Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162

Abstract

Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Å resolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.

Going Forward and Back: The Complex Evolutionary History of the GPx

“…The animal GPx common ancestor would give rise to three different types of GPx. The closest to non-animal GPx, GPx04, retained its monomeric structure and catalytic capacity to reduce lipid peroxides [ 16 , 53 ]. Despite the absence of the amino acid residues responsible to bind to GSH, the presence of selenocysteine in the GPx04 contributed to the use of glutathione as a reducing agent [ 8 , 19 ].…”

Bibliometric Evaluation of 2012–2020 Publications on Ferroptosis in Cancer Treatment

“…Cluster #4 (necroptosis) and Cluster #6 (Fenton reaction) contain fewer nodes on the timeline, suggesting that this is a previous research hotspot or an emerging research direction. Therefore, future research should focus on comprehensive mechanism and clinical application, such as novel tumor therapies based on GPX4 covalent inhibitors, such as ML162 ( Moosmayer et al, 2021 ). In addition, p53-regulated ferroptosis therapy or CD8 + -mediated immunotherapy of cancer also has potential clinical value, which needs further research and mechanism elaboration ( Chu et al, 2019 ; Kang et al, 2019 ; Stockwell and Jiang, 2019 ).…”

A Promising Future of Ferroptosis in Tumor Therapy

“…GPX4 can reduce hydroperoxides to block the lipoxygenase pathway to inhibit ferroptosis. Inactivating GPX4 with RSL3 ( Sui et al, 2018 ), ML162 ( Moosmayer et al, 2021 ), and ML210 ( You et al, 2021 ) could increase the production of lipid ROS, thereby inducing ferroptosis. Friedmann Angeli et al (2014) observed that GPX4 knockout contributed to renal failure in mice, but this injury could be rescued by ferrostatin-1, an agent that could decrease the level of iron to inhibit iron-dependent lipid peroxidation.…”