This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by George, J. Articles by Dougal Burns, D. Search for Related Content PubMed PubMed Citation Articles by George, J. Articles by Dougal Burns, D. Social Bookmarking                   What's this?

Evaluation of an Imaging Platform during the Development of a FRET Protease Assay

Amersham Biosciences UK Limited, Buckinghamshire, UK

Michelle L. Teear

Amersham Biosciences UK Limited, Buckinghamshire, UK

Christopher G. Norey

Amersham Biosciences UK Limited, Buckinghamshire, UK, christopher.norey{at}uk.amershambiosciences.com

D. Dougal Burns

Amersham Biosciences UK Limited, Buckinghamshire, UK

Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from bi-ologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH₂ is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70,000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMTbased fluorescence plate reader and at the same time evaluated a charged couple device (CCD)—based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens. (Journal of Biomolecular Screening 2003:72-80)

Key Words: High-throughput screening • CyDye reagents • fluorescent resonance energy transfer • synthetic peptide substrates

Journal of Biomolecular Screening, Vol. 8, No. 1, 72-80 (2003)
DOI: 10.1177/1087057102239778


CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				T. Naqvi, A. Lim, R. Rouhani, R. Singh, and R. M. Eglen Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology J Biomol Screen, August 1, 2004; 9(5): 398 - 408. [Abstract] [PDF]