Discovery of Fluorescence Polarization Probe for the ELISA-Based Antagonist Screening of ?₁-Adrenergic Receptors.

ABSTRACT: High-throughput screening (HTS) of ligand library to find new active molecules for G protein-coupled receptors is still a major interest, as well as an actual challenge. Fluorescence polarization (FP) assay portrays an essential role in HTS; however, in many cases, it was restricted by the absence of FP probes, the narrow measurement window, and low signal-to-noise (S/N) ratio. Herein, based on the modification of our previous probe 1 (QFL), we discovered an FP probe 3 (QGGFL) for ?₁-adrenergic receptors (?₁-ARs), which has satisfactory fluorescence intensity, specific binding ability to receptors, and suitable fluorescence properties that were compatible with the filters in the FP system. Meanwhile, an "ELISA-like" strategy was designed for FP-based HTS assay in which proteins were adhered into a solid phase to improve the measurement window and S/N ratio. With fluorescent antagonist QGGFL and the ELISA strategy, we succeeded in establishing the first competitive binding FP assay for ?₁-AR antagonists as the alternative of the radioligand binding assay.

SUBMITTER: Ma Z 

PROVIDER: S-EPMC5066147 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature