title: Molecular basis for benzodiazepine agonist action at the type 1 cholecystokinin receptor.
abstract: Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu(7.39) that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a "trigger" for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu(7.39), whereas the role of this residue was less clear for chemically distinct agonists.
authors.affiliation: From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
authors.forename: Kaleeckal G
authors.initials: KG
authors.lastname: Harikumar
authors.affiliation: From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Cawston
authors.affiliation: Molsoft LLC, La Jolla, California 92037.
authors.forename: Polo C H
authors.initials: PCH
authors.lastname: Lam
authors.affiliation: From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
authors.forename: Achyut
authors.initials: A
authors.lastname: Patil
authors.affiliation: Molsoft LLC, La Jolla, California 92037.
authors.forename: Andrew
authors.initials: A
authors.lastname: Orry
authors.affiliation: the Metabolic Pathways and Cardiovascular Therapy Area Unit, GlaxoSmithKline Inc., Research Triangle Park, North Carolina 27709.
authors.forename: Brad R
authors.initials: BR
authors.lastname: Henke
authors.affiliation: Molsoft LLC, La Jolla, California 92037,; the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92037, and.
authors.forename: Ruben
authors.initials: R
authors.lastname: Abagyan
authors.affiliation: the Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville 3052, Australia.
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.affiliation: the Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville 3052, Australia.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259,. Electronic address: miller@mayo.edu.
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001569:Benzodiazepines / Q000737:chemistry / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D012372:ROC Curve; D043886:Receptor, Cholecystokinin A / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D043887:Receptor, Cholecystokinin B / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C488719:GI 181771X; D050505:Mutant Proteins; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D011994:Recombinant Proteins; D001569:Benzodiazepines
keywords: Allosteric Regulation; Benzodiazepine Agonist; Binding; Biological Activity; Calcium Signaling; Cholecystokinin Receptor; G Protein-coupled Receptors (GPCR); Receptor Modification; Signaling