Isoenzyme-specific determination of glutathione peroxidase activity in cell lysates and GPX1/GPX4-HiBiT cellular target engagement
5 affiliations
- 1Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Dr., Rockville, MD, 20850, USA; Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SE-171 77, Sweden
- 2Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Dr., Rockville, MD, 20850, USA
- 3Department of Nutritional Physiology, Institute of Nutritional Sciences, Friedrich Schiller University Jena, Dornburger Str. 24, Jena, 07743, Germany
- 4Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SE-171 77, Sweden; Assays, Profiling, and Cell Sciences, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden
- 5Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SE-171 77, Sweden
Abstract
Glutathione peroxidases (GPX) are primarily selenoproteins that maintain cellular redox balance. GPX1, GPX2 and GPX4 are associated with cancer progression and treatment resistance, but the lack of isoenzyme-specific cellular assays has limited evaluation of GPX-targeting compounds. As GPX1 and GPX2 share high sequence homology, as well as substrate and inhibitor profiles, their individual activities are difficult to distinguish. Here, we present a glutathione reductase (GR)-coupled assay using auranofin to quantify combined GPX1/GPX2 activity and total GPX activity in crude cell lysates. The method was validated using multiple cell lines, with or without selenium supplementation, spiked human recombinant enzymes, and knockdown cells. Combined GPX1/GPX2 activity correlated with GPX1 protein levels and accounted for the majority of GPX activity in selenium-supplemented cells. GPX4's low catalytic turnover limits the feasibility of measuring its activity in cell lysates, so we established a high-throughput cellular thermal shift assay (CETSA) using HiBiT-tagging of GPX1 and GPX4 in HT1080 cells to quantify isoenzyme-specific target engagement. Using this new complementary assay platform, we found that ferroptosis inducers RSL3, PACMA-31 and ML-162 destabilized GPX4 in cells, RSL3 and ML-162 destabilized cellular GPX1, but none of these compounds had a strong effect on either total GPX, or GPX1/GPX2 activities in lysates. Additional reported GPX1/GPX2 or GPX4 inhibitors showed minimal GPX4 target engagement, whereas GPX1 was readily targeted in cells. The FDA-approved drugs, ceftibuten and omapatrilat, inhibited GPX1/GPX2 activity more potently than auranofin in cell lysates, suggesting that GPX1/GPX2 suppression may contribute to their cellular effects. Together, these complementary GPX assays presented here provide practical tools for evaluations of specific GPX isoenzyme cellular activities and development of selective GPX-targeted therapies.
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BibTeX
@article{barrett2026isoenzymespecific,
title = {{Isoenzyme-specific determination of glutathione peroxidase activity in cell lysates and GPX1/GPX4-HiBiT cellular target engagement}},
author = {Madeleine S. Barrett and Jonathan H. Shrimp and Alina Löser and Dorian M. Cheff and Qing Cheng and Anna P. Kipp and Matthew D. Hall and Elias S.J. Arnér},
journal = {Redox Biology},
year = {2026},
month = oct,
doi = {10.1016/j.redox.2026.104445},
url = {https://doi.org/10.1016/j.redox.2026.104445},
note = {CC BY},
}
RIS
TY - JOUR
TI - Isoenzyme-specific determination of glutathione peroxidase activity in cell lysates and GPX1/GPX4-HiBiT cellular target engagement
AU - Madeleine S. Barrett
AU - Jonathan H. Shrimp
AU - Alina Löser
AU - Dorian M. Cheff
AU - Qing Cheng
AU - Anna P. Kipp
AU - Matthew D. Hall
AU - Elias S.J. Arnér
PY - 2026
DA - 2026/10/31
JO - Redox Biology
DO - 10.1016/j.redox.2026.104445
UR - https://doi.org/10.1016/j.redox.2026.104445
AB - Glutathione peroxidases (GPX) are primarily selenoproteins that maintain cellular redox balance. GPX1, GPX2 and GPX4 are associated with cancer progression and treatment resistance, but the lack of isoenzyme-specific cellular assays has limited evaluation of GPX-targeting compounds. As GPX1 and GPX2 share high sequence homology, as well as substrate and inhibitor profiles, their individual activities are difficult to distinguish. Here, we present a glutathione reductase (GR)-coupled assay using auranofin to quantify combined GPX1/GPX2 activity and total GPX activity in crude cell lysates. The method was validated using multiple cell lines, with or without selenium supplementation, spiked human recombinant enzymes, and knockdown cells. Combined GPX1/GPX2 activity correlated with GPX1 protein levels and accounted for the majority of GPX activity in selenium-supplemented cells. GPX4's low catalytic turnover limits the feasibility of measuring its activity in cell lysates, so we established a high-throughput cellular thermal shift assay (CETSA) using HiBiT-tagging of GPX1 and GPX4 in HT1080 cells to quantify isoenzyme-specific target engagement. Using this new complementary assay platform, we found that ferroptosis inducers RSL3, PACMA-31 and ML-162 destabilized GPX4 in cells, RSL3 and ML-162 destabilized cellular GPX1, but none of these compounds had a strong effect on either total GPX, or GPX1/GPX2 activities in lysates. Additional reported GPX1/GPX2 or GPX4 inhibitors showed minimal GPX4 target engagement, whereas GPX1 was readily targeted in cells. The FDA-approved drugs, ceftibuten and omapatrilat, inhibited GPX1/GPX2 activity more potently than auranofin in cell lysates, suggesting that GPX1/GPX2 suppression may contribute to their cellular effects. Together, these complementary GPX assays presented here provide practical tools for evaluations of specific GPX isoenzyme cellular activities and development of selective GPX-targeted therapies.
SN - 2213-2317
ER -