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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

Published on: March 10, 2017

Fluorescence spectroscopic profiling of compound libraries.

Anton Simeonov¹, Ajit Jadhav, Craig J Thomas

¹NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.

Journal of Medicinal Chemistry

|March 28, 2008

Summary

Many compounds used in high-throughput screening (HTS) exhibit fluorescence, leading to inaccurate results. Profiling over 70,000 samples revealed over 5% are highly fluorescent, impacting optical assays.

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Area of Science:

Chemical biology
Assay development
High-throughput screening (HTS)

Background:

Heterocyclic scaffolds and impurities in compound libraries often possess inherent chromo/fluorophoric properties.
These properties can interfere with optical detection methods in high-throughput screening (HTS), causing assay artifacts like false positives and negatives.
Understanding compound autofluorescence is critical for reliable HTS data analysis.

Purpose of the Study:

To systematically characterize the fluorescence profiles of a large compound library across relevant spectral regions for HTS.
To quantify the prevalence and intensity of native compound fluorescence and fluorescent impurities.
To assess the impact of spectral window selection on reducing assay interference.

Main Methods:

Utilized a quantitative high-throughput screening (qHTS) paradigm to assess fluorescence.
Tested over 70,000 samples across multiple concentrations (4-log range) in a 1,536-well format.
Normalized raw fluorescence data against common fluorophore standards (e.g., 4-methylumbelliferone) to determine fluorescence intensity as a function of concentration and spectral region.

Main Results:

Over 5% of the tested library members exhibited fluorescence exceeding 10 nM 4-methylumbelliferone.
A significant reduction in autofluorescence was observed by red-shifting the spectral detection window by approximately 100 nm.
Identified native compound fluorescence, fluorescent impurities, and novel fluorescent compounds within the library.

Conclusions:

Compound autofluorescence is a widespread issue in HTS libraries, potentially compromising assay reliability.
Strategic selection of spectral detection windows, such as red-shifting, can mitigate interference from compound fluorescence.
Fluorescence profiling data is essential for accurate interpretation of HTS results and for identifying problematic compounds.