A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3)
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Po-Chang Shih1, Yiwen Yang1, Gary N. Parkinson1, Andrew Wilderspin1 and Geoffrey Wells1
1UCL School of Pharmacy, University College London, Bloomsbury, London WC1N 1AX, UK
Geoffrey Wells, email: [email protected]
Keywords: STAT3; signal transducer and activator of transcription; high-throughput; DNA-binding domain; fluorescence polarization
Received: March 27, 2018 Accepted: July 31, 2018 Published: August 24, 2018
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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