Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

• Published in: The Journal of biological chemistry Vol. 288; no. 29; pp. 21082 - 21095
• Main Authors: Harikumar, Kaleeckal G., Cawston, Erin E., Lam, Polo C.H., Patil, Achyut, Orry, Andrew, Henke, Brad R., Abagyan, Ruben, Christopoulos, Arthur, Sexton, Patrick M., Miller, Laurence J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.07.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Allosteric Regulation; Amino Acid Sequence; Animals; Benzodiazepine Agonist; Benzodiazepines - chemistry; Benzodiazepines - pharmacology; Binding; Biological Activity; Calcium Signaling; CHO Cells; Cholecystokinin Receptor; Cricetinae; Cricetulus; G Protein-coupled Receptors (GPCR); Models, Molecular; Molecular Sequence Data; Mutant Proteins - agonists; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Receptor Modification; Receptor, Cholecystokinin A - agonists; Receptor, Cholecystokinin A - chemistry; Receptor, Cholecystokinin A - metabolism; Receptor, Cholecystokinin B - chemistry; Receptor, Cholecystokinin B - metabolism; Recombinant Proteins - agonists; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; ROC Curve; Sequence Alignment; Signal Transduction; Signaling; Binding; Signaling; G Protein-coupled Receptors (GPCR); Benzodiazepine Agonist; Biological Activity; Allosteric Regulation; Receptor Modification; Cholecystokinin Receptor; Calcium Signaling
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.480715

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 Leu7.39 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 Leu7.39, whereas the role of this residue was less clear for chemically distinct agonists. Background: Cholecystokinin receptor type 1 (CCK1R) stimulates satiety. Results: Binding and activity of a CCK1R agonist/CCK2R antagonist are studied at wild-type and chimeric receptors, and ligand-guided model refinement is utilized. Conclusion: The small molecule agonist docking site is distinct from the antagonist site, with benzodiazepines docked with consistent pose, including approximation with Leu7.39. Significance: The molecular model and determinants for small molecule agonist action should facilitate drug development.