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

G. L. Lokesh, Aparna Rachamallu, G. D.Kishore Kumar, Amarnath Natarajan

Research output: Contribution to journalArticle

27 Scopus citations

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 G2 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 (Kd) 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.

Original languageEnglish (US)
Pages (from-to)135-141
Number of pages7
JournalAnalytical Biochemistry
Volume352
Issue number1
DOIs
StatePublished - May 1 2006

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Keywords

  • BRCT-BACH1 inhibitors
  • Fluorescence polarization assay
  • High-throughput screening

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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