GPX4[1]

Researchers 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].

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 novel 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].

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.
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.

---

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.
For lysate CETSA experiments, lysate from untreated cells was prepared as described in the intact-cell CETSA protocol and diluted with PBS (pH 7.4) to a total protein concentration of 1 mg/mL. Samples were treated with 10 μM compound (or 0.1% v/v DMSO control) for 1.5 h at 37 °C. After compound treatment, samples were aliquoted (50 μL per sample) into PCR tubes processed as described above for intact-cell CETSA experiments.

---

In-cell GPX4 mass spectrometry binding assay.[1]
HEK293–6E cells were transfected with GPX4_WT_FLAG-pTT5/SPB2-pTT5 in 24-well plates. Cells were harvested 72 h following seeding and compounds were added 1, 4 or 24 h before cell harvest. For each time-point, compounds were added at the following concentrations: 1, 10, or 20 μM. Viability of transfected and compound-treated cells was monitored. Cells were harvested by centrifugation and lysed in lysis buffer (a pH 7.4 solution of 50 mM sodium phosphate, 300 mM NaCl, and 0.1% NP-40 supplemented with Roche Complete protease inhibitor cocktail). GPX4 was purified by anti-FLAG chromatography as described for the large-scale preparation of FLAG-GPX4WT. Denaturing MS analysis of purified GPX4 samples was performed with a SYNAPT G2-S quadrupole time-of-flight mass spectrometer connected to a nanoAcquity UPLC system (Waters). Samples were loaded on a 2.1 × 5 mm mass prep C4 guard column and desalted with a short gradient (3 min) of increasing concentrations of acetonitrile at a flow rate of 100 μL/min. Spectra were analyzed by using MassLynx v4.1 software and deconvoluted with the MaxEnt1 algorithm.

---

GPX4 mass spectrometry binding assay with purified GPX4.[1]
Recombinant FLAG-GPX4WT protein (10 μM in pH 7.4 buffer containing 50 mM sodium phosphate and 300 mM NaCl) was incubated with 100 μM compound (1% v/v final DMSO concentration) at room temperature for 2 h. The reaction was quenched by adding 1 μL 5% v/v TFA to 15 μL reaction volume and was subjected to LC-MS analysis as described for the in-cell GPX4 MS binding assay. Binding experiments with GPX4U46C allCys(−) were performed as described previously

Cell treatment for GPX4 pulldown and ABPP experiments.
LOX-IMVI cells were seeded in 6-well plates in RPMI media supplemented with 10% FBS. Alkyne affinity probes were added to the cells and incubated for 1 h at 37 °C (10 mM stocks in DMSO, final alkyne concentration = 10 μM). Media was removed and cells were washed once with ice-cold PBS (pH 7.4). Cells were then lysed in PBS containing 1% Triton X-100 and protease inhibitors (Roche Complete) for 20 min on ice. Samples were cleared by centrifugation (20,000 × g, 4 °C). Total protein content of lysates was assessed with a Pierce Coomassie Plus Bradford assay kit and adjusted to 2 mg/mL.
For lysate pulldown experiments, LOX-IMVI cell lysates were prepared as described above from untreated cells and adjusted to a total protein concentration of 2 mg/mL. Lysates were treated with the indicated alkyne probe (10 μM, 1 h) or DMSO at ambient temperature and then used immediately for subsequent click chemistry reactions.

---

Sample processing for GPX4 pulldown assay.
Lysates were subjected to copper-catalyzed azide-alkyne cycloaddition (CuAAC) conditions with azide-PEG3-biotin conjugate. Typical reactions were performed with a final volume of 120 μL consisting of 100 μL lysate (2 mg/mL; final concentration = 1.67 mg/mL), 2.4 μL SDS (10%; final concentration = 0.2%), 2.4 μL azide-PEG3-biotin conjugate (5 mM in DMSO; final concentration = 100 μM), and 15.2 μL of catalyst mix (final concentration = 1.3 mM Cu2SO4, 1.3 mM TCEP, and 75 μM TBTA). The catalyst mix stock was prepared by mixing 3 parts tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-amine (TBTA; 1 mM in 1:4 DMSO/tBuOH), 1 part 50 mM Cu2SO4 in water, and 1 part tris(2-carboxyethyl)phosphine (TCEP; 50 mM in water, pH 7.0). After addition of all components, CuAAC reactions were vortexed and allowed to react at ambient temperature for 1 h and then diluted with 120 μL 0.2% SDS in PBS. A 40 μL aliquot was removed and quenched with 6x SDS sample buffer as an input control. Pierce highcapacity streptavidin agarose beads were added to the remaining sample and rotated overnight at 4 °C. Beads were then separated by centrifugation and washed sequentially with 1% SDS (3 × 1 mL) and PBS (2 × 1 mL). Proteins were eluted by boiling the beads in 75 μL of 2x SDS sample buffer for 10 minutes and analyzed by SDS-PAGE and western blotting.

Pharmacokinetic (PK) studies in mice.[1]
To determine the pharmacokinetic properties of JKE-1674 in SCID mice after oral administration, blood samples for analysis of plasma were taken from three animals/time point at 1, 3, 6 and 24 h after single oral dose of JKE-1674 at 50 mg/kg formulated in PEG400/Ethanol (90/10, v/v). Blood was collected into tubes containing lithium heparin, centrifuged to isolate plasma, precipitated with acetonitrile (1/5, v/v) and analyzed by LC-MS/MS.

---

Toxicity assessment in mice.[1]
Tolerability of JKE-1674 was assessed preceding pharmacokinetic measurements. Over a period of 7 consecutive dosing days, average body weight loss in mice which received 50 mg/kg active compound did not exceed 10%. Doses higher than 50 mg/kg were not tolerated in this experimental setting.

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

GPX4 is a selenoprotein that plays a critical role in protecting cells from lipid peroxidation and ferroptosis by reducing lipid hydroperoxides to their corresponding lipid alcohols. GPX4 is unique in its ability to reduce complex lipid hydroperoxides and is the sole mammalian protein known thus far to be capable of performing this crucial function5,6. The broad substrate scope of GPX4 likely derives from its monomeric structure and the relatively flat surface adjacent to the catalytic selenocysteine residue in the active site[1].
Unfortunately, these same structural features also make GPX4 a challenging target for the development of small-molecule inhibitors. The lack of a drug-like binding pocket and reliance on a nucleophilic selenocysteine residue for enzymatic activity suggest that covalent inhibitors may be necessary for inhibition of cellular GPX4. Indeed, all known direct and cell-active GPX4 inhibitors are alkylating agents that covalently bind the selenocysteine residue via an activated alkyl chloride (Fig. 1a and Supplementary Fig. 1). Such inhibitors exhibit low selectivity and poor pharmacokinetic properties. This limits their utility as tool compounds in vitro, hinders their use in vivo, and makes them undesirable starting points for the development of drug-like GPX4 inhibitors. It is further notable that cellular and biochemical screening efforts have not identified other common electrophiles, such as acrylamides, that can serve as selective GPX4-targeting warheads. Likewise, replacing the chloroacetamide warheads with less reactive electrophiles produces analogs that no longer inhibit GPX4[1].
The toolkit of GPX4 inhibitors uncovered by our mechanistic studies represents an unprecedented wealth of diverse GPX4-targeting chemotypes (nitroisoxazoles, α-nitroketoximes, and nitrolic acids). These compounds promise to overcome a major shortcoming of the GPX4-biology field, namely, reliance on a single non-specific chemotype (chloroacetamides) to perform and interpret biological experiments. We therefore encourage scientists probing GPX4 and ferroptosis biology to incorporate into their experimental designs the chemically diverse GPX4 inhibitors we have illuminated. This approach promises to mitigate chemotype-specific off-target effects and enable biological discoveries that require highly selective ferroptosis induction such as that achieved by masked nitrile-oxide GPX4 inhibitors[1].
Finally, we expect that the numerous tool compounds, mechanistic insights, cellular target engagement assays, and biochemical data we make available will be catalytic for the development of improved GPX4-inhibiting small molecules by the academic and pharmaceutical communities.

C25H22CL2N2O3S

501.424783229828

500.072

C, 59.88; H, 4.42; Cl, 14.14; N, 5.59; O, 9.57; S, 6.39

2883115-46-4

145865959

White to off-white solid powder

5.3

1

4

10

33

694

0

AFUOBFISLHQJJJ-UHFFFAOYSA-N

InChI=1S/C25H22Cl2N2O3S/c1-2-14-32-21-11-10-19(16-20(21)27)29(23(30)17-26)24(22-9-6-15-33-22)25(31)28-13-12-18-7-4-3-5-8-18/h1,3-11,15-16,24H,12-14,17H2,(H,28,31)

2-(3-chloro-N-(2-chloroacetyl)-4-prop-2-ynoxyanilino)-N-(2-phenylethyl)-2-thiophen-2-ylacetamide

ML162-yne; 2883115-46-4; 2-Chloro-N-(3-chloro-4-(prop-2-yn-1-yloxy)phenyl)-N-(2-oxo-2-(phenethylamino)-1-(thiophen-2-yl)ethyl)acetamide; 2-{2-chloro-N-[3-chloro-4-(prop-2-yn-1-yloxy)phenyl]acetamido}-N-(2-phenylethyl)-2-(thiophen-2-yl)acetamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (199.43 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.99 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.

---

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