HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.
HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.
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HTRF：一项针对药物发现量身定制的技术 - 对理论方面和最新应用的回顾。
DOI：
10.2174/1875397300903010022
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发表时间：
2009-05-28
期刊：
影响因子：
--
通讯作者：
Xie B
中科院分区：
文献类型：
--
作者：
Degorce F;Card A;Soh S;Trinquet E;Knapik GP;Xie B
HTRF（均相时间分辨荧光）是最常用的通用分析技术，用于以均相形式测量分析物，这是高通量筛选（HTS）中用于药物靶点研究的理想平台。该技术结合了荧光共振能量转移技术（FRET）和时间分辨测量（TR）。在TR-FRET测定中，当供体和受体分子彼此紧密接近时，通过供体和受体分子之间的荧光共振能量转移产生信号。缓冲液和介质干扰通过双波长检测显著降低，并且最终信号与产物形成的程度成比例。HTRF测定通常是灵敏和稳健的，可以小型化到384和1536孔板格式中。该检测技术已应用于许多基于抗体的检测，包括GPCR信号（cAMP和IP-One），激酶，细胞因子和生物标志物，生物过程（抗体和蛋白质生产），以及蛋白质-蛋白质，蛋白质肽和蛋白质-DNA/RNA相互作用的检测。自从十多年前HTRF被引入药物筛选领域以来，研究人员已经使用HTRF来加速GPCR、激酶、新生物标志物、蛋白质-蛋白质相互作用和其他感兴趣的靶点的研究。HTRF也被用作生物过程监测的替代方法。第一代HTRF技术使用铕穴状化合物作为荧光供体来监测生物分子之间的反应，2008年通过引入第二代供体铽穴状化合物（Tb）来扩展该技术，从而提高了筛选性能。与铕相比，铽穴状化合物具有不同的物理化学性质，包括增加的量子产率和更高的摩尔消光系数。除了与铕所用的相同受体荧光团兼容外，它还可以作为发绿光荧光团的供体荧光团，因为它具有多个发射峰，包括490 nm处的一个发射峰。此外，所有铽HTRF检测都可以在与铕HTRF检测相同的HTRF兼容仪器上读取。总体而言，HTRF是一种高度灵敏、稳健的体外分子相互作用检测技术，广泛用于药物开发的初级和二级筛选阶段。本文综述了HTRF的一般原理及其在药物发现中的应用。
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions. Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays. Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.