title: Development of a highly selective allosteric antagonist radioligand for the type 1 cholecystokinin receptor and elucidation of its molecular basis of binding.
abstract: Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 cholecystokinin receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to the closely related type 2 cholecystokinin receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser(208) at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met(121) in TM3 and Arg(336) in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes.
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Maoqing
authors.identifier: http://orcid.org/0000-0002-0775-6762
authors.initials: M
authors.lastname: Dong
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Ashton M
authors.initials: AM
authors.lastname: Vattelana
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Polo C-H
authors.initials: PC
authors.lastname: Lam
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Orry
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Ruben
authors.initials: R
authors.lastname: Abagyan
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: Patrick M
authors.identifier: http://orcid.org/0000-0001-8902-2473
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.).
authors.forename: David R
authors.initials: DR
authors.lastname: Haines
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (M.D., L.J.M.); Department of Chemistry, Wellesley College, Wellesley, Massachusetts (A.M.V., D.R.H.); Molsoft LLC, La Jolla, California (P.C.-H.L., A.J.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C., P.M.S.) miller@mayo.edu.
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D003412:Cricetulus; D006801:Humans; D007211:Indoles / Q000737:chemistry* / Q000494:pharmacology; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D043886:Receptor, Cholecystokinin A / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D043887:Receptor, Cholecystokinin B / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D007211:Indoles; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; C103722:T 0632