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Assay Development
Assay Development:
Identification of a screening target and development of an assay format and methodology with the
eventual goal of assay miniaturization into a format suitable for HTS.
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Chemistry validation/confirmationChemistry validation/confirmation: The structure and activity of
HTS hits are validated by various methods, including obtaining dry compound from a vendor or the
MLSMR for re-analysis, confirming the compound structure using LC-MS and/or NMR, etc., and
direct re-synthesis of the compound.
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Cherry pickingCherry picking: Selection of individual compounds from a compound
library for further analysis.
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Confirmatory AssaysConfirmatory Assays: Includes replicate runs and dose response using
the primary screening assay to confirm primary hits. This process may involve “cherry picking”
hits from primary screen.
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Hit“Hit”: A compound that yielded a positive response in a
primary HTS assay. Hits are confirmed or rejected via subsequent confirmatory and secondary assays
to determine whether they are true hits.
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HTS ImplementationHTS Implementation:
Following the assay development phase, implementation involves HTS modification, miniaturization,
and optimization of the assay for HTS. Metrics used to evaluate and HTS assay’s
performance include the Z’ factor, signal-to-noise and signal-to-background ratio,
and intra- and inter-day experiment variability
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HTS ValidationHTS Validation: Replicate pilot screens of small compound libraries
to evaluate a newly-optimized HTS assay’s performace before large-scale screens.
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Primary screening Primary screening: Initial screen of compound library,
usually at a single dose, to identify active compounds
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Profiling Assays and Counterscreens Profiling Assays and Counterscreens:
Using an HTS assay to screen a related target (e.g. other members of receptor or kinase family) to
evaluate the selectivity/specificity of primary HTS hit compounds
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Secondary bioassays Secondary bioassays: measure activity of target in different assay
than primary screen, assess agonism vs antagonism, confirm rank order of
compound potencies/affinities (e.g. primary: binding assay; secondary: signaling assay)
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Z’ factorZ’ factor: A metric used to determine the robustness of an
HTS assay. The Z’ factor is calculated as follows using the mean (M) and standard deviation
(SD) of the positive control and background values:
A Z’ factor greater than 0.5 is considered suitable for HTS.
