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 Citations (Scopus)

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

Fingerprint

Fluorescence Polarization
Neoplasm Genes
Assays
Genes
Fluorescence
Throughput
Polarization
Breast Neoplasms
Molecules
Phosphoserine
Cell Cycle
Screening
BRCA1 Protein
Cells
Phosphothreonine
G2 Phase
Nuclear Proteins
Dimethyl Sulfoxide
Phenylalanine
Cell Division

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1. / Lokesh, G. L.; Rachamallu, Aparna; Kumar, G. D.Kishore; Natarajan, Amarnath.

In: Analytical Biochemistry, Vol. 352, No. 1, 01.05.2006, p. 135-141.

Research output: Contribution to journalArticle

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