High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1

Abstract

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G 2 phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)–BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide ( BDP1) containing the critical phosphoserine, a phenylalanine at (P + 3), and a GST–BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant ( K d) of 1.58 ± 0.01 μM and a dynamic range (ΔmP) of 164.9 ± 1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z′ factor of 0.87 was achieved in a 384-well format for high-throughput screening.