Sills Matthew A, Weiss Donna, Pham Quynhchi, Schweitzer Robert, Wu Xiang, Wu Jinzi J
Novartis Institute for Biomedical Research, Summit, NJ 07901, USA.
J Biomol Screen. 2002 Jun;7(3):191-214. doi: 10.1177/108705710200700304.
In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.
在当今的高通量筛选（HTS）环境中，越来越多的检测技术被常规用于先导化合物发现项目。由于其中几种技术具有相对广泛的适用性，人们常常面临选择哪种技术用于特定检测的问题。本研究的目的是解决在三种不同版本的HTS检测中筛选一组样品时是否会鉴定出相同化合物的问题。在此，使用闪烁邻近检测（SPA）、均相时间分辨荧光共振能量转移（HTR-FRET）和荧光偏振（FP）技术建立了三种不同版本的酪氨酸激酶检测。在本研究中，在激酶检测的每个版本中，对30000种化合物进行了初筛、去卷积和剂量反应实验评估。通过这项工作，在去卷积实验后鉴定出的活性化合物只有很小程度的重叠。当所有活性化合物随后在所有三种检测中进行分析时，在HTR-FRET和FP检测中分别鉴定出100种和101种活性化合物。相比之下，在激酶检测的SPA版本中鉴定出40种化合物，而所有这些化合物在HTR-FRET检测中均被检测到，只有35种在FP检测中具有活性。尽管在HTR-FRET和FP检测中获得的IC50值之间具有良好的相关性，但与SPA检测中获得的IC50值相关性较差。这些发现表明，根据所使用的检测技术，HTS可能会观察到显著差异，特别是在高命中率的检测中。
