qHTS Assay for Inhibitors of BRCT-Phosphoprotein Interaction (Red Fluorophore)
Inhibitors of the BRCT:pBACH1 interaction should prove useful in studies of BRCA1's tumor suppression role and to potentially sensitive tumors to chemotherapeutic agents. A complex of BRCT (C-terminal portion of BRCA1, MW ~35 kDa, His-tagged) and fluorescently labeled pBACH1 phosphorylated 10 aa peptide fragment of BACH1, a helicase implicated in DNA repair, is incubated with library members. more ..
BioActive Compounds: 20
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: MH081227-01
PI Name: Natarajan, Amarnath; University of Texas
Inhibitors of the BRCT:pBACH1 interaction should prove useful in studies of BRCA1's tumor suppression role and to potentially sensitive tumors to chemotherapeutic agents. A complex of BRCT (C-terminal portion of BRCA1, MW ~35 kDa, His-tagged) and fluorescently labeled pBACH1 phosphorylated 10 aa peptide fragment of BACH1, a helicase implicated in DNA repair, is incubated with library members. Inhibitors of BRCT-pBACH1 binding are detected by a decrease in the fluorescence polarization (FP) of the fluorophore. In order to minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the compound library members were incubated in separate reactions with rhodamine-labeled probe-protein complexes. Protein and labeled probes were supplied by Dr. Amarnath Natarajan, Department of Pharmacology and Toxicology, University of Texas Medical Branch Galveston, TX.
Assay Buffer: 16mM Na2HPO4, 4 mM NaH2PO4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.02% NaN3, and 0.01% Tween 20 at pH 7.3.
Assay Controls: 100 nM TAMRA labeled decapeptide dispensed into columns 3 and 4 to generate negative control (full inhibition of
binding, -100 % activity). 100 nM labeled decapeptide/100 nM protein in columns 1, 2, 5 - 48. Titration of unlabeled decapeptide control (from 10 mM, then 1:2 dilution in duplicate) pin-transferred to column 2, rows 1 to 16. Column 1 is neutral.
Three uL of reagents were dispensed to 1536-well Greiner black solid-bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 12 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using BODIPY for the rhodamine-labeled decapeptide probe polarization filter sets. During dispense, reagent bottles were kept submerged into 4 deg C water bath and all liquid lines were covered with aluminum foil to minimize probe and protein degradation. All screening operations were performed on a Kalypsys robotic system (Kalypsys, Inc., San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms. The timing and order of assay plates passing through the screening system were adjusted such that each compound library plate was assayed against the fluorescein- and rhodamine-labeled assay systems at immediately adjacent time points.
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, brct, brca1
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)