title: 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+/-0.01microM and a dynamic range (DeltamP) 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.
authors.affiliation: Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: G L
authors.initials: GL
authors.lastname: Lokesh
authors.forename: Aparna
authors.initials: A
authors.lastname: Rachamallu
authors.forename: G D Kishore
authors.initials: GD
authors.lastname: Kumar
authors.forename: Amarnath
authors.initials: A
authors.lastname: Natarajan
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019313:BRCA1 Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism; D005260:Female; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015927:Skin Test End-Point Titration
chemical_list: D000970:Antineoplastic Agents; C102108:BACH1 protein, human; D019313:BRCA1 Protein; D050976:Basic-Leucine Zipper Transcription Factors; D051856:Fanconi Anemia Complementation Group Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins