GPX4/Glutathione Peroxidase 4

In a panel of 821 cancer cells (WM88, LOX-IMVI, CJM, U257, CAKI2, A498, HT1080, MC38, PANC02), ML-210 shown cell-killing ability. One prodrug that needs cells is ML-210. ML-210 has an IC50 of 71 nM, 272 nM, and 107 nM (without HRASV12) against BJeLR (HRASV12), BJeH-LT, and DRD cell lines, respectively [2].

Given that traditional anticancer therapies fail to significantly improve the prognoses of triple negative breast cancer (TNBC), new modalities with high efficiency are urgently needed. Herein, by mixing the metal-phenolic network formed by tannic acid (TA), bleomycin (BLM), and Fe3+ with glutathione peroxidase 4 (GPX4) inhibitor (ML210) loaded hollow mesoporous Prussian blue (HMPB) nanocubes, the HMPB/ML210@TA-BLM-Fe3+ (HMTBF) nanocomplex is prepared to favor the ferroptosis/apoptosis synergism in TNBC. During the intracellular degradation, Fe3+ /Fe2+ conversion mediated by TA can initiate the Fenton reaction to drastically upregulate the reactive oxygen species level in cells, subsequently induce the accumulation of lipid peroxidation, and thereby cause ferroptotic cell death; meanwhile, the released ML210 efficiently represses the activity of GPX4 to activate ferroptosis pathway. Besides, the chelation of Fe2+ with BLM leads to in situ BLM toxification at tumor site, then triggers an effective apoptosis to synergize with ferroptosis for tumor therapy. As a result, the superior in vivo antitumor efficacy of HMTBF is corroborated in a 4T1 tumor-bearing mice model regarding tumor growth suppression, indicating that the nanoformulations can serve as efficient ferroptosis and apoptosis inducers for use in combinatorial TNBC therapy.https://pubmed.ncbi.nlm.nih.gov/34623753/

[1]. Targeting a Therapy-Resistant Cancer Cell State Using Masked Electrophiles as GPX4 Inhibitors. Biorxiv. 2018.

[2]. Development of small-molecule probes that selectively kill cells induced to express mutant RAS. Bioorg Med Chem Lett. 2012 Feb 15;22(4):1822-6.

Synthetic lethal screening is a chemical biology approach designed to identify small molecules that can selectively kill cell lines expressing oncogenes, thereby discovering signaling pathways that can serve as anticancer targets. We performed high-throughput screening of 303,282 compounds from the NIH-MLSMR library of molecular libraries in the United States, targeting immortalized BJ fibroblasts expressing HRAS(G12V). Subsequently, we performed reverse screening on a series of homologous cells lacking the HRAS(G12V) oncogene to verify the activity of these lethal compounds. This study ultimately identified two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potency and 4-23 times selectivity, which are expected to be used to identify oncogene-specific pathways and targets in cancer cells. [2] We recently discovered that inhibition of lipid peroxidase GPX4 can selectively kill drug-resistant cancer cells by inducing ferroptosis. Despite the lack of a traditional drug-binding pocket for GPX4, covalent small-molecule inhibitors have overcome this challenge by eliminating the enzyme's activity through a reaction with the catalytic selenocysteine residues of GPX4. However, all currently reported GPX4 inhibitors are active via an active chloroacetamide group. We demonstrate that such chloroacetamide compounds are not ideal starting points for further development due to their substrate heterogeneity, poor stability, and low bioavailability. Developing more effective GPX4 inhibitors, including those with therapeutic potential, requires the identification of novel electrophilic types and mechanisms of action to overcome these limitations. Here, we report our findings: nitrile oxide electrophilic reagents and a series of remarkable chemical transformations that generate these reagents intracellularly from masked precursors provide an effective strategy for selectively targeting GPX4. Our results, including structural resolution, target binding experiments, and a variety of GPX4 inhibitor tool compounds, provide key insights that promise to drive the development of more effective compounds capable of elucidating the fundamental biological characteristics of GPX4 and its therapeutic potential in inducing ferroptosis. In addition, we found that nitrile oxide electrophiles can engage in highly selective cell interactions and have bioavailability in a masked form, which may be related to targeting other proteins that are currently undrugable, such as those revealed by recent proteome ligand accessibility studies. [1]

C22H20CL2N4O4

475.326

474.086

C, 55.59; H, 4.24; Cl, 14.92; N, 11.79; O, 13.46

1360705-96-9

1360705-96-9;

49766530

White to light yellow solid powder

1.4±0.1 g/cm3

644.7±55.0 °C at 760 mmHg

343.7±31.5 °C

0.0±1.9 mmHg at 25°C

1.636

2.39

0

6

4

32

635

0

VIBHJPDPEVVDTB-UHFFFAOYSA-N

InChI=1S/C22H20Cl2N4O4/c1-14-20(28(30)31)19(25-32-14)22(29)27-12-10-26(11-13-27)21(15-2-6-17(23)7-3-15)16-4-8-18(24)9-5-16/h2-9,21H,10-13H2,1H3

[4-[Bis(4-chlorophenyl)methyl]piperazin-1-yl]-(5-methyl-4-nitro-1,2-oxazol-3-yl)methanone

CID 49766530; ML-210; CID-49766530; [4-[bis(4-chlorophenyl)methyl]piperazin-1-yl]-(5-methyl-4-nitro-1,2-oxazol-3-yl)methanone; ML210; (4-(bis(4-chlorophenyl)methyl)piperazin-1-yl)(5-methyl-4-nitroisoxazol-3-yl)methanone; CHEMBL1951048; BRD7528; ML 210; CID49766530; ML210.

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 : ~25 mg/mL (~52.60 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.26 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.38 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 20.8 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.)