ACS Chem. Biol. 15 (7) 1842-1851 [2020-07-17; online 2020-07-02]
Signal transducer and activator of transcription (STAT) proteins have important biological functions; however, deregulation of STAT signaling is a driving force behind the onset and progression of inflammatory diseases and cancer. While their biological roles suggest that STAT proteins would be valuable targets for developing therapeutic agents, STAT proteins are notoriously difficult to inhibit using small drug-like molecules, as they do not have a distinct inhibitor binding site. Despite this, a multitude of small-molecule STAT inhibitors have been proposed, primarily focusing on inhibiting STAT3 protein to generate novel cancer therapies. Demonstrating that inhibitors bind to their targets in cells has historically been a very challenging task. With the advent of modern target engagement techniques, such as the cellular thermal shift assay (CETSA), interactions between experimental compounds and their biological targets can be detected with relative ease. To investigate interactions between STAT proteins and inhibitors, we herein developed STAT CETSAs and evaluated known STAT3 inhibitors for their ability to engage STAT proteins in biological settings. While potent binding was detected between STAT proteins and peptidic STAT inhibitors, small-molecule inhibitors elicited variable responses, most of which failed to stabilize STAT3 proteins in cells and cell lysates. The described STAT thermal stability assays represent valuable tools for evaluating proposed STAT inhibitors.