Early identification of false positives in high-throughput screening for activators of p53-DNA interaction

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Mutant p53 proteins often lose their DNA-binding ability, hindering their tumor-suppressive functions.
• Reactivating latent p53 DNA binding is a promising strategy for developing novel cancer therapies.
• Identifying small molecules that restore p53 function is a critical challenge in oncology.

Purpose of the Study:

• To develop and validate a homogeneous fluorescent assay for characterizing the binding affinities of wild-type p53.
• To compare the efficacy of fluorescence correlation spectroscopy (FCS) and 2D-FIDA anisotropy for p53-DNA binding analysis.
• To establish a robust high-throughput screening (HTS) method for identifying p53-reactivating compounds.

Main Methods:

• Utilized DNA(*)spec(26) and the antibody PAb421 to differentiate between latent and activated p53 binding states.
• Employed fluorescence correlation spectroscopy (FCS) and 2D-FIDA anisotropy for quantitative binding affinity measurements.
• Implemented a novel data evaluation algorithm for hit validation in a 25,000-compound HTS campaign.

Main Results:

• Demonstrated high correlation between FCS and 2D-FIDA anisotropy in measuring p53 DNA binding affinities (K(D) ~25-30 nM).
• Observed a significant 10-fold increase in binding affinity with the addition of poly(dI-dC) and PAb421 (K(D) ~3.3 nM).
• 2D-FIDA anisotropy proved to be the most accurate readout, leading to its selection for HTS.

Conclusions:

• The developed homogeneous fluorescent assay, particularly with 2D-FIDA anisotropy, provides an accurate method for p53 binding studies.
• The HTS campaign successfully identified potential drug candidates by employing a rigorous hit qualification process.
• This approach has the potential to accelerate the discovery of p53-reactivating cancer therapeutics and reduce attrition rates in drug discovery.