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An Ultra-High Throughput Screening Approach for an Adenine Transferase Using Fluorescence Polarization

Profiling and Screening Department, Aventis Pharma, Bridgewater, NJ

Shujaath Mehdi

CNS Disease Group, Aventis Pharma, Bridgewater, NJ

Indravadan Patel

Biotechnology Department, Aventis Pharma, Bridgewater, NJ

John Kawooya

Biotechnology Department, Aventis Pharma, Bridgewater, NJ

Madeline Judkins

Profiling and Screening Department, Aventis Pharma, Bridgewater, NJ

Weihua Zhang

Biotechnology Department, Aventis Pharma, Bridgewater, NJ

Katrina Diener

Biotechnology Department, Aventis Pharma, Bridgewater, NJ

Anthony Lozada

Profiling and Screening Department, Aventis Pharma, Bridgewater, NJ

Damien Dunnington

Profiling and Screening Department, Aventis Pharma, Bridgewater, NJ

We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated to adenine of the donor substrate through a C₆ spacer arm. As a result of the transfer of the adenine-containing moieties to the acceptor protein substrate, the rotational correlation time of the Texas Red conjugate increased, hence increasing the degree of fluorescence polarization. The pharmacological profile and kinetics of the enzyme measured according to the FP method were consistent with those determined previously by conventional analysis. We have successfully executed a 250,000-compound high throughput screening program based on the FP assay method. The quality and validity of the assay were verified by a variety of statistical analyses.

Journal of Biomolecular Screening, Vol. 5, No. 1, 31-37 (2000)
DOI: 10.1177/108705710000500107


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