Mechanism-based peroxiredoxin 3 inhibitors exploit a covalent warhead for cancer therapy

Our plain-English summary of Mechanism-based peroxiredoxin 3 inhibitors exploit a covalent warhead for cancer therapy, by K.J. Nelson, T.L. Smalley, T. Messier, et al (Science Advances, 2025).

The study

• Authors: K.J. Nelson, T.L. Smalley, T. Messier, R. Gumpena, U. Gandhi, S. Milczarek, A. Habibovic, H. Hoffman, V. Gibson, R.J. Hondal, W.T. Lowther, B. Cunniff
• Journal: Science Advances
• Published: 2025;11(44):eady4492
• Cited by: 4 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Covalent inhibitors that are approved and marketed drugs exploit a wide array of warheads and reactions with amino acid side chain-based nucleophiles. Thiostrepton (TS) inhibits the peroxidase activity of the mitochondrial antioxidant protein peroxiredoxin 3 by forming a covalent crosslink between the two active site cysteine residues. Peroxiredoxin 3 inactivation increases reactive oxygen species levels, induces cancer cell death in preclinical models, and shows promise in an ongoing clinical trial for malignant mesothelioma using direct pleural infusion. We report the identification of the minimal fragment of TS that contains tandem dehydro-alanine (DHA) moieties and maintains anticancer activity while losing interactions with three alternative targets of intact TS. Biochemical, kinetic, cellular, and structural studies demonstrate that this fragment is a mechanism-based peroxiredoxin inhibitor. These findings represent a promising start toward a pro-oxidant approach for cancer therapy. Moreover, the data support that the DHA moiety should be added to the covalent warhead arsenal.

In plain English

This laboratory study tested synthetic fragments of thiostrepton (TS), which inhibits the mitochondrial antioxidant enzyme peroxiredoxin 3 (PRX3), a target in an ongoing clinical trial for malignant mesothelioma. Using biochemical, kinetic, cellular and structural methods, the authors found that WF-097, the fragment carrying both tandem dehydro-alanine (DHA) groups, crosslinked PRX3 like intact TS, while derivatives lacking one or both DHA groups did not. Adduction of the peroxidatic Cys108 (about 0.04 per minute) matched crosslinking and loss of peroxidase activity; adduction of the resolving Cys229, measured alone, was slower (0.007 per minute), and the authors inferred that reaction with CysR proceeds rapidly once CysP is adducted. A 2.30 Angstrom structure of an engineered PRX1 dimer showed the CysP residue bound to DHA1. After 48 hours in H-MESO-1 mesothelioma cells, EC50 values were 4.0 plus or minus 0.4 micromolar (WF-097), 0.86 plus or minus 0.06 (WF-242) and 0.91 plus or minus 0.03 (TS). Unlike TS, the fragments did not inhibit the proteasome, FOXM1 DNA binding, or growth of E. hirae at 20 micromolar; neither TS nor WF-242 inhibited recombinant TR1 or TR2.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory study combining biochemical and kinetic assays with recombinant proteins, mass spectrometry, x-ray crystallography and cultured human cancer cell experiments
• Who was studied: recombinant engineered human PRX3 dimer (PRX3-EE) and Cys variants, an engineered PRX1 dimer for crystallography, human malignant mesothelioma cell lines H-MESO-1 and H2373, immortalized nontumorigenic mesothelial cells (LP9), other cancer cell lines, and Enterococcus hirae cultures
• Exposure: thiostrepton (TS) and synthetic TS fragments WF-097 (both DHA groups), WF-160, WF-196, WF-292 (DHA groups removed) and WF-242, at concentrations stated for each assay
• Main results:
  • With recombinant PRX3, WF-097 reacted with CysP108 at about 0.04 per minute, matching the crosslink formation rate and loss of peroxidase activity; reaction with CysR229 measured in the CysP-mutant variant was about 0.007 per minute
  • The authors inferred from the slow CysR229-only adduction rate that, once the CysP adduct has formed, reaction with CysR is rapid
  • A 2.30 Angstrom crystal structure of an engineered PRX1 dimer showed the peroxidatic Cys crosslinked to DHA1 of WF-097, with DHA2 positioned for attack by the resolving Cys
  • In H-MESO-1 mesothelioma cells treated for 48 hours, WF-097 gave an EC50 of 4.0 plus or minus 0.4 micromolar versus 0.91 plus or minus 0.03 micromolar for TS; compounds with one or both DHA groups substituted had no cytotoxic activity
  • The analog WF-242 gave an EC50 of 0.86 plus or minus 0.06 micromolar in H-MESO-1 cells
  • WF-242 showed a five- to sixfold increase in crosslinking for PRX3 compared with PRX1 or PRX2 in H-MESO-1 cells
  • Relative potency depended on exposure time: EC50 values of WF-242 and TS were similar after 24 and 48 hours, but WF-242 was more potent than TS at 3- and 6-hour exposures
  • LP9 nontumorigenic mesothelial cells were about two- to threefold less sensitive to TS and WF-242 than the mesothelioma lines H-MESO-1 and H2373
  • TS reduced the activity of both the purified 20S proteasome and the cellular proteasome by more than 70%
  • WF-097 and WF-242 did not inhibit proteasome activity in either the purified or cellular assay, even at 200 micromolar
  • TS at 200 micromolar decreased FOXM1-DNA binding, whereas WF-097 and WF-242 showed no inhibition at the same concentration
  • Neither TS nor WF-242 at 50 micromolar inhibited recombinant human TR1 or mouse TR2
  • Both TS and WF-242 (1 to 2.5 micromolar) gave modest inhibition of global TR1/TR2 activity in cell lysates, which the authors said was complicated to interpret
  • Against Enterococcus hirae, the minimal inhibitory concentration for TS was 0.045 micromolar
  • No inhibition of E. hirae growth was seen with 20 micromolar WF-097 or WF-242
  • TS, WF-097 and WF-242 did not react with the deprotonated thiolate TNB− over 22 hours at 37°C, whereas the iodoacetamide control reacted completely
• The authors' conclusion: The authors concluded that the minimal TS fragment acts as a mechanism-based PRX3 inhibitor that raises ROS and kills cancer cells without blocking the alternative targets of intact TS, and that WF-242 is a starting point for developing PRX3 inhibitors for cancer treatment
• Limitations the authors state:
  • Inhibition of thioredoxin reductases seen in cell lysates was difficult to interpret because other lysate proteins could act as substrates
  • The crystal structure used an engineered PRX1 variant because crystallizing WF-097-crosslinked PRX3 failed
  • The recombinant mouse TR2 used in some assays contained Cys rather than seleno-Cys in its active site

Topics

Medical terms in this study, explained in our Lexicon: Reactive Oxygen Species (ROS), Clinical Trial, Mesothelioma.

Related studies

• Preclinical characterization and phase 1 clinical testing of targeting mitochondrial peroxiredoxin 3 in cancer — V. Gibson, J. Dzialo, et al. (2026)
• Peroxiredoxin 3 levels regulate a mitochondrial redox setpoint in malignant mesothelioma cells — B. Cunniff, A.N. Wozniak, et al. (2014)
• Mitochondrial-targeted nitroxides disrupt mitochondrial architecture and inhibit expression of peroxiredoxin 3 and FOXM1 in malignant mesothelioma cells — B. Cunniff, K. Benson, et al. (2013)
• Peroxiredoxin 3 is a redox-dependent target of thiostrepton in malignant mesothelioma cells — K. Newick, B. Cunniff, et al. (2012)
• SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma — I.C. Salaroglio, D.C. Belisario, et al. (2022)
• Design of pH responsive clickable prodrugs applied to histone deacetylase inhibitors: a new strategy for anticancer therapy — R. Delatouche, I. Denis, et al. (2013)

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