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

Desroses, Matthieu; Busker, Sander; Astorga‐Wells, Juan; Attarha, Sanaz; Kolosenko, Iryna; Zubarev, Roman A.; Helleday, Thomas; Grandér, Dan; Page, Brent D. G.

doi:10.1016/j.jpba.2018.07.018

Cited by 14 publications

(31 citation statements)

References 25 publications

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“…Recombinant human mitochondrial ATP synthase subunit α (α-F1-ATP synthase) and β (β-F1-ATP synthase) were purchased from CusaBio (Houston, TX, USA). Binding of CADD522 to α or β subunit was examined experimentally using DSF, which evaluates changes in the target protein melting temperature (Tm) due to interactions with the test compound [ 64 , 114 , 115 ]. SYPRO orange (Thermo Fisher Scientific) diluted 1:1000 in 10 mM HEPES, 150 mM NaCl (pH 7.5), and 1.5 μM recombinant proteins were added to 96-well PCR plates.…”

Section: Methodsmentioning

confidence: 99%

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis

Kim

Gernapudi

Cedeno³

et al. 2020

Oncotarget

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Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-ofconcept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth.

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Section: Methodsmentioning

confidence: 99%

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis

Kim

Gernapudi

Cedeno³

et al. 2020

Oncotarget

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“…Band intensities were normalized to the sample containing 25 nM selenite, as this was the concentration used throughout this work to ensure adequate selenium supplementation. (12)]. These truncations were used because of difficulties producing and working with recombinant fulllength protein and to assess whether the compound was able to bind preferentially with the SH2 domain of STAT3, which is a primary target for many known STAT3 inhibitors (12).…”

Section: Small-molecule Inhibitors Of Stat3 Transcriptional Activitymentioning

confidence: 99%

“…(12)]. These truncations were used because of difficulties producing and working with recombinant fulllength protein and to assess whether the compound was able to bind preferentially with the SH2 domain of STAT3, which is a primary target for many known STAT3 inhibitors (12). The resulting mixtures were analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE), and the dansyltagged proteins could be detected under ultraviolet (UV) ex citation, as illustrated in Fig.…”

Section: Small-molecule Inhibitors Of Stat3 Transcriptional Activitymentioning

confidence: 99%

Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death

et al. 2020

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Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.

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“…Although their chemical structures vary, many of these compounds aim to bind the same site of STAT3, its Src Homology 2-domain (SH2-domain) [ 9 , 10 ]. Binding to the SH2 domain is believed to block STAT3 phosphorylation, dimerization and thereby its transcriptional function [ 11 , 12 ]. These small molecules were often confirmed to target STAT3 using biochemical assays, such as fluorescence polarization assay (FPA), electrophoretic mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA) [ 10 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

“…Binding to the SH2 domain is believed to block STAT3 phosphorylation, dimerization and thereby its transcriptional function [ 11 , 12 ]. These small molecules were often confirmed to target STAT3 using biochemical assays, such as fluorescence polarization assay (FPA), electrophoretic mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA) [ 10 , 12 ]. Additional cellular and in vivo experiments have also typically been employed to demonstrate inhibition of STAT3 phosphorylation, down-regulation of STAT3-dependent gene expression, or blockade of other STAT3-related cellular processes [ 9 , 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

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

2020

Self Cite

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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.

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

Cited by 14 publications

References 25 publications

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis

Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death

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

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