(1354-C) Optimization of high-throughput human glutathione peroxidase GPx1 and GPx4 assays for the discovery of small molecule inhibitors
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Cancer cells rely heavily on glutathione peroxidase (GPx) antioxidant enzymes which reduce harmful reactive oxygen species (ROS), specifically hydroperoxides. GPx enzymes, such as GPx1 and GPx4, are selenoproteins. They have emerged as attractive targets for treating cancer as they have been linked to treatment resistance and are overexpressed in some cancer types. GPx4 is the only GPx that can reduce lipid hydroperoxides, and absence of GPx4 triggers a non-apoptotic cell death from the accumulation of lipid hydroperoxides called ferroptosis. GPx enzymes reduce hydroperoxides to alcohol by oxidizing the co-factor glutathione (GSH). GSH is recycled by glutathione reductase (GR) in conjunction with the oxidation of NADPH. Here, we have optimized a high-throughput 1536-well GR-coupled assay for the detection of GPx1 and GPx4 activity using an NADPH readout with pure recombinant selenoprotein (selenocysteine-containing) GPx enzymes. This assay will enable the discovery of novel, specific GPx inhibitors, alongside a counter-assay for off-target GR inhibition. Reagent concentration (GSH, cumene hydroperoxide and GR) was re-optimized for the GPx1 and GPx4 recombinant enzymes in the present assay. This optimization accounted for an updated recombinant production method for GPx4. Additionally, enzyme kinetics were re-examined. We validated the inhibitory properties of previously published GPx1 inhibitors, including auranofin, tenatoprazole, omapatrilat, and many cephalosporin antibiotics, and GPx4 inhibitors such as lusutrombopag, PACMA 31, VU0661013, and navitoclax (PMID: 37244126). GPx4 prior art inhibitors RSL3, ML162 and ML210 were confirmed to not directly inhibit the enzyme in the biochemical assay (PMID: 37087975). These compounds were found to be cytotoxic in vitro, triggering ferroptosis which was rescued by inhibition of lipid peroxidation using the ROS scavenger ferrostatin-1. Identifying novel GPx1 and GPx4 inhibitors using this GPx screening pipeline remains of great interest as many marketed GPx inhibitors are promiscuous and may only indirectly inhibit different GPx isoforms or other cellular targets.