Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep:36:101646.
doi: 10.1016/j.redox.2020.101646. Epub 2020 Jul 17.

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

Affiliations

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

Sander Busker et al. Redox Biol. 2020 Sep.

Abstract

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.

Keywords: Electrophiles; Redox; Selenocysteine; Signal transducer and activator of transcription 3; Small molecule inhibitors; Thioredoxin reductase 1.

PubMed Disclaimer

Conflict of interest statement

BDGP is listed as an inventor on a patent describing the STAT3 inhibitors BP1-102 and SH-4-054 (WO2013177534) and ESJA has several patents on specific TrxR1 inhibitors. The authors declare no other competing interests with regard to the presented data.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Chemical structures of explored small molecule STAT3 inhibitors. Compounds S3I-1757, BP1-102, SH-4-54 and S3I-201 all contain the same scaffold. LLL12, Napabucasin, STA-21, Cryptotanshinone and Atovaquone all contain a quinone structure. See Table 1 for further details.
Fig. 2
Fig. 2
In vitro and cellular inhibition of TrxR1 activity by small molecule inhibitors of STAT3. (A) TrxR1 (12.5 nM) activity was assessed in vitro after 40 min incubation with the compounds in presence of NADPH (250 μM), using an enzymatic DTNB reduction assay. (B) Selenium-supplemented A549 cells were incubated with compounds at the indicated concentration for 4 h before harvesting. Cellular TrxR1 activity was analyzed using the Trx1-linked insulin reduction endpoint assay. Color-coding was used to highlight compounds used in Fig. 3C–E. P-values are displayed as * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and **** = p ≤ 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Compounds that are both inhibitor and substrate of TrxR1 affect its thermal stability. (A) TrxR1 (1 μM) was incubated for 40 min with compounds and NADPH (500 μM) before running DSF. Stattic, LLL12, Cpd188, Napabuscasin, Cryptotanshinone and Disulfiram thermally stabilize TrxR1. (B) TrxR1 (1 μM) was incubated for 40 min with only compounds in the absence of NADPH. Solely Disulfiram thermally destabilized TrxR1. (C) TrxR1 (1 μM) was incubated with compounds and NADPH [0.5 or 5 mM] for either 5 or 40 min. (D) NADPH consumption by TrxR1 (12.5 nM) during incubation of compounds tested in (C) over the course of 90 min. All compounds that thermally stabilize TrxR1 in DSF are also substrates of TrxR1. (E) TrxR1 (315 nM) was incubated in vitro for 40 min with compounds and NADPH (200 μM) before desalting. TrxR1 activity was measured with aliquots taken before and after desalting. All tested compounds that are substrates of TrxR1 also irreversibly inhibit Sec-dependent reduction of DTNB. Tm calculations of BP1-102 were excluded, because the background fluorescence from the compound interfered significantly with Sypro Orange™, which caused high fluorescent signal already at very low temperatures (Fig. S2). P-values are displayed as * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and **** = p ≤ 0.0001.

References

    1. Yu H., Jove R. The STATs of cancer — new molecular targets come of age. Nat. Rev. Canc. 2004;4:97–105. doi: 10.1038/nrc1275. - DOI - PubMed
    1. Yu H., Pardoll D., Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Canc. 2009;9:798–809. doi: 10.1038/nrc2734. - DOI - PMC - PubMed
    1. Hillmer E.J., Zhang H., Li H.S., Watowich S.S. STAT3 signaling in immunity. Cytokine Growth Factor Rev. 2016;31:1–15. doi: 10.1016/j.cytogfr.2016.05.001. - DOI - PMC - PubMed
    1. Wake M.S., Watson C.J. STAT3 the oncogene - still eluding therapy? FEBS J. 2015;282:2600–2611. doi: 10.1111/febs.13285. - DOI - PubMed
    1. Chen J., Jiang C.C., Jin L., Zhang X.D. Regulation of PD-L1: a novel role of pro-survival signalling in cancer. Ann. Oncol. 2016;27:409–416. doi: 10.1093/annonc/mdv615. - DOI - PubMed

Publication types

LinkOut - more resources