qHTS for Inhibitors of the Phosphatase Activity of Eya2: Confirmatory Assay 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: 462
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). Inhibitors found against Eya2 could serve as potential therapeutic tools to inhibit Six1-mediated breast tumorigenesis and to increase the efficiency of radiation and certain chemo therapy. This assay assessed the activity of the primary screen cherry-picked compounds against Eya2. Inhibition is the desired outcome for this assay.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA030559
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
Eya2 enzyme and 3-O-methyl-fluorescein phosphate (OMFP) substrate working solutions were reconstituted in the buffer reagent consist of 50 mM HEPES pH 7.5, 50 mM NaCl, 5 uM MgCl2, 0.05% BSA and 1 mM DTT. One and a half microliter of 0.2 uM Eya2 working solution was dispensed in a 1536 well Greiner plate (black, medium bind). Twenty-three nanoliter of test compounds and control compound EGTA was transferred to the assay plate using the Kalypsys robotic system equipped with a 1536 pin head. The plate was incubated at room temperature for 10 minutes. One and a half microliter of the 50 uM the OMFP substrate was then dispensed to the assay plate and incubated at room temperature for additional 10 min. The fluorescence signal was read using the PerkinElmer ViewLux plate reader using a 485 nm excitation and 525 nm emission filters. The data was normalized to the positive control 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)