Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists.
Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.
Exelixis, South San Francisco, CA 94083-0511, USA. swu@exelixis.com
Xiang
X
Wu
J Fraser
JF
Glickman
Benjamin R
BR
Bowen
Matthew A
MA
Sills
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D008024:Ligands; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000099150:Receptor, Farnesoid X-Activated
D004268:DNA-Binding Proteins; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated