Schuck Brittany W, MacArthur Ryan, Inglese James
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland.
National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland.
Curr Protoc Neurosci. 2017 Apr 10;79:5.32.1-5.32.27. doi: 10.1002/cpns.27.
Reporter-biased artifacts-i.e., compounds that interact directly with the reporter enzyme used in a high-throughput screening (HTS) assay and not the biological process or pharmacology being interrogated-are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe and therapeutic development. Furthermore, narrow or single-concentration HTS perpetuates false negatives during primary screening campaigns. Titration-based HTS, or quantitative HTS (qHTS), and coincidence reporter technology can be employed to reduce false negatives and false positives, respectively, thereby increasing the quality and efficiency of primary screening efforts, where the number of compounds investigated can range from tens of thousands to millions. The three protocols described here allow for generation of a coincidence reporter (CR) biocircuit to interrogate a biological or pharmacological question of interest, generation of a stable cell line expressing the CR biocircuit, and qHTS using the CR biocircuit to efficiently identify high-quality biologically active small molecules. © 2017 by John Wiley & Sons, Inc.
报告偏倚性假象——即直接与高通量筛选（HTS）分析中使用的报告酶相互作用，而非与正在研究的生物学过程或药理学相互作用的化合物——如今已被广泛认识到会降低用于化学探针和治疗药物开发的HTS的效率和质量。此外，窄范围或单一浓度的HTS会在初次筛选过程中持续产生假阴性结果。基于滴定的HTS，即定量HTS（qHTS），以及巧合报告技术可分别用于减少假阴性和假阳性，从而提高初次筛选工作的质量和效率，在此过程中所研究的化合物数量可从数万到数百万不等。这里描述的三种方案可用于生成一个巧合报告（CR）生物电路以探究感兴趣的生物学或药理学问题、生成表达CR生物电路的稳定细胞系，以及使用CR生物电路进行qHTS以有效鉴定高质量的生物活性小分子。© 2017约翰威立国际出版公司
