Fan Frank, Wood Keith V
Promega Corporation, Madison, WI 53711, USA.
Assay Drug Dev Technol. 2007 Feb;5(1):127-36. doi: 10.1089/adt.2006.053.
In the development of high throughput screening (HTS) as a central paradigm of drug discovery, fluorescence has generally been adopted as the favored methodology. Nevertheless, luminescence has maintained a prominent position among certain assay formats, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a wider range of applications due to their sensitivity, broad linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by the development of several new assay designs for diverse targets such as kinases, cytochrome p450s, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for HTS, highlighting new detection capabilities brought about by engineering luciferase genes, enzymes, and substrates. In genetic reporter applications, modifications to the luciferase genes have improved assay sensitivity by substantially increasing expression efficiency and enhanced response dynamics by reducing expression lifetime. The performance of assays based on detection of ATP and luciferin has been enhanced by modifications to the luciferase enzyme that increase its chemical and physical stability. Detection of ATP allows rapid analysis of cell metabolism and enzymatic processes coupled to ATP metabolism. Because luciferins are not naturally associated with mammalian physiology, assays for luciferin detection utilize synthetic derivatives designed to yield luminescence only when coupled with specific target enzymes. Finally, new methods for modulating the specific activity of luciferases are leading to the development of intracellular biosensors for dynamic detection of physiological processes.
在高通量筛选（HTS）作为药物发现核心范式的发展过程中，荧光通常被用作首选方法。然而，发光在某些检测形式中一直占据突出地位，最显著的是基因报告检测。最近，由于其灵敏度、宽线性范围以及对文库化合物和复杂生物样品的稳健性，发光检测在更广泛的应用中越来越受到青睐。针对多种靶点（如激酶、细胞色素P450、蛋白酶、细胞凋亡和细胞毒性）的几种新检测设计的开发推动了这一趋势。本综述阐述了在高通量筛选中使用生物发光检测所取得的最新进展，重点介绍了通过改造荧光素酶基因、酶和底物带来的新检测能力。在基因报告应用中，对荧光素酶基因的修饰通过大幅提高表达效率提高了检测灵敏度，并通过缩短表达寿命增强了响应动力学。基于ATP和荧光素检测的检测方法通过对荧光素酶进行修饰提高了其化学和物理稳定性，从而提升了性能。ATP检测可快速分析细胞代谢以及与ATP代谢相关的酶促过程。由于荧光素与哺乳动物生理学并无天然关联，荧光素检测方法利用的是仅在与特定靶酶偶联时才产生发光的合成衍生物。最后，调节荧光素酶比活性的新方法正促使开发用于动态检测生理过程的细胞内生物传感器。
