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Updated: Sep 8, 2025
Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays
Published on: August 9, 2024
STAT Protein Thermal Shift Assays to Monitor Protein-Inhibitor Interactions
Petar Iliev1, Danielle Hanke1, Brent D G Page1
1Faculty of Pharmaceutical Sciences, University of British Columbia, 2405 Wesbrook Mall, Vancouver, V6T 1Z3, Canada.
Abstract:
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.
Insights
Many STAT3 inhibitors are not specific. A new assay revealed common inhibitors non-specifically alkylate STAT proteins and other targets, highlighting the need for precise drug development tools.
Area of Science:
- Biochemistry
- Chemical Biology
- Molecular Biology
Background:
- Signal transducer and activator of transcription 3 (STAT3) is a crucial target in cancer therapy.
- Existing STAT3 inhibitors (STAT3i) may lack specificity, acting indirectly on STAT signaling pathways.
- Novel methods are needed to accurately assess drug target engagement for STAT proteins.
Purpose of the Study:
- To introduce the STAT protein thermal shift assay (PTSA) as a novel tool for assessing STAT protein interactions.
- To evaluate the target engagement of known STAT3 inhibitors using PTSA.
- To investigate the mechanism of action of STAT3 inhibitors, including potential off-target effects.
Main Methods:
- STAT protein thermal shift assay (PTSA) was employed to assess the binding of STAT3 inhibitors to STAT3 and STAT1.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein modifications.
- Mass spectrometry was utilized to identify the specific modifications and proteins affected by the inhibitors.
Main Results:
- PTSA revealed that STATTIC, BP-1-102, and Cpd188 destabilized both STAT3 and STAT1 proteins.
- Unique SDS-PAGE migratory patterns suggested covalent modifications of STAT proteins by these compounds.
- Mass spectrometry confirmed that these compounds non-specifically alkylated STAT proteins and the unrelated protein NUDT5.
Conclusions:
- The STAT protein thermal shift assay (PTSA) is an effective tool for investigating STAT protein interactions and target engagement.
- Several known STAT3 inhibitors exhibit non-specific alkylating activity, potentially leading to off-target effects.
- The findings underscore the importance of rigorous validation of STAT3 inhibitors and highlight the utility of PTSA for chemical biology research.
