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Use of Red-Shifted Dyes in a Fluorescence Polarization AKT Kinase Assay for Detection of Biological Activity in Natural Product Extracts

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ tammy.turek-etienne{at}spcorp.com

Ming Lei

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ

Joseph S. Terracciano

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ

Erik F. Langsdorf

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ

Robert W. Bryant

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ

Richard F. Hart

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ

Ann C. Horan

Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, NJ

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^TM or Cy5^TM), 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. (Journal of Biomolecular Screening 2004:52-61)

Key Words: fluorescence polarization • red-shifted Cy^TM dyes • kinase assay • natural product extracts

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