Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles

Abstract

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.

Figure 1.. ML210 is a selective covalent inhibitor of cellular GPX4

(A) Chemical structures of RSL3, ML162, and ML210 (1–3). Chloroacetamide groups are shown in blue and the nitroisoxazole group is colored red. (B) ML210 exhibits cell-killing activity similar to RSL3 and ML162 across a panel of 821 cancer cell lines. Each dot represents a single cancer cell line. Data plotted reflect an area-under-the-curve (AUC) metric of cell-line sensitivity to small molecules. (C) Treatment of cells with RSL3, ML162, or ML210 (10 μM, 90 min) leads to accumulation of lipid hydroperoxides in LOX-IMVI cells as assessed by fluorescence imaging with C11-BODIPY 581/591. The C11-BODIPY dye emission shifts from orange to green upon oxidation. Scale bars, 50 μM. See also Supplementary Fig. 3a,b. (D) Chemical structures of GPX4-inhibitor affinity probes RSL3-yne, ML162-yne, and ML210-yne (4–6) with alkyne groups shown in blue. (E) RSL3-yne, ML162-yne, and ML210-yne (10 μM, 1 h) pull down GPX4 from LOX-IMVI cells. Full-length western blots are shown in Supplementary Fig. 4. (F) GPX4 CETSA profiles for intact HCC4006 cells treated with DMSO (black), RSL3 (blue; destabilizing), or ML210 (red; stabilizing). Cells were treated with 10 μM compound for 1 h. Data are plotted as mean ± s.e.m., n ≥ 4 biologically independent samples. Representative western blots are shown in Supplementary Fig. 4. (G) Competitive affinity enrichment between ML210-yne probe (10 μM, 30 min) and chloroacetamide GPX4 inhibitors (10 μM, 30 min pretreatment in LOX-IMVI cells). Full-length western blots are shown in Supplementary Fig. 4. (H) Fluorescent labeling of proteins modified by RSL3-yne, ML162-yne, and ML210-yne probes (10 μM, 1 h) in LOX-IMVI cells. See also Supplementary Fig. 4a. (I) Co-treatment with fer-1 (1.5 μM) rescues the cell-killing effects of RSL3, ML162, and ML210 in LOX-IMVI cells. Data are plotted as mean ± s.e.m., n = 4 technical replicates.

Figure 2.. ML210 requires the intact cell context to bind GPX4

(A) RSL3, ML162, and ML210 (10 μM, 24 h) show evidence of covalent interaction with FLAG-GPX4^WT in HEK293–6E cells by intact protein mass spectrometry. Covalent adduct peaks are marked with a red asterisk (*). See also Supplementary Fig. 7b. (B) RSL3 and ML162, but not ML210, (100 μM, 2 h) covalently bind purified FLAG-GPX4^WT (10 μM) as assessed by intact protein mass spectrometry. Adduct peaks are marked with a red asterisk (*). (C) GPX4 CETSA profiles for cell lysates (HCC4006) treated with DMSO (black), RSL3 (blue), or ML210 (red). Cells were treated with 10 μM compound for 1 h. Data are plotted as mean ± s.e.m., n = 3 biologically independent samples. Representative western blots are shown in Supplementary Fig. 8a (D) RSL3-yne, but not ML210-yne, pulls down GPX4 from cell lysate. Lysates were treated for 1 h with the indicated alkyne probe (10 μM). Full gel image is shown in Supplementary Fig. 8b.

Figure 3.. ML210 requires conversion in cells to the α-nitroketoxime JKE-1674

(A) Summary of ML210 nitroisoxazole group SAR studies. Chemical structures of compounds 7–36 are shown in Supplementary Fig. 9. (B) Chemical structure of isopropyl-ML210 (27). (C) Treatment of HEK293–6E cells with isopropyl-ML210 (10 μM, 24 h) produces the same covalent GPX4 adduct mass increase (+434 Da) as ML210. (D) Scheme showing proposed ML210 hydrolysis and structure of JKE-1674 with α-nitroketoxime group highlighted in blue. (E) GPX4 CETSA of intact cells (LOX-IMVI) treated with JKE-1674 (red, 10 μM, 1 h) reveals thermal stabilization of GPX4 compared to treatment with DMSO (black). Data are plotted as mean ± s.e.m., n ≥ 4 biologically independent samples. Representative western blots are shown in Supplementary Fig. 12. (F) Treatment of cells with JKE-1674 (10 μM, 1 h) produces the same covalent GPX4 adduct (+434 Da) as ML210. (G) Co-treatment with fer-1 rescues the LOX-IMVI cell-killing effects of JKE-1674 and ML210 to a similar extent. Data are plotted as mean ± s.e.m., n = 4 technical replicates. See also Supplementary Fig. 10f.

Figure 4.. Dehydration of JKE-1674 yields a nitrile-oxide electrophile that binds GPX4

(A) Proposed structure of JKE-1777 (40) with nitrile-oxide group shown in blue. (B) Co-treatment with fer-1 rescues the cell-killing effects of JKE-1777 in LOX-IMVI cells. Data are plotted as two individual technical replicates. (C) JKE-1777 (50 μM, 1 h) is able to form a +434 Da covalent adduct with purified GPX4^U46C allCys(−) (5 μM). Adduct peak is marked with a red asterisk (*). (D) Proposed cellular transformation of masked nitrile-oxide GPX4 inhibitors ML210 and JKE-1674 into JKE-1777.

Figure 5.. Diverse masked nitrile oxides target GPX4

(A) Structure of JKE-1708 (55) with nitrolic acid group shown in blue. (B) GPX4 CETSA with intact cells (LOX-IMVI) treated with JKE-1708 (red, 10 μM, 1 h) reveals thermal stabilization of GPX4 compared to treatment with DMSO (black). Data are plotted as mean ± s.e.m., n ≥ 3 biologically independent samples. DMSO data is reproduced from Fig. 3e. Representative western blots are shown in Supplementary Fig. 18a. (C) JKE-1708 forms covalent adduct when incubated with purified GPX4 protein. Adduct peak is marked with a red asterisk (*). A second peak (denoted with red **) indicates covalent protein adduct after benzhydryl group fragmentation. See also Supplementary Fig. 11c. (D) Co-treatment with fer-1 (1.5 μM) rescues the cell-killing effects of JKE-1708 in LOX-IMVI cells. Data are plotted as mean ± s.e.m., n = 4 technical replicates. See also Supplementary Fig. 16f,g. (E) Inactive nitroalkane 50 can be synthetically transformed into the GPX4-inhibiting nitrolic acid JKE-1716 (56). (F) Viability measurements of cells treated with inactive nitroalkane 50 (red) and active nitrolic acid JKE-1716 (blue). Data are plotted as mean ± s.e.m., n = 4 technical replicates. See also Supplementary Fig. 17a. (G) Pretreatment of LOX-IMVI cells with nitrolic acids (10 μM, 30 min) prevents GPX4 pulldown by ML162-yne. Nitrolic acid-containing compounds are denoted with blue labels. Full-length western blots are shown in Supplementary Fig. 18b.

Figure 6.. Profiling of structurally diverse GPX4 inhibitors in cellular and pharmacokinetic assays

(A) GPX4 inhibitors bearing chloroacetamide, nitroisoxazole, and α-nitroketoxime warheads exhibit a similar pattern of cell killing across a range of cancer cell lines relative to control lethal agents. Nitrile-oxide precursors show enhanced rescuability by fer-1 (1.5 μM) relative to chloroacetamide GPX4 inhibitors. WM88, LOX-IMVI, CJM and U257 are human melanoma cell lines. CAKI2 and A498 are human renal cell carcinoma cell lines. HT1080 is a human fibrosarcoma cell line. MC38 is a mouse colon cancer cell line and PANC02 is a mouse pancreatic cancer cell line. (B) Compared to GPX4-targeting chloroacetamides, ML210 and JKE-1674 exhibit fewer off-target effects in LOX-IMVI cells that cannot be rescued by ferroptosis inhibitors. Data are plotted as two individual technical replicates. (C) Summary of hits identified in ML210 and RSL3 genome-wide CRISPR suppressor screens. See also Supplementary Tables 1,2. (D) In vivo PK assessment of JKE-1674 in SCID mice. Plasma concentration of JKE-1674 was determined by LCMS after oral administration of JKE-1674 (50 mg/kg) aver a 24-hour period. Data are plotted as mean ± s.d., n = 4 biologically independent samples.