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Published on: September 13, 2014
Validating Signal Transducer and Activator of Transcription (STAT) Protein-Inhibitor Interactions Using Biochemical
Sanaz Attarha1,2, Anja Reithmeier2,3,4, Sander Busker3
1Department of Oncology and Pathology, Karolinska Institutet, 171 65, Karolinska vägen A2:07, Solna 171 64, Sweden.
Abstract:
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.
Insights
Evaluating STAT protein inhibitors is crucial for cancer and inflammatory disease therapies. New STAT CETSA assays reveal that while peptide inhibitors bind effectively, many small-molecule STAT inhibitors fail to engage their targets in cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Signal transducer and activator of transcription (STAT) proteins are vital in cellular functions.
- Dysregulated STAT signaling contributes to inflammatory diseases and cancer.
- Targeting STAT proteins is challenging due to the lack of distinct binding sites.
Purpose of the Study:
- To develop and validate STAT cellular thermal shift assays (CETSA) for assessing STAT protein engagement by inhibitors.
- To evaluate the target engagement of known STAT inhibitors, particularly STAT3 inhibitors, in cellular and cell lysate settings.
Main Methods:
- Development of STAT-specific CETSA protocols.
- Application of STAT CETSA to test known peptidic and small-molecule STAT inhibitors.
- Assessment of STAT protein stabilization in response to inhibitor treatment.
Main Results:
- STAT CETSA successfully detected interactions between STAT proteins and peptidic inhibitors.
- Small-molecule STAT inhibitors showed variable target engagement, with many failing to stabilize STAT3.
- The assays demonstrated the utility of CETSA for evaluating STAT inhibitor efficacy.
Conclusions:
- STAT CETSA are valuable tools for validating the cellular target engagement of potential STAT inhibitors.
- The findings highlight the need for rigorous validation of small-molecule STAT inhibitors.
- Improved methods for evaluating STAT inhibitor binding are essential for developing new therapeutics.
