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 homogenous proximity assays, and (c)
counter-screens and other strategies to classify compound-mediated assay
interferences in homogenous proximity assays. This information should
be useful for prioritizing bioactive compounds in homogenous proximity
assays for drug and chemical probe discovery