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Intermolecular interactions were determined between a synthetic peptide corresponding to the third extracellular loop and several residues from the adjoining sixth and seventh transmembrane domains of the human cholecystokinin-1 receptor, CCK(1)-R(329-357), and the synthetic agonists Ace-Trp-Lys[NH(epsilon)CONH-o-(MePh)]-Asp-MePhe-NH(2) (GI5269) and the C1 N-isopropyl-N-(4-methoxyphenyl)acetamide derivative of 3-(1H-Indazol-3ylmethyl)-3-methyl-5-pyridin-3-yl-1,5-benzodiazepine (GI0122), using high-resolution nuclear magnetic resonance spectroscopy and computer simulations. Addition of the ligands to CCK(1)-R(329-357) in an aqueous solution of DPC micelles produced a number of intermolecular nuclear Overhauser enhancements (NOEs) to residues in TMs 6 and 7 of the receptor fragment. NOE-restrained molecular models of the GI5269 and GI0122/CCK(1)-R complexes provide evidence for overlapping ligand-binding sites for peptidic and nonpeptidic agonists. The proposed binding modes of GI5269 and GI0122 are supported by the structure-activity relationship of analogues and mutagenesis data for the CCK(1)-R selective antagonist L-364,718.

Analogs of the CCK-A receptor selective agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623) were prepared in which the lysine residue was replaced with L-4-aminophenylalanine and D-or L-3-aminophenylalanine. These new analogs were moderately potent antagonists of CCK-8 in the isolated guinea pig gallbladder with exceptional CCK-A receptor selectivity as evaluated in membrane preparations from CHO K1 cells stably transfected with human CCK-A and CCK-B receptors.

Three new analogues of N alpha-hydroxysulfonyl-[Nle28,31]CCK26-33 are reported in which the C-terminal L-Phe33 residue has been replaced by L-Leu, D-Phe or N-methyl-L-Phe. Biological evaluation in a series of binding and bioassays demonstrates that both L-stereochemistry and an aromatic side chain at position-33 are essential for full agonist activity. While the L-Leu33 and D-Phe33 analogues had reduced potencies in stimulating contraction of the guinea pig ileum or gall bladder, the D-Phe33 analogue was fourfold selective for the ileum. This latter analogue also exhibited apparent partial agonism in the rat pancreatic amylase release assay. The N-methyl-L-Phe33 analogue was almost equipotent to the parent analogue in all bioassays, suggesting that this modification might be useful for introducing enzymatic stability in CCK analogues.

The downfield shift of the tyrosyl proton resonances and an increased chemical shift difference between the resonances for the 2',6' and 3',5' hydrogens in a series of deamino-oxytocin analogs modified in the disulfide bridge provide evidence for aromatic-sulfur interactions in d6-dimethylsulfoxide solutions.