Reducing False Positives through the Application of Fluorescence Lifetime Technology: A Comparative Study Using TYK2...

Area of Science:

• Biochemistry
• Drug Discovery
• Biotechnology

Background:

• Fluorescence-based assays are standard for enzyme inhibitor identification in early drug discovery.
• Fluorescence intensity, while common, suffers from false-positive readouts.
• Confirming hits requires costly and time-consuming secondary assays.

Purpose of the Study:

• To evaluate fluorescence lifetime technology (FLT) as a superior alternative to time-resolved fluorescence resonance energy transfer (TR-FRET).
• To compare FLT with label-free methods like RapidFire mass spectroscopy (RF-MS).
• To assess FLT's efficacy in identifying inhibitors for tyrosine kinase 2 (TYK2).

Main Methods:

• Developed three equivalent assays targeting TYK2.
• Screened annotated compound sets using FLT and TR-FRET.
• Conducted cellular screening to validate inhibitor activity and downstream effects.

Main Results:

• FLT demonstrated a significant reduction in false-positive hits compared to TR-FRET.
• Identified compounds that directly interact with TYK2.
• Confirmed inhibition of downstream signal transducer and activator of transcription 4 (STAT4) phosphorylation.

Conclusions:

• FLT offers a more reliable readout than TR-FRET for enzyme inhibitor screening.
• FLT shows promise as a valuable tool in early-stage drug discovery, potentially reducing costs and time.
• FLT can effectively identify true inhibitors and their biological activity in cellular contexts.