title: Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor.
abstract: The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH2 (Eu-DTPA-PEGO-CCK4), a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe was prepared by solid phase synthesis. A Kd value of 17±2nM was determined by means of saturation binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties, and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-throughput screening of compounds in development targeted to cholecystokinin receptors.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0041, USA.
authors.forename: N G R Dayan
authors.initials: NG
authors.lastname: Elshan
authors.affiliation: Department of Physiology, University of Arizona, Tucson, AZ 85724-5051, USA.
authors.forename: Thanuja
authors.initials: T
authors.lastname: Jayasundera
authors.affiliation: Department of Physiology, University of Arizona, Tucson, AZ 85724-5051, USA.
authors.forename: Craig S
authors.initials: CS
authors.lastname: Weber
authors.affiliation: Department of Physiology, University of Arizona, Tucson, AZ 85724-5051, USA; The Bio5 Institute, University of Arizona, Tucson, AZ 85721-0240, USA.
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Lynch
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0041, USA. Electronic address: emash@email.arizona.edu.
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Mash
mesh_terms: D001667:Binding, Competitive; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D043887:Receptor, Cholecystokinin B / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D043887:Receptor, Cholecystokinin B
keywords: Cholecystokinin 2 receptor; Dissociation-enhanced lanthanide fluoroimmunoassay; High-throughput screening; Multivalent binding; Time-resolved fluorescence