p97/VCP ATPase complex (specifically p97-associated deubiquitination activity), Sec61 translocon. Eeyarestatin I inhibits p97-associated deubiquitination (PAD) by associating with the p97 complex, and also inhibits co-translational protein translocation into the endoplasmic reticulum by targeting the Sec61 complex. No specific IC₅₀ or Kᵢ values were reported in these studies. [2,3]
Endoplasmic reticulum-associated protein degradation (ERAD)

Inhibition of protein secretion: In HepG2 cells treated with 8 μM EerI for 1 hour, secretion of glycoproteins was almost completely blocked, as determined by ConA lectin binding assay. Immunoprecipitation of α1-antitrypsin from the media confirmed this effect. The effect was more pronounced than that of brefeldin A (5 μg/mL). [3]
Inhibition of ER translocation in cultured cells: In HeLa cells expressing TCRα, treatment with 8 μM EerI for 1 hour substantially reduced the level of N-glycosylated TCRα, accompanied by an increase in non-glycosylated protein. This was confirmed using an in vitro translocation assay with semi-permeabilized cells, where EerI pretreatment inhibited signal sequence cleavage of preprolactin (pPL) and N-glycosylation of a glycosylated proteolipid protein (gPLP). [3]
Inhibition of co-translational translocation in microsomes: In canine pancreatic rough microsomes treated with 250 μM EerI, the translocation of pPL was almost completely blocked, as determined by signal sequence cleavage and protease protection assays. EerI inhibited translocation of a wide range of membrane proteins (ASGPr, SPP, US11, P2X₂, PL, HA-TCRα, gCytB5) that utilize the Sec61-dependent co-translational pathway, but did not affect the Sec61-independent post-translational integration of a glycosylated cytochrome b5 (gCytB5) variant. [3]
Inhibition of p97-associated deubiquitination (PAD): In 293T cells treated with EerI (10 μM, 14 h), p97 immunoprecipitates contained significantly more polyubiquitinated proteins. In vitro deubiquitination assays showed that p97-associated substrates from EerI-treated cells were only reduced by ~10% after incubation, compared to ~60% from control cells. EerI also inhibited ataxin-3 (atx3)-mediated deubiquitination of p97-associated substrates, but did not affect HAUSP-associated deubiquitination. [2]
Accumulation of polyubiquitinated proteins: In A9 cells expressing US11 and HA-tagged MHC class I heavy chain (HC), treatment with EerI (10 μM, 14 h) led to accumulation of polyubiquitinated HC in the cytosol. Similar accumulation was observed for TCRα-GFP and a soluble cytosolic proteasome substrate, GFPµ (a GFP fusion with a degradation signal derived from the µ chain of IgM). The degradation of GFPµ, which is atx3-dependent, was delayed by EerI, whereas the degradation of Ub-R-GFP (atx3-independent) was not affected. [2]
Mechanism of action on p97 complex: EerI did not disrupt the interaction of p97 with Derlin-1, VIMP, Ufd1, or atx3. However, an unknown ~180 kDa protein was found to interact with p97 in untreated cells but not in EerI-treated cells, and this interaction was restricted to the membrane fraction. EerI was shown to associate with the p97 complex, as fluorescence emitted from EerI (the compound is fluorescent) was detected in p97 and atx3 immunoprecipitates from EerI-treated cells. [2]

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A549 and H358 cells treated with eeyarestatin I (2.5–40 μM; 48 hours) experience quantitative essential cell death [1]. Treatment of A549 and H358 cells with eeyarestatin I (2.5–40 μM; 48 hours) resulted in a rise in endoplasmic reticulum (ER) intermediate markers, including Bip and CHOP at as low as 20 μM. Important proteins, such as IRE1α and PERK, are ubiquitinated in a dose-dependent manner when treated with eeyarestatin I[1]. Treatment with eeyarestatin I (20 μM; 48 hours; A549 and H358 cells) causes cell invasion and migration [1]. By most likely deactivating the Sec61 complex, eeyarestatin 1 inhibits the transfer of immature proteins from membrane complexes to the ER translocation machinery [2].

Metabolic labeling and secretion assay: HepG2 cells were treated with EerI (8 μM, 1 hour), starved in methionine/cysteine-free medium, and pulse-labeled with [³⁵S]Met/Cys (22 μCi/mL) for 30 minutes. After a 90-minute chase, media were collected and glycoproteins were isolated using ConA-Sepharose and analyzed by SDS-PAGE and autoradiography. [3]
Immunoprecipitation of specific proteins: Cells expressing HA-tagged MHC class I heavy chain or FLAG-tagged atx3 were lysed under denaturing conditions (0.2% SDS, 5 mM NEM, heated at 95°C) to preserve ubiquitination. Proteins were immunoprecipitated with anti-HA or anti-FLAG antibodies and analyzed by immunoblotting with anti-ubiquitin antibodies. [2]
Subcellular fractionation: A9 cells treated with EerI (10 μM, 14 h) were permeabilized with 0.028% digitonin in the presence of an ATP-regenerating system. Samples were separated into membrane pellet (P) and supernatant (S) fractions by centrifugation. HC was immunoprecipitated from each fraction under denaturing conditions. [2]
In vitro deubiquitination assay: p97 or atx3 complexes were immunoprecipitated from control or EerI-treated 293T cells. The immunoprecipitates were incubated at 37°C for 0–60 minutes in the presence or absence of ATP. The reaction was stopped by adding SDS sample buffer, and samples were analyzed by immunoblotting with anti-ubiquitin antibodies to monitor the loss of polyubiquitinated substrates. [2]
In vitro translocation assay using semi-permeabilized cells: HeLa cells treated with EerI (8 μM, 1 hour) were harvested, washed, and semi-permeabilized with 20 μg/mL digitonin. These cells were then used as a source of ER membranes in an in vitro translation reaction containing rabbit reticulocyte lysate, [³⁵S]Met/Cys, and mRNA encoding preprolactin or glycosylated proteolipid protein (gPLP). Translocation was assessed by signal sequence cleavage (pPL) or N-glycosylation (gPLP). [3]
Protease protection assay: In vitro translation reactions containing ER microsomes were treated with 500 μg/mL proteinase K for 1 hour on ice, in the presence or absence of 1% Triton X-100. The reaction was stopped with 1 mM PMSF, and samples were analyzed by SDS-PAGE. [3]
Cross-linking analysis of nascent chain transfer: Truncated P2X₂ [Q56C] ribosome-nascent chain (RNC) complexes were synthesized in rabbit reticulocyte lysate and added to ER membranes treated with DMSO or 250 μM EerI. Cross-linking was induced with 1 mM bismaleimidohexane (BMH). After immunoprecipitation with anti-HA antibodies, cross-linked adducts between the nascent P2X₂ chain and Sec61α or SRP54 were analyzed. [3]
EerI fluorescence detection: EerI is a yellow compound that emits fluorescence when excited at 488 nm. This property was used to detect EerI association with p97 complexes by measuring fluorescence in immunoprecipitates from EerI-treated cells. [2]

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Viability assay [1]
Cell Types: A549 and H358 cells
Tested Concentrations: 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Caused dose-dependent cell death of A549 and H358 cells.

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Western Blot Analysis[1]
Cell Types: A549 and H358 Cell
Tested Concentrations: 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Increased ER stress markers including Bip and CHOP.

[1]. Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress. Oncotarget. 2015 Jul 10;6(19):17725-37.

[2]. Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum. J Cell Sci. 2009 Dec 1;122(Pt 23):4393-400.

[3]. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J Biol Chem. 2008 Mar 21;283(12):7445-54.

[4]. ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in cancer cells. Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2200-5.

[5]. Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway. Int J Mol Sci. 2021 Nov 5;22(21):12007.

Dual mechanism of action: Eeyarestatin I is a small molecule with at least two distinct cellular targets. It inhibits co-translational protein translocation into the ER by targeting the Sec61 translocon, preventing the transfer of nascent polypeptides from the SRP targeting complex to the Sec61 complex. It also inhibits ER-associated degradation (ERAD) by targeting p97-associated deubiquitination (PAD), specifically affecting ataxin-3-mediated deubiquitination of substrates extracted by p97. [2,3]
Substrate specificity of translocation inhibition: Unlike the cyclodepsipeptides CAM741 and cotransin, which show substrate-specific inhibition, EerI broadly inhibits the translocation of many Sec61-dependent substrates, including ASGPr, SPP, US11, P2X₂, PL, and HA-TCRα, while having no effect on the Sec61-independent integration of gCytB5. [3]
Requirement for cellular metabolism/activation: EerI appears to require cellular metabolism to become active, as it failed to inhibit retrotranslocation when added directly to permeabilized US11 cells, and trypsin-treated cells no longer responded to EerI. The compound itself is fluorescent and can be taken up by cells, but its active species remains to be identified. [2]
Relationship between ERAD and translocation inhibition: The accumulation of polyubiquitinated MHC class I heavy chain in the cytosol of EerI-treated cells suggests that EerI inhibits a step after dislocation, rather than blocking dislocation itself. This contrasts with a previous report that proposed EerI blocks dislocation. The discrepancy may be due to different experimental approaches (steady-state vs. pulse-chase analysis). [2]
Potential therapeutic application: EerI has been shown to induce ER stress and may have anti-cancer activity similar to the proteasome inhibitor bortezomib. Its ability to inhibit p97-associated deubiquitination and Sec61-mediated translocation makes it a potentially useful tool for studying protein homeostasis and a candidate for further development as an anti-cancer agent. [2,3]

C27H25CL2N7O7

630.436103582382

490.136

C, 51.44; H, 4.00; Cl, 11.25; N, 15.55; O, 17.76

412960-54-4

5003929

Yellow to orange solid powder

1.4±0.1 g/cm3

1.643

3.63

3

8

8

43

1090

0

ClC1C=CC(=CC=1)N1C(N(CC(NN=CC=CC2=CC=C([N+](=O)[O-])O2)=O)C(C)(C)C1N(C(NC1C=CC(=CC=1)Cl)=O)O)=O

JTUXTPWYZXWOIB-UHFFFAOYSA-N

InChI=1S/C27H25Cl2N7O7/c1-27(2)24(35(40)25(38)31-19-9-5-17(28)6-10-19)34(20-11-7-18(29)8-12-20)26(39)33(27)16-22(37)32-30-15-3-4-21-13-14-23(43-21)36(41)42/h3-15,24,40H,16H2,1-2H3,(H,31,38)(H,32,37)

2-[3-(4-chlorophenyl)-4-[(4-chlorophenyl)carbamoyl-hydroxyamino]-5,5-dimethyl-2-oxoimidazolidin-1-yl]-N-[3-(5-nitrofuran-2-yl)prop-2-enylideneamino]acetamide

Eeyarestatin I; 412960-54-4; DTXSID20849579; RefChem:339570; DTXCID701323513;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: 25~85 mg/mL (39.7~134.8 mM)
Ethanol: ~7 mg/mL (~11.1 mM)

Solubility in Formulation 1: 1.25 mg/mL (1.98 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 12.5 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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 (Please use freshly prepared in vivo formulations for optimal results.)