To inhibit TrxR1 is to inactivate STAT3-Inhibition of TrxR1 enzymatic function by STAT3 small molecule inhibitors.

The transcription factor STAT3 plays a key role in cancer and immunity, being widely explored as a potential drug target for the development of novel immunomodulatory or anticancer therapeutics. The mechanisms of small molecule-derived inhibition of STAT3 appear, however, to be more complex than initially perceived. Our recent discovery, that some novel STAT3 inhibitors were bona fide inhibitors of the cytosolic selenoprotein oxidoreductase TrxR1 (TXNRD1), led us to explore the effects of a wide array of previously described STAT3 inhibitors on TrxR1 function. We found that 17 out of 23 tested STAT3 small molecule inhibitors indeed inhibited purified TrxR1 at the reported concentrations yielding STAT3 inhibition. All tested compounds were electrophilic as shown by direct reactivities with GSH, and several were found to also be redox cycling substrates of TrxR1. Ten compounds previously shown to inhibit STAT3 were here found to irreversibly inhibit cellular TrxR1 activity (Auranofin, Stattic, 5,15-DPP, Galiellalactone, LLL12, Napabucasin, BP1-102, STA-21, S3I-201 and Degrasyn (WP1130)). Our findings suggest that targeting of TrxR1 may be a common feature for many small molecules that inhibit cellular STAT3 function. It is possible that prevention of STAT3 activation in cells by several small molecules classified as STAT3 inhibitors can be a downstream event following TrxR1 inhibition. Therefore, the relationship between TrxR1 and STAT3 should be considered when studying inhibition of either of these promising drug targets.

Validating Signal Transducer and Activator of Transcription (STAT) Protein-Inhibitor Interactions Using Biochemical and Cellular Thermal Shift Assays.

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.

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions.

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided.

Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.