Abstract |
Anti- cancer drug discovery efforts to directly inhibit the signal transducer and activator of transcription 3 (STAT3) have been active for over a decade following the discovery that 70% of cancers exhibit elevated STAT3 activity. The majority of research has focused on attenuating STAT3 activity through preventing homo-dimerization by targeting the SH2 or transcriptional activation domains. Such dimerization inhibitors have not yet reached the market. However, an alternative strategy focussed on preventing STAT3 DNA-binding through targeting the DNA-binding domain (DBD) offers new drug design opportunities. Currently, only EMSA and ELISA-based methods have been implemented with suitable reliability to characterize STAT3 DBD inhibitors. Herein, we present a new orthogonal, fluorescence polarization (FP) assay suitable for high-throughput screening of molecules. This assay, using a STAT3127-688 construct, was developed and optimized to screen molecules that attenuate the STAT3: DNA association with good reliability (Z' value > 0.6) and a significant contrast (signal-to-noise ratio > 15.0) at equilibrium. The assay system was stable over a 48 hour period. Significantly, the assay is homogeneous and simple to implement for high-throughput screening compared to EMSA and ELISA. Overall, this FP assay offers a new way to identify and characterize novel molecules that inhibit STAT3: DNA association.
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Authors | Po-Chang Shih, Yiwen Yang, Gary N Parkinson, Andrew Wilderspin, Geoffrey Wells |
Journal | Oncotarget
(Oncotarget)
Vol. 9
Issue 66
Pg. 32690-32701
(Aug 24 2018)
ISSN: 1949-2553 [Electronic] United States |
PMID | 30220975
(Publication Type: Journal Article)
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