Autophagy

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.

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.

[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 striking feature of EACC-mediated block of autophagic flux is impaired Stx17 loading onto autophagosomes. To the best of our knowledge, there is no other report suggesting any chemical modulator of autophagy that can selectively prevent Stx17 translocation thereby rendering autophagosomes “fusion incompetent.” The exact mechanism by which Stx17 is translocated onto complete autophagosomes is not very clear. A recent report suggested that Stx17 recruitment to autophagosomes occurs via interaction with a small GTPase IRGM and mammalian ATG8 proteins (Kumar et al., 2018). Although we have not checked whether EACC can affect interaction between Stx17 and IRGM, we propose that identification of Stx17-binding partners in the presence or absence of EACC could give a clue regarding the target of EACC as well as help in identification of any other accessory factors that might be involved in Stx17 recruitment on autophagosomes. Furthermore, we also showed that the action of EACC is reversible. The block in autophagic flux is eliminated after washing out EACC because Stx17 is now able to translocate to autophagosomes and participate in further fusion events. Hence, due to the reversible nature of its action, EACC can be used as a useful tool to study Stx17 trafficking. [1]
To determine the rate of autophagic flux, lysosomal inhibitors like BafA1 and chloroquine are routinely used. Unfortunately, these treatments are not ideal as they not only can impair lysosomal function but impede all other lysosomal pathways including the endo-lysosomal trafficking. Our results also show that the action of EACC is specific to autophagosomes and it does not affect lysosomal pH, function, or endocytic trafficking. It also does not affect the localization of lysosomal SNAREs or RABs. Additionally, even the well-known early inhibitors of autophagy such as wortmannin and 3-methyl adenine are promiscuous as they block all phosphatidylinositol 3-kinase–dependent signaling pathways thereby resulting in a plethora of side effects. In such scenarios, inhibiting Stx17 translocation by using EACC, which leads to a specific block in autophagy, might be a cleaner way to perform autophagic flux experiments. In fact, silencing Stx17 expression is recommended as a desired attribute for selectively inhibiting autophagic flux (Hegedus et al., 2013). In conclusion, molecules like EACC can fill the lacuna that exists in the field due to lack of specific autophagy inhibitors. [1]

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.)