Development of a Highly Selective Allosteric Antagonist Radioligand for the Type 1 Cholecystokinin Receptor and Elucidation of Its Molecular Basis of Binding

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 Ser208 at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met121 in TM3 and Arg336 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.

[1]  Adrien B. Larsen,et al.  Structure and dynamics of G-protein coupled receptors. , 2014, Advances in experimental medicine and biology.

[2]  J. Wess,et al.  Activation and allosteric modulation of a muscarinic acetylcholine receptor , 2013, Nature.

[3]  R. Abagyan,et al.  Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor* , 2013, The Journal of Biological Chemistry.

[4]  E. Hulme,et al.  GPCR activation: a mutagenic spotlight on crystal structures. , 2013, Trends in pharmacological sciences.

[5]  Cheng Zhang,et al.  High-resolution Crystal Structure of Human Protease-activated Receptor 1 Bound to the Antagonist Vorapaxar Hhs Public Access , 2022 .

[6]  Brian K Shoichet,et al.  Structure-based drug screening for G-protein-coupled receptors. , 2012, Trends in pharmacological sciences.

[7]  R. Abagyan,et al.  Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor* , 2012, The Journal of Biological Chemistry.

[8]  L. Miller,et al.  Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol , 2012, Journal of Lipid Research.

[9]  Ron O. Dror,et al.  High-resolution crystal structure of human Protease-Activated Receptor 1 bound to the antagonist vorapaxar , 2012, Nature.

[10]  M. Congreve,et al.  Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine. , 2011, Structure.

[11]  Ruben Abagyan,et al.  Molecular Basis of Secretin Docking to Its Intact Receptor Using Multiple Photolabile Probes Distributed throughout the Pharmacophore* , 2011, The Journal of Biological Chemistry.

[12]  Ruben Abagyan,et al.  GPCR 3D homology models for ligand screening: Lessons learned from blind predictions of adenosine A2a receptor complex , 2010, Proteins.

[13]  Ruben Abagyan,et al.  Elucidation of the molecular basis of cholecystokinin Peptide docking to its receptor using site-specific intrinsic photoaffinity labeling and molecular modeling. , 2009, Biochemistry.

[14]  L. Miller,et al.  Synthesis and in vitro characterization of radioiodinatable benzodiazepines selective for type 1 and type 2 cholecystokinin receptors. , 2009, Journal of medicinal chemistry.

[15]  Wendy A. Warr,et al.  ChEMBL. An interview with John Overington, team leader, chemogenomics at the European Bioinformatics Institute Outstation of the European Molecular Biology Laboratory (EMBL-EBI) , 2009, J. Comput. Aided Mol. Des..

[16]  Viktor Hornak,et al.  Helix Movement is Coupled to Displacement of the Second Extracellular Loop in Rhodopsin Activation , 2009, Nature Structural &Molecular Biology.

[17]  John P. Overington.  ChEMBL. An interview with John Overington, team leader, chemogenomics at the European Bioinformatics Institute Outstation of the European Molecular Biology Laboratory (EMBL-EBI). Interview by Wendy A. Warr. , 2009, Journal of computer-aided molecular design.

[18]  R. Abagyan,et al.  GPCR 3 D homology models for ligand screening : Lessons learned from blind predictions of adenosine A 2 a receptor complex , 2009 .

[19]  P. Sexton,et al.  Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor. , 2008, Bioorganic & medicinal chemistry letters.

[20]  L. Miller,et al.  Structural basis of cholecystokinin receptor binding and regulation. , 2008, Pharmacology & therapeutics.

[21]  L. Miller,et al.  Insights into the structural basis of endogenous agonist activation of family B G protein-coupled receptors. , 2008, Molecular endocrinology.

[22]  Maxim Totrov,et al.  Atomic Property Fields: Generalized 3D Pharmacophoric Potential for Automated Ligand Superposition, Pharmacophore Elucidation and 3D QSAR , 2007, Chemical biology & drug design.

[23]  R. Abagyan,et al.  Role of lysine187 within the second extracellular loop of the type A cholecystokinin receptor in agonist-induced activation. Use of complementary charge-reversal mutagenesis to define a functionally important interdomain interaction. , 2007, Biochemistry.

[24]  Christopher I. Bayly,et al.  Evaluating Virtual Screening Methods: Good and Bad Metrics for the "Early Recognition" Problem , 2007, J. Chem. Inf. Model..

[25]  T. Lybrand,et al.  Transmembrane segment peptides can disrupt cholecystokinin receptor oligomerization without affecting receptor function. , 2006, Biochemistry.

[26]  T. Lybrand,et al.  Novel benzodiazepine photoaffinity probe stereoselectively labels a site deep within the membrane-spanning domain of the cholecystokinin receptor. , 2006, Journal of medicinal chemistry.

[27]  M. Martín-Martínez,et al.  Combination of molecular modeling, site-directed mutagenesis, and SAR studies to delineate the binding site of pyridopyrimidine antagonists on the human CCK1 receptor. , 2005, Journal of medicinal chemistry.

[28]  L. Miller,et al.  Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling. , 2005, Biochemistry.

[29]  L. Miller,et al.  Distinct Molecular Mechanisms for Agonist Peptide Binding to Types A and B Cholecystokinin Receptors Demonstrated Using Fluorescence Spectroscopy* , 2005, Journal of Biological Chemistry.

[30]  L. Miller,et al.  Key Differences in Molecular Complexes of the Cholecystokinin Receptor with Structurally Related Peptide Agonist, Partial Agonist, and Antagonist , 2004, Molecular Pharmacology.

[31]  L. Miller,et al.  Heterodimerization of Type A and B Cholecystokinin Receptors Enhance Signaling and Promote Cell Growth* , 2003, Journal of Biological Chemistry.

[32]  L. Miller,et al.  Disulfide bond structure and accessibility of cysteines in the ectodomain of the cholecystokinin receptor: specific mono-reactive receptor constructs examine charge-sensitivity of loop regions. , 2003, Receptors & channels.

[33]  T. Lybrand,et al.  Moleular models for cholecystokinin-A receptor. , 2002, Pharmacology & toxicology.

[34]  T. Lybrand,et al.  Refinement of the conformation of a critical region of charge-charge interaction between cholecystokinin and its receptor. , 2002, Molecular pharmacology.

[35]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[36]  X. Dumont,et al.  Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S. , 2000, European journal of pharmacology.

[37]  B. Maigret,et al.  Arginine 197 of the cholecystokinin‐A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin , 2008, Protein science : a publication of the Protein Society.

[38]  B. Maigret,et al.  Arginine 336 and Asparagine 333 of the Human Cholecystokinin-A Receptor Binding Site Interact with the Penultimate Aspartic Acid and the C-terminal Amide of Cholecystokinin* , 1999, The Journal of Biological Chemistry.

[39]  S. Silvente-Poirot,et al.  Role of the extracellular domains of the cholecystokinin receptor in agonist binding. , 1998, Molecular pharmacology.

[40]  B. Maigret,et al.  Met-195 of the Cholecystokinin-A Receptor Interacts with the Sulfated Tyrosine of Cholecystokinin and Is Crucial for Receptor Transition to High Affinity State* , 1998, The Journal of Biological Chemistry.

[41]  Jie Ren,et al.  First Intracellular Loop of the Human Cholecystokinin-A Receptor Is Essential for Cyclic AMP Signaling in Transfected HEK-293 Cells* , 1997, The Journal of Biological Chemistry.

[42]  L. Miller,et al.  Molecular basis and species specificity of high affinity binding of vasoactive intestinal polypeptide by the rat secretin receptor. , 1996, The Journal of pharmacology and experimental therapeutics.

[43]  L. Miller,et al.  Relationship Between Native and Recombinant Cholecystokinin Receptors: Role of Differential Glycosylation , 1996, Pancreas.

[44]  S. Silvente-Poirot,et al.  A Segment of Five Amino Acids in the Second Extracellular Loop of the Cholecystokinin-B Receptor Is Essential for Selectivity of the Peptide Agonist Gastrin* , 1996, The Journal of Biological Chemistry.

[45]  H. Taniguchi,et al.  Pharmacological profile of T-0632, a novel potent and selective CCKA receptor antagonist, in vitro. , 1996, European journal of pharmacology.

[46]  H. Taniguchi,et al.  Pharmacological profile of T-0632, a novel potent and selective CCKA receptor antagonist, in vivo. , 1996, European journal of pharmacology.

[47]  Ruben Abagyan,et al.  ICM—A new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation , 1994, J. Comput. Chem..

[48]  R. Abagyan,et al.  Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. , 1994, Journal of molecular biology.

[49]  J. Ballesteros,et al.  Analysis and refinement of criteria for predicting the structure and relative orientations of transmembranal helical domains. , 1992, Biophysical journal.

[50]  L. Miller,et al.  Use of N,O-bis-Fmoc-D-Tyr-ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides. , 2009, International journal of peptide and protein research.

[51]  D Rodbard,et al.  Ligand: a versatile computerized approach for characterization of ligand-binding systems. , 1980, Analytical biochemistry.

[52]  N. Metropolis,et al.  Equation of State Calculations by Fast Computing Machines , 1953, Resonance.