Shedding light on the STAT3 small molecule inhibitor field

Abstract

STAT3 is one of the seven family members of the STAT transcription factor family. STAT3 has become a very attractive target for cancer therapy and other diseases. It has a prominent role in cancer development as well as progression. However, there are still no STAT3 inhibitors applied in the clinic. One of the underlying reasons might be the complicated regulation of STAT3 function. Not only do many signaling pathways converge on STAT3, it is also a redox sensitive transcription factor. Therefore, STAT3 activity is determined by a plethora of different intracellular and extracellular signals. STAT3 also affects many different cellular functions and has a role in each hallmark of cancer, either through its role as a transcription factor or through one of its diverse non-transcriptional functions. This thesis focuses on the STAT3 small molecule inhibitor field, by trying to further explore the mechanism of action of novel and widely used STAT3 inhibitors. In addition, we developed a new method to discover specific STAT3 inhibitors. Interestingly, we found that STAT3 was not targeted directly, but rather STAT3 is inactivated due to oxidation of cysteine residues on the protein.

In Paper I, we developed a cell-based high-throughput screening system to evaluate STAT3 transcriptional activity. This was used to screen 28.000 compounds, where after several additional screening steps four lead compounds were selected for further evaluation. All four compounds inhibited STAT3 transcriptional activity. Their mechanisms of action however appeared to be diverse. One compound, KI16, was found to preferentially inhibit STAT3 function compared to STAT1, and inhibited STAT3-driven phenotypes. In Paper II, we used differential scanning fluorimetry (DSF) to highlight some of the shortcomings of other in vitro STAT3 inhibitor methods. Two STAT3 protein truncations were used to evaluate SH2 domain binders of STAT3 as well as binding specificity of wellknown STAT3 small molecule inhibitors. Phosphopeptides were able to specifically bind to the STAT3 SH2 domain and increase protein thermal stability. While two small molecule inhibitors did not affect thermal stability of either truncation, two other small molecules, Stattic and BP1-102 decreased stability of both truncations. Which was an indication that both small molecules are not specific SH2 domain binders, but rather target STAT3 in multiple sites leading to its destabilization.

In Paper III, we performed structure-activity relationship (SAR) on a selected series of compounds that were identified in the high-throughput screening in Paper I. Sub 1 μM compounds were generated that potently inhibited STAT3 transcriptional activity. These compounds were found to have electrophilic properties, which were essential for STAT3 inhibition. With the use of a fluorescently-tagged compound we were able to pinpoint the cellular protein target, thioredoxin reductase 1 (TrxR1). The top compounds as well as Stattic were potent inhibitors of TrxR1 function, and were found to induce oxidative stress. Vice versa, TrxR1 inhibitors were also able to inhibit STAT3 transcriptional activity. Oxidative stress induction leads to the oxidation of Prx2, which can relay its oxidative equivalents to STAT3 that forms oxidized dimers and is transcriptionally inactivated. TrxR1 is the main reductase that reduces Prx2 and STAT3 through consumption of NADPH. However due to the compounds inhibiting its function, the proteins remain oxidized, and eventually leads to cell death.