Quantum Dot‐Based Screening System for Discovery of G Protein‐Coupled Receptor Agonists

Abstract

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.

Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors

“…[251][252][253] Many applications can avoid cellular delivery of QDs because several pathogen receptors are expressed on the outer membrane of cells. Those receptors can be targeted using ligands, [254][255][256] aptamers, 257,258 or antibodies. [259][260][261] Because primary antibodies are relatively expensive compared to secondaries, most fluorescence imaging methods rely on a combination of primaries for receptor binding and fluorophore-conjugated secondaries (against the primaries) for fluorescence staining.…”

Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging

“…This format is potentially useful for high-throughput screening of agonists for drug discovery (GPCRs are involved in several major diseases). 241 Spectral Imaging. Spectral imaging includes both the hyperspectral and multispectral varieties, and offers superior informing power compared with conventional optical filter-based imaging.…”

“…240 Lee and coworkers used confocal imaging with QDs to observe agonist-induced endocytosis of two types of tagged G protein-coupled receptors (GPCRs): (i) influenza hemagglutinin (HA) peptide-tagged k-OR and (ii) green fluorescent protein (GFP)-tagged A3AR (adenosine receptors). 241 These GPCRs are overexpressed on the membrane of human osteosarcoma cells. The k-OR receptor was labeled with QD-anti-HA (antibody) conjugates, and the QD and GFP PL signals allowed real-time parallel visualization (Fig.…”

“…This format is potentially useful for high-throughput screening of agonists for drug discovery (GPCRs are involved in several major diseases). 241…”

One step synthesis of quantum dot–magnetic nanoparticle heterodimers for dual modal imaging applications

“…1,2 In addition, QDs serve as substrates in reactions that lead to the introduction of surface functional groups that enable conjugation with various biomolecules. 3 This capability enhances their use as bioimaging agents 4,5 and biosensors. 6,7 Because of the high potential applications of QDs in a wide range of therapeutic situations, improvement in the efficiency of their binding to a wide range of biomolecules remains a challenge.…”