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Early Identification of False Positives in High-Throughput Screening for Activators of p53-DNA Interaction

Evotec AG, Hamburg, Germany

Nicholas Hunt

Genteq GmbH, Hamburg, Germany

Joern Jungmann

Evotec AG, Hamburg, Germany

Dirk Ullmann

Evotec AG, Hamburg, Germany

Naturally occurring mutant forms of p53 are deficient for specific DNA binding. However, their specific DNA binding can be reactivated. The search for small molecules that reactivate latent p53 is considered to be a cornerstone in cancer therapy. The authors describe a new homogeneous fluorescent assay approach for the characterization of binding affinities of human wild-type latent and activated p53 using DNA^*spec(26), with and without the addition of the antibody PAb421, respectively, and fluorescence correlation spectroscopy (FCS)/2-dimensional fluorescence-intensity distribution analysis anisotropy as the detection methods. FCS was compared with 2D-FIDA anisotropy, and a very good correlation of the results with both readouts was observed (K_Ds for nonspecific DNA binding of 24.4 ± 3.5 nM with 2D-FIDA anisotropy and of 29.5 ± 5.5 nM with FCS). The presence of poly(dI-dC) led to a 10-fold increase of binding affinity (K_D of 3.3 ± 0.5 nM in the presence of PAb421). 2D-FIDA anisotropy was demonstrated to be the most accurate readout; hence, this detection technology was selected for a 25,000 compound member high-throughput screening (HTS) campaign. The hits obtained were qualified using a novel data evaluation algorithm that identifies false positives and moreover assesses the validity of true hits in the presence of the deteriorating artifact. This process step is of utmost importance for decreasing the attrition in fluorescence-based HTS.

Key Words: p53 activators • p53 protein purification • false positives • autofluorescent compounds • confocal spectroscopy • fluorescence polarization • high-throughput screening

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