qHTS for Inhibitors of the Phosphatase Activity of Eya2: Carboxyl-terminal binding protein (CtBP) Counterscreen for Cherry-picked Compounds
The Eya protein family is an essential co-activator of the Six1 transcription factor. Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. The Eya family of protein is also found to contain a unique HAD phosphatase domain with protein tyrosine more ..
BioActive Compounds: 416
The Eya protein family is an essential co-activator of the Six1 transcription factor. Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. The Eya family of protein is also found to contain a unique HAD phosphatase domain with protein tyrosine phosphatase activity which can potentially play a role in Six1-mediated breast tumorigenesis. Recently, Eya2 is found to dephosphorylate the histone variant H2AX and direct cells towards DNA repair instead of apoptosis in the event of DNA damage.
A 1536 well format, fluorescence based qHTS assay was developed and used to target the phosphatase activity of Eya2 (AID 488837). In addition, a CtBP counterscreen was developed using the AlphaScreen technology to evaluate compound selectivity to target Eya1. The data herein reports the activity of the cherry-picked compounds against the CtBP AlphaScreen. Compounds that are active or have > 100 fold selectivity vs. the Eya2 activity were excluded for further characterization. Inactivity is the desired outcome for the CtBP counterscreen.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA030559
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
Four microliters of reagent (25 nM GST-E1A, 25 nM CtBP-6x HIS) was dispensed in a 1536 well Greiner plates. Twenty-three nanoliter of test compounds (final concentration of 2.5 nM - 46 uM) and positive control E1A peptide inhibitor (final concentration of 0.14 nM - 4.6 uM) were added to the plates using the Kalypsys pintool equipped with a 1536 pin head. The plates was then incubated at room temperature for 2 hours followed by addition of 1 uL Alpha bead donor / acceptor mix (20 ug/mL final concentration). The assay plates were incubated for additional 1 hour at room temperature and protected from light. The AlphaScreen signal was obtained using the PerkinElmer EnVision plate reader using the 680 nm excitation and 520 - 620 nm emission filters. The data was normalized to the E1A peptide inhibitor for maximum inhibition and DMSO treated wells as negative control.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)