GPX4/glutathione peroxidase 4

In cellular target engagement experiments, JKE-1674 and ML210 showed identical action, including the generation of the same +434Da GPX4 adduct in cells. Similar to ML210, JKE-1674 killed LOX-IMVI cells, however ferroptosis inhibitors totally prevented this death. In cells, JKE-1674 creates oxynitrile electrophiles. JKE-1674 dehydrogenases to form an oxidized nitrile electrophile that binds GPX4. Compared to inhibitors of chloroacetamide, JKE-1674 has greater stability [1].

JKE-1674 can be found in the serum of mice treated the drug orally (50 mg/kg; oral) [1].

GPX4 activity assay.[1]
A mass spectrometry-based GPX4 enzymatic activity assay was adapted from a previously described procedure4. LOX-IMVI cells were treated with indicated compounds (10 μM) or DMSO for 1 h at 37 °C. Cells were washed with PBS and lysed by freeze-thaw method (x3) in GPX4 reaction buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 1 mM EDTA, 0.1 mM DFO; pH 7.4). Lysates were cleared by centrifugation (10 min, 20000 × g, 4 °C) and total protein concentration was adjusted to 1.67 mg/mL. Typical enzymatic activity assay mixtures were prepared as follows: 200 μL lysate (1.67 mg/mL in GPX4 reaction buffer), 2 μL of PCOOH in MeOH, and 20 μL GSH solution (100 mM; ~5 mM final concentration). Reactions were vortexed briefly and incubated at 37 °C for 15 min. Reaction mixtures were then extracted using 250 μL of a 2:1 chloroform/methanol (v/v) solution. Extracts were dried under a stream of nitrogen and reconstituted in methanol before analysis. [1]
LC-MS analysis was performed with Acquity RP UPLC system coupled to a Xevo G2XS QToF mass spectrometer. Reconstituted extract was separated on a Waters Acquity RP UPLC BEH-C18 column (2.1 × 50 mm; 1.7 μm particle size; 45 °C). Mobile phase consisted of 10 mM aqueous ammonium acetate (solvent A) and 95:5 acetonitrile/10 mM ammonium acetate (solvent B). The total run time was 8 minutes. UPLC eluate was introduced into the mass spectrometer by positive mode electrospray ionization. Source settings were 120 °C, 50 V cone voltage, 1 kV capillary voltage, 500 °C desolvation temperature, and 1100 L/h desolvation gas flow. Mass spectrometry experiments were performed in sensitivity mode with a resolution of 20,000 and a mass accuracy of <1 ppm. The lockmass (Leu-Enk, m/z 556.2771) was infused continuously at 5 μL/minute and sampled every 15 seconds. MassLynx and TargetLynx software were used for analysis of mass spectra, PCOOH identification, and chemical formula confirmation analysis.

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Cellular thermal shift assay (CETSA).[1]
For intact-cell CETSA experiments, cells were pretreated with 10 μM compound or DMSO control (0.1%, v/v) for 1 h at 37 °C. Media was then aspirated and cells were washed with PBS (pH 7.4). Adherent cells were detached from the flask with trypsin-EDTA and pelleted by centrifugation (500 × g, 5 min). Cells were aliquoted into PCR tubes (50 μL volume, ~1 million cells/condition) for heating at different temperatures (typically 40–67 °C in 3 °C increments) in a thermocycler for 3 minutes. Samples were allowed to cool to room temperature for an additional 3 minutes. Cells were lysed by either three freeze-thaw cycles in liquid nitrogen, or by the addition of Triton X-100 solution (1% final TX-100 volume, PBS pH 7.4) and subsequent incubation on ice for 20 minutes with occasional vortexing. After lysis, cells were centrifuged (20 minutes at 20,000 rcf, 4 °C) to remove insoluble material. The soluble fraction was carefully separated and diluted with 6x SDS loading buffer for SDS-PAGE and western blotting analysis.

Cell viability experiments were performed by seeding 1000 cells/well (30 μL volume) in opaque white 384-well plates. Cells were allowed to adhere for 24 h after which they were exposed to compounds for 72 h. DMSO stock solutions of compounds were added to cells using an CyBio Well Vario liquid dispenser. Cellular ATP levels were measured using CellTiter-Glo as a surrogate for viability. Rescue experiments were performed using ferrostatin-1 (fer-1; 1.5 μM), liproxstatin-1 (lip-1; 1 μM), deferoxamine (DFO; 50 μM), or zileuton (10 μM) added to cells at the time of seeding. [1]

Animal/Disease Models: SCID (severe combined immunodeficient) mouse [1]
Doses: 50 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: Po
Experimental Results: It can be detected in the serum of mice that took the compound orally.

[1]. Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles. Nat Chem Biol. 2020;16(5):497-506.

[2]. A masked zinger to block GPX4. Nat Chem Biol. 2020;16(5):482-483.

[3]. Unraveling Masked GPX4 Inhibitors. Nat. Chem. Biol. 2020, 16, 497–506.

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.[1]

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Ferroptosis induced by GPX4 inhibition offers promise for killing drug-resistant cancer cells, yet current GPX4 inhibitors lack selectivity. The discovery of masked nitrile oxide electrophiles as selective prodrug inhibitors of GPX4 points to an attractive path for chemically inducing ferroptosis.[2]

C20H20CL2N4O4

451.303202629089

450.086

C, 53.23; H, 4.47; Cl, 15.71; N, 12.41; O, 14.18

2421119-60-8

145865941

Off-white to light yellow solid powder

1.4±0.1 g/cm3

595.5±60.0 °C at 760 mmHg

313.9±32.9 °C

0.0±1.8 mmHg at 25°C

1.651

3.35

1

6

5

30

599

0

C1CN(CCN1C(C2=CC=C(C=C2)Cl)C3=CC=C(C=C3)Cl)C(=O)/C(=N/O)/C[N+](=O)[O-]

XIOHKIAPXVDWCP-PTGBLXJZSA-N

InChI=1S/C20H20Cl2N4O4/c21-16-5-1-14(2-6-16)19(15-3-7-17(22)8-4-15)24-9-11-25(12-10-24)20(27)18(23-28)13-26(29)30/h1-8,19,28H,9-13H2/b23-18+

(2E)-1-[4-[bis(4-chlorophenyl)methyl]piperazin-1-yl]-2-hydroxyimino-3-nitropropan-1-one

JKE-1674; (E)-1-(4-(bis(4-chlorophenyl)methyl)piperazin-1-yl)-2-(hydroxyimino)-3-nitropropan-1-one; 2421119-60-8; (2E)-1-[4-[bis(4-chlorophenyl)methyl]piperazin-1-yl]-2-hydroxyimino-3-nitropropan-1-one; SCHEMBL25854799; JKE1674;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~221.58 mM)
Ethanol :≥ 50 mg/mL (~110.79 mM)

Solubility in Formulation 1: 5 mg/mL (11.08 mM) in 10% EtOH + 90% PEG400 (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)