qHTS Assay for Inhibitors of the CtBP/E1A Interaction
Carboxyl-terminal binding protein (CtBP) is a co-repressor for many transcription factors. CtBP was initially recognized as an adenoviral E1A-binding protein and its over-activation, in combination with mutated Ras, leads to tumorigenesis and metastasis, suggesting CtBP plays a critical role in oncogenesis. Our studies have shown that CtBP is overexpressed in multiple human cancers, including more ..
BioActive Compounds: 1652
Carboxyl-terminal binding protein (CtBP) is a co-repressor for many transcription factors. CtBP was initially recognized as an adenoviral E1A-binding protein and its over-activation, in combination with mutated Ras, leads to tumorigenesis and metastasis, suggesting CtBP plays a critical role in oncogenesis. Our studies have shown that CtBP is overexpressed in multiple human cancers, including lung, breast, and head and neck cancers. In particular, it is over-expressed in 50% of primary lung cancer and 90% of metastatic lesions. CtBP suppresses epithelial-specific genes as well as genes critical for apoptosis, providing the underlying molecular mechanism for its role in tumorigenesis. Moreover, we found that CtBP-knockdown in lung cancer cells induced p53-independent apoptosis, and suppressed human tumor growth in mouse model. Based on the above data, we hypothesize that 1) CtBP suppresses the pro-apoptotic and epithelial genes, therefore overexpressed CtBP in human cancer cells increases EMT and cancer survival; 2) decreasing CtBP's function represents a targeted therapy for lung cancer and potentially multiple cancer types. As a co-repressor, CtBP binds to E1A and other transcription factors through a conserved peptide motif to carry out its action. Inhibiting this interaction using small molecules can potentially serve as effective cancer therapeutics.
To this end, we have developed a homogenous AlphaScreen assay targeting the CtBP/E1A interaction. We performed a large scale HTS of the Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the CtBP/E1A interaction as potential anti-cancer therapeutics. Compounds identified can also be used as chemical probes to better understand the biological functions of CtBP.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA033982
Assay Submitter (PI): Rui Zhao, University of Colorado Denver
The first step of the 5uL 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 (125 nM GST-E1A protein + 125 nM His-tagged CtBP 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 and the plate is incubated with a lid at 37 deg C in the dark for two hours. 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)