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A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3)

Shih, PC; Yang, Y; Parkinson, GN; Wilderspin, A; Wells, G; (2018) A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3). Oncotarget , 9 (66) pp. 32690-32701. 10.18632/oncotarget.26013. Green open access

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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 STAT3127-688construct, 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-tonoise 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.

Type: Article
Title: A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3)
Open access status: An open access version is available from UCL Discovery
DOI: 10.18632/oncotarget.26013
Publisher version: https://doi.org/10.18632/oncotarget.26013
Language: English
Additional information: Copyright: Shih et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0 - http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: STAT3; signal transducer and activator of transcription; high-throughput; DNA-binding domain; fluorescence polarization
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharma and Bio Chemistry
URI: https://discovery.ucl.ac.uk/id/eprint/10055582
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