Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion.

Area of Science:

• Biochemistry
• Biotechnology
• Pharmacology

Background:

• Caspase proteases are crucial in apoptosis and drug discovery.
• Conventional fluorescence assays suffer from biological autofluorescence.
• Upconverting phosphors (UCPs) offer a solution by emitting light at near-infrared excitation, avoiding autofluorescence.

Purpose of the Study:

• To develop a novel, sensitive, and robust assay for detecting caspase-3 inhibitors.
• To eliminate autofluorescence interference in enzyme-activity assays.
• To enable efficient high-throughput screening of potential drug candidates.

Main Methods:

• Utilized UCPs as donors and a fluorophore as an acceptor on a caspase-3 substrate peptide.
• Incorporated a quencher molecule to attenuate fluorescence in intact peptides.
• Enzyme activity releases the fluorophore, enabling signal detection.

Main Results:

• The assay successfully detected and characterized a known caspase-3 inhibitor (Z-DEVD-FMK) with IC50 values around 13 nM.
• Achieved high signal-to-background ratios (approx. 100) due to autofluorescence and background reduction.
• Demonstrated applicability for high-throughput screening with reduced reagent consumption.

Conclusions:

• UCPs provide a viable method for fluorescence-quenching-based enzyme assays, eliminating autofluorescence.
• The developed assay is sensitive, robust, and suitable for high-throughput screening of caspase-3 inhibitors.
• This approach facilitates cost-effective instrumentation and reduced reagent usage in drug discovery.