Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts.
J Biomol Screen
; 9(1): 52-61, 2004 Feb.
Article
in En
| MEDLINE
| ID: mdl-15006149
ABSTRACT
Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B or Cy5), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes.
Search on Google
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biological Factors
/
Proto-Oncogene Proteins
/
Protein Serine-Threonine Kinases
/
Fluorescence Polarization
/
Fluorescent Dyes
Type of study:
Diagnostic_studies
/
Prognostic_studies
Language:
En
Journal:
J Biomol Screen
Journal subject:
BIOLOGIA MOLECULAR
Year:
2004
Document type:
Article
Affiliation country:
United States