qHTS Assay for Inhibitors of the Six1/Eya2 Interaction
The Six1 homeobox gene encodes a transcription factor that is crucial for the development of many organs but is down-regulated after organ development is complete. Its expression is low or undetectable in normal adult breast tissue but the gene is over-expressed in 50% of primary breast tumors and 90% of metastatic lesions. Our analysis of public microarray databases containing 535 breast more ..
BioActive Compounds: 2025
The Six1 homeobox gene encodes a transcription factor that is crucial for the development of many organs but is down-regulated after organ development is complete. Its expression is low or undetectable in normal adult breast tissue but the gene is over-expressed in 50% of primary breast tumors and 90% of metastatic lesions. Our analysis of public microarray databases containing 535 breast cancer samples demonstrates that Six1 levels correlate significantly with shortened time to relapse, shortened time to metastasis, and decreased survival. Using transgenic and xenograft mouse models, we have demonstrated that mammary specific overexpression of Six1 results in enhanced proliferation, transformation, increased tumor volume, and metastasis. Importantly, RNA interference against Six1 significantly decreases breast cancer associated metastasis. Together these data demonstrate that Six1 is necessary and sufficient to induce breast cancer metastasis. Six1 does not have an intrinsic activation or repression domain and requires the Eya coactivator proteins to activate transcription. Similar to Six1, Eya2 is also not expressed in most normal adult tissues, but is reexpressed in cancers. The Six1-Eya interaction is essential for proliferation during embryonic development, and both Six1 and Eya2 have been independently implicated in the same types of cancer. Because Six1 and Eya2 are embryonic genes with little expression in adult tissues, inhibitors of their expression/activity are likely to have limited side effects. Our goal is to identify inhibitors of the Six1/Eya interaction for potential cancer therapy.
To this end, we have developed a homogenous AlphaScreen assay targeting the Six1/Eya interaction. We performed a large scale HTS of the Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the Six1/Eya interaction as potential anti-cancer therapeutics. Compounds identified can also be used as chemical probes to better understand the biological functions of Six1 and Eya.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA033147
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
The first step of the 5 uL AlphaScreen assay is to dispense assay buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.05% BSA, 0.02% Tween 20, 1 mM TCEP) into a Greiner 1536 white solid bottom microtiter plate (789175-F). Five microliters is added to Column 1, 4 uL is added to Column 2 and 3 uL is added to Columns 3 through 48. A 5X protein solution (250 nM GST-Six1 protein + 500 nM His-tagged Eya2 protein) is mixed and 1 uL is dispensed to Columns 3-48. Using a 1536 pintool, 23 nL of compound is transferred to the assay plate. A solution of 5X AlphaScreen beads is mixed (50 ug/mL Glutathione linked donor beads + Nickel chelated acceptor beads) and 1 uL is dispensed to Columns 2-48. The assay plate is incubated for one hour, with a lid, at 37 deg C in the dark. The plate is read using a Perkin Elmer Envision using an AlphaScreen protocol.
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.
Data Table (Concise)