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Salicylates are interference compounds in TR-FRET assays
Ronan P Hanley1, Shanti Horvath1, Jianghong An2
1Department of Chemistry, University of Victoria, PO Box 3065 STN CSC, Victoria, BC V8W 3V6, Canada.
Bioorganic & Medicinal Chemistry Letters
|January 7, 2016
Summary
Salicylic acids, common in drug discovery screening libraries, were found to interfere with time-resolved Förster resonance energy transfer (TR-FRET) assays. This interference, likely due to interaction with the FRET donor, can lead to false positives in high-throughput screening.
Area of Science:
- Biochemistry
- Medicinal Chemistry
- Assay Development
Background:
- High-throughput screening (HTS) is vital for drug discovery, but assay interference from false positives wastes resources.
- Time-resolved Förster resonance energy transfer (TR-FRET) assays are widely used but susceptible to interfering compounds.
- Identifying interfering compounds is crucial to ensure the reliability of HTS results and focus on genuine drug leads.
Purpose of the Study:
- To identify common compound classes that interfere with TR-FRET assay readouts.
- To investigate the interference of salicylic acids, a prevalent chemical subclass in screening libraries, with TR-FRET assays.
- To elucidate the potential mechanism behind salicylate-induced interference in TR-FRET assays.
Main Methods:
- Utilized TR-FRET assays to study the PD1-PDL2 interaction.
- Screened various compound subclasses, including salicylic acids, for assay interference.
- Analyzed assay signal changes in the presence of salicylic acids to infer interference mechanisms.
Main Results:
- Salicylic acids were identified as a common compound subclass that interferes with TR-FRET assays.
- The interference was observed despite the specific target interaction being studied (PD1-PDL2).
- Preliminary data suggest the interference mechanism involves salicylate interaction with the cryptand-ligated europium FRET donor.
Conclusions:
- Salicylic acids pose a significant challenge in TR-FRET based HTS, potentially leading to false positives.
- The interaction of salicylic acid with the FRET donor is the likely cause of assay signal alteration.
- Awareness and mitigation strategies for salicylate interference are necessary for accurate drug discovery screening.
