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Validating Signal Transducer and Activator of Transcription (STAT) Protein–Inhibitor Interactions Using Biochemical and Cellular Thermal Shift Assays
journal contribution
posted on 2020-07-03, 02:13 authored by Sanaz Attarha, Anja Reithmeier, Sander Busker, Matthieu Desroses, Brent D. G. PageSignal 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.