Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3

Area of Science:

• Biochemistry
• Drug Discovery
• Assay Development

Background:

• Proteases are key targets in drug discovery, necessitating sensitive screening methods.
• Existing homogeneous protease assays lack the speed and sensitivity required for high-throughput screening.
• Caspases and viral proteases represent important therapeutic targets.

Purpose of the Study:

• To develop a novel homogeneous time-resolved fluorescence quenching assay for protease inhibition.
• To establish a sensitive and high-throughput compatible assay using caspase-3 as a model.
• To evaluate the assay's performance against commercial assays and for ADME studies.

Main Methods:

• Utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) approach.
• Designed a peptide substrate labeled with a europium chelate and a quencher.
• Developed a homogeneous assay where caspase-3 cleavage separates the quencher, restoring fluorescence.

Main Results:

• Achieved high sensitivity: 1 pg/microl for active caspase-3 and 200 pM for substrate.
• Successfully screened 9600 small-molecule compounds in a high-throughput format.
• Demonstrated utility in cell lysate-based absorption/distribution/metabolism/excretion (ADME) assays.
• Outperformed a commercial fluorescence caspase-3 assay.

Conclusions:

• The developed homogeneous time-resolved fluorescence quenching assay is sensitive and suitable for high-throughput screening.
• This assay technology offers a valuable tool for protease inhibitor drug discovery.
• The assay's adaptability for ADME studies enhances its utility in pharmaceutical research.