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In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/microl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
October 2004,
Clinical chemistry,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
May 2012,
Analytica chimica acta,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
March 2011,
Journal of biomolecular screening,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
December 2003,
Combinatorial chemistry & high throughput screening,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
April 1999,
Analytical biochemistry,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
June 2006,
Journal of pharmaceutical and biomedical analysis,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
January 2013,
Methods in enzymology,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
August 2014,
Acta pharmacologica Sinica,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
February 2000,
Analytical biochemistry,
Jarkko Karvinen, and
Pertti Hurskainen, and
Sujatha Gopalakrishnan, and
David Burns, and
Usha Warrior, and
Ilkka Hemmilä
January 2013,
Methods in molecular biology (Clifton, N.J.),
