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 to identify small molecules that selectively kill oncogene-expressing cell lines with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the National Institutes of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) against immortalized BJ fibroblasts expressing HRAS(G12V) followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the HRAS(G12V) oncogene. This effort led to the identification of two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potencies and 4-23-fold selectivities, which can potentially be used for identifying oncogene-specific pathways and targets in cancer cells.[2]

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We recently discovered that inhibition of the lipid peroxidase GPX4 can selectively kill cancer cells in a therapy-resistant state through induction of ferroptosis. Although GPX4 lacks a conventional druggable pocket, covalent small-molecule inhibitors are able to overcome this challenge by reacting with the GPX4 catalytic selenocysteine residue to eliminate enzymatic activity. Unfortunately, all currently-reported GPX4 inhibitors achieve their activity through reactive chloroacetamide groups. We demonstrate that such chloroacetamide-containing compounds are poor starting points for further advancement given their promiscuity, instability, and low bioavailability. Development of improved GPX4 inhibitors, including those with therapeutic potential, requires the identification of new electrophilic chemotypes and mechanisms of action that do not suffer these shortcomings. Here, we report our discovery that nitrile oxide electrophiles, and a set of remarkable chemical transformations that generates them in cells from masked precursors, provide an effective strategy for selective targeting of GPX4. Our results, which include structural insights, target engagement assays, and diverse GPX4-inhibitor tool compounds, provide critical insights that may galvanize development of improved compounds that illuminate the basic biology of GPX4 and therapeutic potential of ferroptosis induction. In addition, our discovery that nitrile oxide electrophiles engage in highly selective cellular interactions and are bioavailable in their masked forms may be relevant for targeting other currently undruggable proteins, such as those revealed by recent proteome-wide ligandability 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.)