Mechanisms of action of thioredoxin reductase inhibitors in the context of cancer

The increased understanding of the role of redox homeostasis in cancer survival and progression has placed a spotlight on studying the perturbations in redox signaling as cancer vulnerabilities. These vulnerabilities are in part under the control of antioxidant systems such as the thioredoxin system, with its main effector, the selenoprotein thioredoxin reductase (TXNRD). The selenocysteine- containing (Sec) active site of TXNRD is highly susceptible to alkylation by electrophiles and this has in fact been shown to be a part of the mechanism of action of a range of anticancer compounds in the clinic and in trials, including cisplatin and auranofin. Discovering and developing compounds with a focused target profile on TXNRD has been a major aim of the Arner group, so as to limit the side effects seen with drugs with broader target profiles and to study the precise downstream effects of TXNRD inhibition on redox signaling in cancer. These efforts led to the discovery of thioredoxin reductase inhibitor 1 (TRi-1), a highly selective TXNRD inhibitor with good antitumoral activity in mouse models. This thesis has focused on further characterizing the effects of TRi-1 in cancer cells and better understanding the role of TXNRD as a cancer vulnerability.

Paper I further confirmed the narrow target profile of TRi-1 in comparison to auranofin on a proteomics scale by combining thermal proteome profiling, protein expression and redox proteomics approaches. It also established that TRi-1 does not result in a stable downregulation of cellular TXNRD activity, but instead causes rapid inhibition followed by a compensatory increase.

In Paper II, we explored the activities of TRi-1 analogs with functionalizations allowing for increased mitochondrial uptake or differential electrophilicity of the selenocysteine-interacting moiety, both against cytosolic and mitochondrial TXNRD isoforms. We arrived at a diverse collection of novel compounds with a range of efficacies in recombinant systems and cancer cell lines, including scaffolds with improved activity compared to TRi-1.

Paper III identified surprising connections between TXNRD and ferroptosis by confirming ferroptosis inducers as inhibitors of TXNRD and by establishing that TRi-1-induced cell death cannot be classified as ferroptosis and indeed cannot be rescued by any of the most common death inhibitors. We also reported differential glutathione peroxidase 4 migration patterns in response to ferroptosis inducing TXNRD1 inhibitors and TRi-1, hinting their effects on antioxidant systems in recombinant and cellular contexts are more complex than previously anticipated.

Paper IV characterized in detail the kinetics of cellular events taking place after TRi-1 and thereby comprehensively portrayed the mechanisms of action of this compound in cancer cells. We established that short term exposure of the cells, in the order of one hour, to the compound is in every way equivalent to a continuous exposure and showed the kinetics of TRi-1 derivatization of the TXNRD selenopeptide in cells. The anticancer effects were predominantly caused by an arrest in cell cycle progression characterized by a downregulation of CDK1/2. Cells experienced peroxiredoxin dimerization and overoxidation, suggestive of increased oxidative stress, but without major perturbations in protein or nucleotide homeostasis.

Paper V established the efficacy of TRi-1 in parasite TXNRD isoforms with unusual structural domains, suggesting that a broader application of this compound to pathologies beyond cancer may be possible, much like the broader indications for using the more promiscuous auranofin.

Overall, the papers comprising this thesis have focused on exploring different aspects of the mechanism of action of TRi-1 as a TXNRD inhibitor and have painted a multifaceted picture of the compound's activities extending far beyond its target engagement.

List of scientific papers

I. Pierre Sabatier, Christian M. Beusch, Radosveta Gencheva, Qing Cheng, Roman Zubarev, Elias S.J. Arnér. Comprehensive chemical proteomics analyses reveal that the new TRi-1 and TRi-2 compounds are more specific thioredoxin reductase 1 inhibitors than auranofin. Redox Biol. 2021 Nov 11;48:102184. https://doi.org/10.1016/j.redox.2021.102184

II. Miloš Jović#, Radosveta Gencheva #, Qing Cheng, Karoline C. Scholzen, Života Selaković, Elias S.J. Arnér, Igor M. Opsenica. Development of novel analogs of the TRi-1 and TRi-2 selenoprotein thioredoxin reductase inhibitors with initial assessment of their cytotoxicity profiles. [Submitted]

III. Dorian M. Cheff, Chuying Huang, Karoline C. Scholzen, Radosveta Gencheva, Michael H. Ronzetti, Qing Cheng, Matthew D. Hall, Elias S. J. Arnér. The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD. Redox Biol. 2023 Jun;62:102703. https://doi.org/10.1016/j.redox.2023.102703

IV. Radosveta Gencheva, Giovanni Chiappetta, Karoline C. Scholzen, Praveen Pandey, Zhao Wenchao, Lucia Coppo, Qing Cheng, Joelle Vinh, Andrei Chabes, Elias S.J. Arnér. Insights into the kinetics of thioredoxin reductase derivatization and associated cancer cell death by the small molecule inhibitor TRi-1. [Manuscript]

V. Francesca Fata#, Radosveta Gencheva #, Qing Cheng, Rachel Lullo, Matteo Ardini, Ilaria Silvestri, Federica Gabriele, Rodolfo Ippoliti, Christina A. Bulman, Judy A. Sakanari, David L. Williams, Elias S.J. Arnér, Francesco Angelucci. Biochemical and structural characterizations of thioredoxin reductase selenoproteins of the parasitic filarial nematodes Brugia malayi and Onchocerca volvulus. Redox Biol. 2022 May;51:102278. https://doi.org/10.1016/j.redox.2022.102278