In many forms of
cancer the
signal transducer and activator of transcription 3 (
STAT3) transcription factor remains constitutively active, driving
cancer survival and progression. The critical role of STAT3 in
tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the
DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant
STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the
DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3
DNA-binding domain inhibitors. Here, we report a modified
DNA-binding ELISA to incorporate recombinant
STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that
niclosamide inhibits STAT3 through interaction with the
DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying
DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the
DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors.