Modulation of Cell Surface Expression of Nonactivated Cholecystokinin Receptors Using Bivalent Ligand-Induced Internalization

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, USA.
Journal of Medicinal Chemistry (Impact Factor: 5.45). 03/2010; 53(7):2836-42. DOI: 10.1021/jm100135g
Source: PubMed


CCK(2) receptor antagonists potentiate pain relief by MOP receptor agonists. In an attempt to enhance this effect, we prepared bivalent ligands incorporating CCK(2) receptor antagonist and MOP receptor agonist pharmacophores. (9) Ligands with 16- to 22-atom spacers could simultaneously bind both receptors but provided no advantage in activity over individual ligands. We now examine the effect of these ligands on receptor internalization as a mechanism of receptor regulation. We prepared CHO cell lines expressing nonfluorescent halves (YN and YC) of yellow fluorescent protein attached to each receptor. Spatial approximation of constructs was needed to yield fluorescence. Monovalent MOP agonist 1 signaled normally and internalized the MOP receptor. Monovalent CCK(2) antagonist 2 did not stimulate receptor internalization. In the dual receptor-bearing cells, bivalent ligands 3a-c capable of simultaneously binding both receptors resulted in cell surface fluorescence and internalization of the fluorescent complex in a time- and temperature-dependent manner. Bivalent ligand 4 with spacer too short to occupy both receptors simultaneously yielded no signal. Receptor tethering with appropriate bivalent ligands can down-regulate signaling by moving a nonactivated receptor into the endocytic pathway.

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    • "However, bivalent ligands containing a mu agonist and a CCK2 antagonist did cause a BRET response, suggesting that they bound simultaneously to each component of the dimer to promote oligomerization. Similar results were reported by Harikumar et al. [117] in which a fluorescent complement assay was employed to measure dimer formation and in these studies, the authors were able to fine tune the linker size between the opioid and CCK pharmacophore to measures distances of the ligand parts needed to promote dimer formation. Thus, these studies indicate that dimer formation can be induced by single ligands designed to bind simultaneously to two different receptors and suggest an approach to designing novel drugs based on their selective interaction with oligomers. "
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