Bractoppin

Preparation Method

For the direct binding assay, 10μl of labeled protein at a final concentration of 20nM was mixed with 10μl of test compound( Bractoppin ) and incubated on ice for 10 min. For the competitive assay, 10μl of labeled protein at a final concentration of 20nM was mixed with 2μM cognate peptide substrate at the EC80 concentration determined by prior titration using a 16-point serial dilution by direct-binding MST.

Reaction Conditions

0-10⁵nM Bractoppin

Applications

Bractoppin is a 414-Da compound, with nanomolar potency in displacing cognate BACH1 phosphopeptide substrate from the BRCA1 tBRCT as measured by MST（ IC50 0.074 μM）. Its predicted binding mode to BRCA1 tBRCT reveals favorable hydrophobic interactions in the hydrophobic cavity, as well as a T-shaped, pi-pi stacking interaction with Phe1662, that together significantly contribute toward its activity. Indeed, substitution of the phenyl ring at R1 in Bractoppin with a 4-pyridyl group (CCBT2082) decreased activity by 5-fold by affecting the stacking interaction with Phe1662.

Cell lines

HEK293 cells

Preparation Method

HEK293 cells stably harboring plasmids for Tet-inducible expression of tBRCT domains were seeded. Compound( Bractoppin )addition or inducible expression of BRCA1 tBRCT were performed as indicated, when cells were 30% confluent, before exposure to 1Gy irradiation.

Reaction Conditions

1-100uM Bractoppin for 7 days

Applications

In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51.

References:

[1]. Periasamy J, Kurdekar V, et,al.Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling. Cell Chem Biol. 2018 Jun 21;25(6):677-690.e12. doi: 10.1016/j.chembiol.2018.02.012. Epub 2018 Mar 29. PMID: 29606576; PMCID: PMC6015222.