Compound-Mediated Assay Interferences in Homogeneous Proximity Assays

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.