LAUSR

HIGHLIGHTSTruncated variants of STAT3 allowed the development of conventional STAT3 thermal stability assays.Thermal denaturing of STAT3 can be tracked using Sypro OrangeTM fluorescence, tryptophan fluorescence or light scattering.Novel thermal stability assays confirm that peptide STAT3 inhibitors bind specifically to the STAT3 SH2 domain.Thermal stability assays show that reported small molecule STAT3 inhibitors may not specifically bind the STAT3 SH2 domain.High‐throughput STAT3 thermal stability assays could help to accelerate the development of new STAT3 inhibitors. ABSTRACT STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti‐cancer activity. Because STAT3 signaling is mediated by protein‐protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein‐inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided.