Medicinal Chemistry & Chemical Biology Group, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
Chembiochem. 2012 Jul 9;13(10):1503-8. doi: 10.1002/cbic.201200128. Epub 2012 Jun 12.
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.
细胞成像已成为揭示生物学复杂性和加速药物发现过程的重要工具，包括基于细胞的筛选、靶标识别和作用机制研究。最近，半导体纳米粒子（量子点，简称 QD）因其独特的光物理性质，如大小、可调光学性质、多重检测能力和光稳定性，在细胞成像应用中引起了极大的兴趣。在此，我们展示了 QD 也可应用于测定法开发，最终应用于高通量/大容量筛选（HTS/HCS）以进行药物发现。我们合成了 PEG 修饰的 QD 和一抗，用作基于细胞的 HTS 系统的荧光探针。众所周知，G 蛋白偶联受体（GPCR）家族参与大多数主要疾病。因此，我们构建了人骨肉瘤（U2OS）细胞，这些细胞特异性过表达两种不同标记的 GPCR：流感血凝素（HA）肽标记的κ-阿片受体（κ-OR）和 GFP 标记的 A3 腺苷受体（A3AR）。在这项研究中，我们证明了：1）抗 HA 抗体偶联的 QD 可以特异性标记 HA 标记的 κ-OR；2）随后用 QD 标记的 GPCR 激动剂处理可以实时监测激动剂诱导的转位；3）QD 优异的发射光谱特性可以在一个细胞中同时检测两种 GPCR；4）QD-抗体偶联物的强大成像能力可以从高内涵细胞图像中获得可重复的定量数据。这些结果表明，目前基于 QD 的 GPCR 抑制剂筛选系统可以成为进一步药物筛选应用的有前途的平台。
