Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction

Borchert, Astrid; Kalms, Jacqueline; Roth, Sophia Regina; Rademacher, Marlena; Schmidt, Andrea; Holzhütter, Hermann‐Georg; Kühn, Hartmut; Scheerer, Patrick

doi:10.1016/j.bbalip.2018.06.006

Cited by 59 publications

(62 citation statements)

References 62 publications

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“…Historically, structural studies of GPX4 have been possible only with U46C and U46G active-site mutants 38,52,54,55 . The structure of a selenocysteine-containing GPX4 (though not wild type) was recently reported 49 , but to date, structural studies of covalent GPX4 inhibitors have not been successful 5 . Co-crystal structures of GPX4 U46C in complex with reversible peptide binders have been reported, but their relevance remains unknown given that these compounds are unable to inhibit cellular GPX4 38 .…”

Section: Extended Discussionmentioning

confidence: 99%

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Targeting a Therapy-Resistant Cancer Cell State Using Masked Electrophiles as GPX4 Inhibitors

Eaton

Furst

et al. 2018

Preprint

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We recently discovered that inhibition of the lipid peroxidase GPX4 can selectively kill cancer cells in a therapy-resistant state through induction of ferroptosis. Although GPX4 lacks a conventional druggable pocket, covalent small-molecule inhibitors are able to overcome this challenge by reacting with the GPX4 catalytic selenocysteine residue to eliminate enzymatic activity. Unfortunately, all currently-reported GPX4 inhibitors achieve their activity through reactive chloroacetamide groups. We demonstrate that such chloroacetamide-containing compounds are poor starting points for further advancement given their promiscuity, instability, and low bioavailability. Development of improved GPX4 inhibitors, including those with therapeutic potential, requires the identification of new electrophilic chemotypes and mechanisms of action that do not suffer these shortcomings. Here, we report our discovery that nitrile oxide electrophiles, and a set of remarkable chemical transformations that generates them in cells from masked precursors, provide an effective strategy for selective targeting of GPX4. Our results, which include structural insights, target engagement assays, and diverse GPX4-inhibitor tool compounds, provide critical insights that may galvanize development of improved compounds that illuminate the basic biology of GPX4 and therapeutic potential of ferroptosis induction. In addition, our discovery that nitrile oxide electrophiles engage in highly selective cellular interactions and are bioavailable in their masked forms may be relevant for targeting other currently undruggable proteins, such as those revealed by recent proteome-wide ligandability studies.

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Section: Extended Discussionmentioning

confidence: 99%

“…Biochemical and structural studies of GPX4 and its inhibitors have been hindered by the lack of an efficient source of wild-type GPX4 protein 5,34,35 . The recombinant expression of mammalian selenoproteins in bacterial protein production systems is often difficult due to low efficiency of selenocysteine incorporation [36][37][38] .…”

Section: Extended Discussionmentioning

confidence: 99%

Targeting a Therapy-Resistant Cancer Cell State Using Masked Electrophiles as GPX4 Inhibitors

Eaton

Furst

et al. 2018

Preprint

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“…Crystal structures of a mutant (U46C) GPX4 and a recombinant selenium‐containing GPX4 have been solved to 1.55 and 1.3 Å, respectively . GPX4 is composed of a thioredoxin motif made of four solvent‐exposed alpha helices and seven beta strands, five of which create a central beta sheet.…”

Section: Gpx4 Structure and Functionmentioning

confidence: 99%

“…Mutation of selenocysteine to cysteine diminishes GPX4 activity by 90% and heightens sensitivity to redox stress . The inability of cysteine to compensate for selenocysteine is likely because of differences in pK a between selenocysteine (5.2) and cysteine (8.2) . Since the active site of GPX4 is near the protein surface, selenocysteine is more likely to be in a deprotonated state at neutral pH, which is necessary for its catalytic function.…”

Section: Gpx4 Structure and Functionmentioning

confidence: 99%

“…A crystal structure of seleno–GPX4 observed the presence of a seleninic acid (Se–OO — ) in the enzyme active site . It is therefore possible that GPX4 goes through low oxidation cycles of selenol to selenenic acid and high oxidation cycles of selenenic acid to seleninic acid, depending on the oxidative stress burden of a cell.…”

Section: Gpx4 Structure and Functionmentioning

confidence: 99%

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GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis

2019

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Oxygen is necessary for aerobic metabolism but can cause the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe2+)‐dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to lipid peroxidation and can result in the induction of ferroptosis, an iron‐dependent, non‐apoptotic form of cell death. This review describes the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation, and the induction of ferroptosis.

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Probing the Formation of a Seleninic Acid in Living Cells by the Fluorescence Switching of a Glutathione Peroxidase Mimetic

et al. 2019

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Glutathione peroxidase (GPx) is as elenoenzyme that protects cells against oxidative damage.A lthough the formation of aseleninic acid (-SeO 2 H) by this enzyme during oxidative stress has been proposed, aselenic acid has not been identified in cells.H erein, we report that the formation of as eleninic acid can be monitored in living cells by using ar edox-active ebselen analogue with an aphthalimide fluorophore.T he probe reacts with H 2 O 2 to generate the highly fluorescent seleninic acid. The electron withdrawing nature of the -SeO 2 Hm oiety and strong Se···O interactions,w hich prevent the photoinduced electron transfer,a re responsible for the fluorescence.

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Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction

Cited by 59 publications

References 62 publications

Targeting a Therapy-Resistant Cancer Cell State Using Masked Electrophiles as GPX4 Inhibitors

Targeting a Therapy-Resistant Cancer Cell State Using Masked Electrophiles as GPX4 Inhibitors

GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis

Probing the Formation of a Seleninic Acid in Living Cells by the Fluorescence Switching of a Glutathione Peroxidase Mimetic

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