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

Po-Chang Shih,Yiwen Yang,Gary N. Parkinson,Andrew Wilderspin,Geoffrey Wells

doi:10.18632/oncotarget.26013

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.

Highlights

STAT3, signal transducer and activator of transcription 3, is a key component in several signalling pathways [1, 2], is over-activated in approximately 70% of cancers [3] and plays critical roles in cell proliferation, cell survival, angiogenesis, immune invasion and metastasis [4]
The conditions utilized in the subsequent STAT3127-688:DNA fluorescence polarization (FP) assays require the lowering of the salt and DTT concentrations by diafiltration using a 50 kDa concentrator to a final NaCl concentration < 200 μM
Since STAT3 is highly involved in oncogenic pathways, there has been significant research directed towards finding inhibitors that interact with STAT3 or its upstream effectors

Summary

Introduction

STAT3, signal transducer and activator of transcription 3, is a key component in several signalling pathways [1, 2], is over-activated in approximately 70% of cancers [3] and plays critical roles in cell proliferation, cell survival, angiogenesis, immune invasion and metastasis [4]. During the discovery of STAT3 dimerization inhibitors, numerous cell-free and cell-based assays were developed to validate the inhibitory effect of selective inhibitors on dimerization. The FP, AlphaScreenTM, ELISA and SPR assays are all applicable to high-throughput screening and are cell-free, while the remaining techniques are applicable to highthroughput approaches, and are cell-based The application of these assays as screening platforms resulted in the discovery and validation of STAT3 dimerization inhibitors JSI-124 [5], bendamustine [6], piperlongumine [8] and static [10]. The assays were utilized as tools to support the discovery of other STAT3 dimerization www.oncotarget.com inhibitors such as STX-0119 [9], STA-21 [11] and LLL-12 [12] which were all initially identified by in-silico highthroughput screening, and applied to LY5 [13], shikonin derivatives [14], “Compound 9” [15], HJC1-30 [24] and HJC0123 [16] and FLLL32 [17] (Figure 1) which were designed based on previously published chemical structures

Methods

Results

Conclusion