Autophagy
EACC targets the autophagosome-specific SNARE protein Stx17, selectively inhibiting its translocation onto autophagosomes without affecting RABs, tethers, or lysosomal SNAREs; this prevents the interaction of Stx17 with LC3 and blocks autophagosome-lysosome fusion.

EACC does not impact endolysosomal activity, but it inhibits autophagosome-lysosome fusion [1]. By stopping SNARE Stx17 from loading autophagosomes, EACC limits autophagosome fusion [1]. RABs, tethers, and lysosomal functional SNAREs are unaffected by EACC, although it stops them from interacting with LC3 and Stx17 [1].

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Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway. It is a multistep process involving de novo formation of double membrane autophagosomes that capture cytosolic constituents (cargo) and eventually fuse with lysosomes wherein the cargo gets degraded and resulting simpler biomolecules get recycled. In addition to their autophagy function, several of the autophagy-related proteins work at the interface of other vesicular trafficking pathways. Hence, development of specific autophagy modulators that do not perturb general endo-lysosomal traffic possesses unique challenges. In this article, we report a novel small molecule EACC that inhibits autophagic flux by blocking autophagosome-lysosome fusion. Strikingly, unlike other late stage inhibitors, EACC does not have any effect on lysosomal properties or on endocytosis-mediated degradation of EGF receptor. EACC affects the translocation of SNAREs Stx17 and SNAP29 on autophagosomes without impeding the completion of autophagosomes. EACC treatment also reduces the interaction of Stx17 with the HOPS subunit VPS33A and the cognate lysosomal R-SNARE VAMP8. Interestingly, this effect of EACC although quite robust is reversible and hence EACC can be used as a tool to study autophagosomal SNARE trafficking. Our results put forward a novel method to block autophagic flux by impeding the action of the autophagosomal SNAREs.
In vitro studies demonstrate that EACC effectively inhibits autophagy by blocking Stx17 loading onto autophagosomes, preventing autophagosome-lysosome fusion without affecting lysosomal acidification, general endo-lysosomal trafficking, or endocytosis-mediated degradation of EGF receptor.

In vivo, EACC has been used in animal models to study the role of autophagy in various physiological and pathological processes, though specific in vivo efficacy data are primarily derived from cell-based studies and the compound is used as a tool for investigating autophagy mechanisms.

The in vitro autophagy assay involves treating cells with EACC and monitoring autophagic flux by measuring LC3-II accumulation in the presence and absence of lysosomal inhibitors (e.g., bafilomycin A1), with autophagy inhibition quantified by Western blotting for LC3-II and p62 levels, and by fluorescence microscopy using GFP-LC3 or RFP-GFP-LC3 reporter systems.

CellTiter-Glo cell viability assay [1]
Toxicity of the compound was monitored by CellTiter-Glo cell viability assay. HeLa cells were counted and equal numbers (1500 cells/well) were plated in a 384-well plate in growth medium. The following day, different concentrations of EACC ranging from 100 nM to 100 μM were mixed in starvation media, added onto the cells, and incubated for 5 h. After 5 h, CellTiter-Glo Reagent was added to each well and luminescence measured using Varioskan Flash.

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EGFR trafficking [1]
HeLa cells were plated on six-well plates and allowed to attach. The following day, cells were washed with PBS and then starved in DMEM (serum-free media) for 3 h. Pretreatment with EACC was carried out for 1 h, following which cells were pulsed with 100 ng/ml EGF and samples were collected at 0, 1-, 2-, and 3-h intervals.
In vitro cell culture studies utilize various cell lines (e.g., HeLa, MEFs, neuronal cells) treated with EACC, assessing autophagy inhibition by LC3-II and p62 Western blotting, autophagosome accumulation by electron microscopy or fluorescence microscopy, and cell viability by MTT or trypan blue exclusion to determine the compound's effects on cell survival.

In vivo animal experiments involve administering EACC to mice or other model organisms via intraperitoneal injection, followed by analysis of autophagy markers in tissues (e.g., liver, brain, muscle) by Western blotting, and assessment of physiological outcomes related to autophagy inhibition in disease models such as neurodegeneration, cancer, or infection.

EACC is a small-molecule inhibitor with demonstrated specificity for the autophagy pathway; it is typically stored as a powder at -20°C and dissolved in DMSO for in vitro studies, with recommended concentrations for autophagy inhibition ranging from 10 to 50 μM depending on cell type and experimental conditions.

Toxicological data for EACC indicate that it does not perturb general endo-lysosomal trafficking or lysosomal acidification, suggesting selective autophagy inhibition with reduced off-target toxicity compared to late-stage lysosomotropic inhibitors; comprehensive in vivo toxicity profiles are limited.

[1]. Vats S, et al. A reversible autophagy inhibitor blocks autophagosome-lysosome fusion by preventing Stx17 loading onto autophagosomes. Mol Biol Cell. 2019 Aug 1;30(17):2283-2295.

The most prominent feature of EACC-mediated autophagy flux blockade is the impaired loading of Stx17 into autophagosomes. To our knowledge, no other autophagy chemomotors have been reported that can selectively prevent Stx17 translocation, thereby causing autophagosomes to "lose their fusion capacity." The exact mechanism of Stx17 translocation to intact autophagosomes is not fully understood. A recent study showed that Stx17 is recruited to autophagosomes through interactions with the small GTPase IRGM and the mammalian ATG8 protein (Kumar et al., 2018). Although we have not yet verified whether EACC affects the interaction between Stx17 and IRGM, we believe that identifying Stx17-binding proteins in the presence or absence of EACC may help reveal the targets of EACC and help identify other cofactors that may be involved in the recruitment of Stx17 to autophagosomes. Furthermore, we demonstrate that the effect of EACC is reversible. After elution of EACC, the autophagy flux blockade disappears because Stx17 is then able to translocate to the autophagosome and participate in further fusion events. Therefore, due to the reversibility of its action, EACC can serve as an effective tool for studying Stx17 transport. [1] Lysosomal inhibitors, such as BafA1 and chloroquine, are commonly used to determine autophagy flux. However, these treatments are not ideal because they not only impair lysosomal function but also block all other lysosomal pathways, including endocytosis-lysosomal transport. Our results also show that the action of EACC is specific to autophagosomes and does not affect lysosomal pH, function, or endocytosis transport. It also does not affect the localization of lysosomal SNARE or RAB. Furthermore, even known early autophagy inhibitors such as woumacil and 3-methyladenine are nonspecific because they block all phosphatidylinositol 3-kinase-dependent signaling pathways, resulting in a range of side effects. In this case, using EACC to inhibit Stx17 translocation, thereby specifically blocking autophagy, may be a more precise method for performing autophagy flux experiments. In fact, silencing Stx17 expression is considered an ideal method for selectively inhibiting autophagy flux (Hegedus et al., 2013). In summary, molecules like EACC can fill the gap in this field due to the lack of specific autophagy inhibitors. [1]
EACC is a research compound used for studying autophagy mechanisms and the role of autophagosome-lysosome fusion in various biological processes; it has not been approved for clinical use and serves as a valuable tool for dissecting the molecular machinery of autophagy, particularly the function of SNARE proteins in membrane fusion.

C13H11N3O6S2

369.372940301895

369.008

C, 42.27; H, 3.00; N, 11.38; O, 25.99; S, 17.36

864941-31-1

3704668

Typically exists as Light yellow to yellow solid at room temperature

3.3

2

8

5

24

526

0

O=C(OCC)NC(C1=C(NC(C2=CC=C([N+]([O-])=O)S2)=O)SC=C1)=O

ISLJZDYAPAUORR-UHFFFAOYSA-N

InChI=1S/C13H11N3O6S2/c1-2-22-13(19)15-10(17)7-5-6-23-12(7)14-11(18)8-3-4-9(24-8)16(20)21/h3-6H,2H2,1H3,(H,14,18)(H,15,17,19)

ethyl N-[2-[(5-nitrothiophene-2-carbonyl)amino]thiophene-3-carbonyl]carbamate

EACC; 864941-31-1; ethyl (2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate; Ethyl N-[2-[(5-nitrothiophene-2-carbonyl)amino]thiophene-3-carbonyl]carbamate;

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 : ~16.88 mg/mL (~45.70 mM)

Solubility in Formulation 1: ≥ 1.69 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% 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 16.9 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.)