STAT Protein Thermal Shift Assays to Monitor Protein‐Inhibitor Interactions

STAT3 protein is a sought‐after drug target as it plays a key role in the progression of cancer. Many STAT3 inhibitors (STAT3i) have been reported, but accumulating evidence suggests many of these act as off‐target/indirect inhibitors of STAT signaling. Herein, we describe the STAT protein thermal shift assay (PTSA) as a novel target engagement tool, which we used to test the binding of known STAT3i to STAT3 and STAT1. This revealed STATTIC, BP‐1‐102, and Cpd188 destabilized both STATs and produced unique migratory patterns on SDS‐PAGE gels, suggesting covalent protein modifications. Mass spectrometry experiments confirmed that these compounds are nonspecifically alkylating STATs, as well as an unrelated protein, NUDT5. These experiments have highlighted the benefits of PTSA to investigate interactions with STAT proteins and have helped reveal the novel reactivity of Cpd188. The described PTSA represents a promising chemical biology tool that could be applied to an array of other protein targets. STAT proteins are a family of multifaceted transcription factors whose deregulated activity is linked to diseases like cancer and arthritis. To interrogate a selection of STAT inhibitors, we have developed a protein thermal shift assay which serves as a steppingstone between biochemical thermal stability assays and cellular thermal shift assays.