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

A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3). Oncotarget. 2018 Aug 24;9(66):32690-32701 Authors: Shih PC, Yang Y, Parkinson GN, Wilderspin A, Wells G 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 per...
Source: Oncotarget - Category: Cancer & Oncology Tags: Oncotarget Source Type: research