We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment ...results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.
Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to ...hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C–SOS1, SOS1, and SOS2. By preventing formation of the KRAS–SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS–SOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations.
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.
The crystal structure of the human selenocysteine‐containing protein glutathione peroxidase 4 (GPX4) was determined at 1.0 Å resolution. A mass‐spectrometry‐based approach was developed to monitor the formation of adducts of the active‐site selenocysteine Sec46 with covalent inhibitors. The crystal structure of Sec46‐containing GPX4 in complex with the covalent inhibitor ML162 (S)‐enantiomer was determined at 1.54 Å resolution.
Microbial U(VI) reduction influences uranium mobility in contaminated subsurface environments and can affect the disposal of high-level radioactive waste by transforming the water-soluble U(VI) to ...less mobile U(IV). The reduction of U(VI) by the sulfate-reducing bacterium Desulfosporosinus hippei DSM 8344T, a close phylogenetic relative to naturally occurring microorganism present in clay rock and bentonite, was investigated. D. hippei DSM 8344T showed a relatively fast removal of uranium from the supernatants in artificial Opalinus Clay pore water, but no removal in 30 mM bicarbonate solution. Combined speciation calculations and luminescence spectroscopic investigations showed the dependence of U(VI) reduction on the initial U(VI) species. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed uranium-containing aggregates on the cell surface and some membrane vesicles. By combining different spectroscopic techniques, including UV/Vis spectroscopy, as well as uranium M4-edge X-ray absorption near-edge structure recorded in high-energy-resolution fluorescence-detection mode and extended X-ray absorption fine structure analysis, the partial reduction of U(VI) could be verified, whereby the formed U(IV) product has an unknown structure. Furthermore, the U M4 HERFD-XANES showed the presence of U(V) during the process. These findings offer new insights into U(VI) reduction by sulfate-reducing bacteria and contribute to a comprehensive safety concept for a repository for high-level radioactive waste.
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•Investigation of speciation-dependent microbial U(VI) reduction•First evidence of U(V) during U(VI) reduction by sulfate-reducing bacteria•The formed U(IV) product cannot be assigned to a uraninite-like structure.•Formation of membrane vesicles as defense mechanism by the cells
Microbial U(VI) reduction influences uranium mobility in contaminated subsurface environments and can affect the disposal of high-level radioactive waste by transforming the water-soluble U(VI) to ...less mobile U(IV). The reduction of U(VI) by the sulfate-reducing bacterium Desulfosporosinus hippei DSM 8344
, a close phylogenetic relative to naturally occurring microorganism present in clay rock and bentonite, was investigated. D. hippei DSM 8344
showed a relatively fast removal of uranium from the supernatants in artificial Opalinus Clay pore water, but no removal in 30 mM bicarbonate solution. Combined speciation calculations and luminescence spectroscopic investigations showed the dependence of U(VI) reduction on the initial U(VI) species. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed uranium-containing aggregates on the cell surface and some membrane vesicles. By combining different spectroscopic techniques, including UV/Vis spectroscopy, as well as uranium M
-edge X-ray absorption near-edge structure recorded in high-energy-resolution fluorescence-detection mode and extended X-ray absorption fine structure analysis, the partial reduction of U(VI) could be verified, whereby the formed U(IV) product has an unknown structure. Furthermore, the U M
HERFD-XANES showed the presence of U(V) during the process. These findings offer new insights into U(VI) reduction by sulfate-reducing bacteria and contribute to a comprehensive safety concept for a repository for high-level radioactive waste.
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