Letavic
authors.forename: Nicholas
authors.initials: N
authors.lastname: Carruthers
authors.forename: Timothy
authors.initials: T
authors.lastname: Lovenberg
authors.forename: Steven W
authors.initials: SW
authors.lastname: Sutton
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D064446:Orexin Receptors; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D064446:Orexin Receptors; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins