PMID- 12855000
OWN - NLM
STAT- MEDLINE
DCOM- 20030827
LR  - 20170214
IS  - 1087-0571 (Print)
IS  - 1087-0571 (Linking)
VI  - 8
IP  - 1
DP  - 2003 Feb
TI  - Evaluation of an imaging platform during the development of a FRET protease 
      assay.
PG  - 72-80
AB  - 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 biologicals 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)-NH(2) 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 70000 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 
      PMT-based 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.
FAU - George, Jeanette
AU  - George J
AD  - Amersham Biosciences UK Limited, United Kingdom.
FAU - Teear, Michelle L
AU  - Teear ML
FAU - Norey, Christopher G
AU  - Norey CG
FAU - Burns, D Dougal
AU  - Burns DD
LA  - eng
PT  - Journal Article
PL  - United States
TA  - J Biomol Screen
JT  - Journal of biomolecular screening
JID - 9612112
RN  - EC 3.4.24.17 (Matrix Metalloproteinase 3)
SB  - IM
MH  - Fluorescence
MH  - Humans
MH  - Matrix Metalloproteinase 3/*analysis
EDAT- 2003/07/12 05:00
MHDA- 2003/08/28 05:00
CRDT- 2003/07/12 05:00
PHST- 2003/07/12 05:00 [pubmed]
PHST- 2003/08/28 05:00 [medline]
PHST- 2003/07/12 05:00 [entrez]
AID - 10.1177/1087057102239778 [doi]
PST - ppublish
SO  - J Biomol Screen. 2003 Feb;8(1):72-80. doi: 10.1177/1087057102239778.