Coussens Nathan P., Auld Douglas, Roby Philippe, Walsh Jarrod, Baell Jonathan B., Kales Stephen, Hadian Kamyar, Dahlin Jayme L.
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
Novartis Institutes for Biomedical Research, Cambridge, MA, USA
Homogeneous proximity assays are widely implemented in high-throughput screening (HTS) of small-molecule libraries for drug and probe discovery. Representative technologies include amplified luminescent proximity homogeneous assays (ALPHA, which is trademarked by PerkinElmer; also informally referred to as Alpha), Förster/fluorescence resonance energy transfer (FRET), time-resolved FRET (TR-FRET) and homogeneous time-resolved fluorescence (HTRF, which is trademarked by CisBio), bioluminescence resonance energy transfer (BRET), and scintillation proximity assays (SPA). While highly useful, these assay technologies are susceptible to a variety of technology-related compound-mediated interferences, most notably signal attenuation (e.g., through quenching, inner-filter effects, light scattering), signal emission (e.g., auto-fluorescence), and disruption of affinity capture components such as affinity tags and antibodies. These assays are also susceptible to more generalized compound-mediated interferences such as nonspecific reactivity and aggregation. This chapter describes (a) the basic principles of proximity assays, (b) common sources of compound-mediated assay interference in homogeneous proximity assays, and (c) counter-screens and other strategies to classify compound-mediated assay interferences in homogeneous proximity assays. This information should be useful for prioritizing bioactive compounds in homogeneous proximity assays for drug and chemical probe discovery.
均相邻近分析广泛应用于小分子文库的高通量筛选（HTS），以发现药物和探针。代表性技术包括放大发光邻近均相分析（ALPHA，由珀金埃尔默公司注册 trademark；也被非正式地称为 Alpha）、Förster/荧光共振能量转移（FRET）、时间分辨 FRET（TR-FRET）和均相时间分辨荧光（HTRF，由 CisBio 公司注册 trademark）、生物发光共振能量转移（BRET）以及闪烁邻近分析（SPA）。虽然这些分析技术非常有用，但它们容易受到各种与技术相关的化合物介导的干扰，最显著的是信号衰减（例如，通过猝灭、内滤效应、光散射）、信号发射（例如，自发荧光）以及亲和力捕获组件（如亲和标签和抗体）的破坏。这些分析还容易受到更普遍的化合物介导的干扰，如非特异性反应性和聚集。本章描述了（a）邻近分析的基本原理，（b）均相邻近分析中化合物介导的分析干扰的常见来源，以及（c）用于在均相邻近分析中分类化合物介导的分析干扰的反筛选和其他策略。这些信息对于在均相邻近分析中对生物活性化合物进行优先级排序以发现药物和化学探针应该是有用的。
