Novel Allosteric Activators for Ferroptosis Regulator Glutathione Peroxidase 4

Abstract

Glutathione peroxidase 4 (GPX4) is essential for cell membrane repair, inflammation suppression, and ferroptosis inhibition. GPX4 upregulation provides unique drug discovery opportunities for inflammation and ferroptosis-related diseases. However, rational design of protein activators is challenging. Until now, no compound has been reported to activate the enzyme activity of GPX4. Here, we identified a potential allosteric site in GPX4 and successfully found eight GPX4 activators using a novel computational strategy and experimental studies. Compound 1 from the virtual screen increased GPX4 activity, suppressed ferroptosis, reduced pro-inflammatory lipid mediator production, and inhibited NF-κB pathway activation. Further chemical synthesis and structure-activity relationship studies revealed seven more activators. The strongest compound, 1d4, increased GPX4 activity to 150% at 20 μM in the cell-free assay and 61 μM in cell extracts. Therefore, we demonstrated that GPX4 can be directly activated using chemical compounds to suppress ferroptosis and inflammation. Meanwhile, the discovery of GPX4 activators verified the possibility of rational design of allosteric activators.

Activation of Glutathione Peroxidase 4 as a Novel Anti-inflammatory Strategy

“…In the previous study, we have identified a potential allosteric site in the crystal structure of human GPX4 U46C mutant (hGPX4-C, PDB entry 2OBI; Scheerer et al, 2007 ) using the binding site detection program CAVITY ( Yuan et al, 2011 , 2013 ) and the allosteric site motion correlation analysis program CorrSite ( Ma et al, 2016 ). We found a series of substituted 4-thioureidobenzenesulfonamide compounds that can activate GPX4 enzyme activity and suppress ferroptosis ( Li et al, 2018 ). In the present study, we used a similar virtual screen procedure and the predicted allosteric site ( Figure 2A ) to screen for compounds with anti-inflammatory activity and novel chemical scaffold.…”

“…The details of allosteric site prediction and virtual screen are described in a recent report ( Li et al, 2018 ). Potential allosteric site in GPX4 was identified using the CAVITY ( Yuan et al, 2011 , 2013 ) and CorrSite ( Ma et al, 2016 ) program, and then applied to screen for potential allosteric activator.…”

“…In our previous study, we have identified human 15-LOX activators and demonstrated their potential in inflammation control ( Meng et al, 2016 ). Recently, we reported the discovery of one series of GPX4 allosteric activator compounds ( Li et al, 2018 ) by using a combining approach of potential binding cavity identification ( Yuan et al, 2011 , 2013 ), allosteric site prediction ( Ma et al, 2016 ), and allosteric ligand virtual screen. In the present study, we identified a new compound with different chemical scaffold that can activate the enzyme activity of GPX4 by more than two folds.…”

Dihydrotanshinone I inhibits human glioma cell proliferation via the activation of ferroptosis

“…Therefore, in the present study, ferroptosis in human glioma cells was assessed by measuring the levels of GPX4, 5-, 12-and 15-HETE. GPX4 is an important membrane lipid peroxidase reductase, and the downregulation of GPX4 causes the accumulation of reactive oxygen species (ROS) on membrane lipids, which leads to ferroptosis (36). The latter three are lipid peroxidation products that are associated with the deposition of ferritin in ferroptosis, and recognized as indicators of ferroptosis levels (20,26).…”

The Investigation of Allosteric Regulation Through Markov State Modeling

“…Understanding allosteric signaling and predicting allosteric binding sites are at the cutting edge of drug discovery [40][41][42][43][44][45] . The Allosteric Database [46][47][48] Action at a distance is a key feature of the allosteric effect; the effector binds a site topographically distinct from the orthosteric site.…”