2. Autophagy
  3. Autophagy
  4. EN6

EN6 is a small-molecule in vivo autophagy activator that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase. EN6-mediated modification of ATP6V1A uncouples v-ATPase from Rag, leading to inhibition of mTORC1 signalling, increased lysosomal acidification, and activation of autophagy. EN6 also scavenges TDP-43 aggregates (causative agents of frontotemporal dementia) in a lysosome-dependent manner.

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EN6 Chemical Structure

EN6 Chemical Structure

CAS No. : 1808714-73-9

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Solid + Solvent
10 mM * 1 mL in DMSO
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Based on 2 publication(s) in Google Scholar

EN6 Related Antibodies

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Description

EN6 is a small-molecule in vivo autophagy activator that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase. EN6-mediated modification of ATP6V1A uncouples v-ATPase from Rag, leading to inhibition of mTORC1 signalling, increased lysosomal acidification, and activation of autophagy. EN6 also scavenges TDP-43 aggregates (causative agents of frontotemporal dementia) in a lysosome-dependent manner[1].

In Vitro

EN6 (50 μM; 1, 4, 8 h) increases the levels of LC3BII, and triggers formation of LC3 puncta in a time- and dose-dependent manner, in HEK293A cells[1].
EN6 leads to significant increases in the number autophagosomes and autolysosomes in HEK293A cells[1].
EN6 (25 μM; 1 h) blocks mTORC1 lysosomal localization and activation in HEK293A cells[1].
EN6 (50 μM; 4 h) activates the v-ATPase and lysosome acidification in HEK293A cells[1].
EN6 (25 μM; 7 h) promotes autophagic clearance of protein aggregates in IPTG-inducible GFP-TDP43 U2OS osteosarcoma cell line model[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

EN6 Related Antibodies

Western Blot Analysis[1]

Cell Line: HEK293A cells
Concentration: 50 μM
Incubation Time: 1, 4, 8 h
Result: Time- and dose-dependently triggered formation of LC3 puncta and increased the levels of LC3BII.

Western Blot Analysis[1]

Cell Line: HEK293A cells
Concentration: 25 μM
Incubation Time: 1 h
Result: Led to complete inactivation of mTORC1 signaling, as shown by reduced levels of phosphorylated canonical substrates, S6 kinase 1 (S6K1), 4EBP1, and ULK1.

Immunofluorescence[1]

Cell Line: HEK293A cells
Concentration: 50 μM
Incubation Time: 4 h
Result: Led to significantly increased acidification of the lysosome in HEK293A cells, and this heightened acidification was blocked by BafA1.

Immunofluorescence[1]

Cell Line: IPTG-inducible GFP-TDP43 U2OS osteosarcoma cell line model
Concentration: 25 μM
Incubation Time: 7 h
Result: Reduced IPTG-induced TDP43 aggregates by 75 %.
In Vivo

EN6 (50 mg/kg; i.p.; single) inhibits mTORC1 and activates autophagy in vivo[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model: Six-week-old male C57BL/6 mice[1].
Dosage: 50 mg/kg
Administration: Intraperitoneal injection; single
Result: Significantly inhibited mTORC1 signaling in both skeletal muscle and heart, as demonstrated by reduced phosphorylation of S6, 4EBP1 and ULK1.
Strongly activated autophagy as shown by heightened LC3BII levels and reduced p62 levels.
Molecular Weight

368.34

Formula

C19H14F2N4O2
CAS No.
Appearance

Solid

Color

White to off-white

SMILES

O=C(C1=CN(C2=CC=CC=C2F)N=C1)NC3=CC=C(F)C(NC(C=C)=O)=C3
Shipping

Room temperature in continental US; may vary elsewhere.
Storage
Powder -20°C 3 years
4°C 2 years
In solvent -80°C 2 years
-20°C 1 year
Solvent & Solubility
In Vitro: 

DMSO : 5 mg/mL (13.57 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Ethanol : 1.11 mg/mL (3.01 mM; ultrasonic and warming and heat to 60°C)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.7149 mL 13.5744 mL 27.1488 mL
5 mM 0.5430 mL 2.7149 mL 5.4298 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

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Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

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Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

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In Vivo:

For the following dissolution methods, please prepare the working solution directly. It is recommended to prepare fresh solutions and use them promptly within a short period of time.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  50% PEG300    50% Saline

    Solubility: 10 mg/mL (27.15 mM); Suspended solution; Need ultrasonic

In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

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Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
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Working solution concentration: mg/mL
Purity & Documentation
References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
Ethanol / DMSO 1 mM 2.7149 mL 13.5744 mL 27.1488 mL 67.8721 mL
DMSO 5 mM 0.5430 mL 2.7149 mL 5.4298 mL 13.5744 mL
10 mM 0.2715 mL 1.3574 mL 2.7149 mL 6.7872 mL
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EN6 Related Classifications

Help & FAQs
  • Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

Keywords:

EN61808714-73-9EN 6EN-6AutophagyautophagyHEK293A cellsU2OS osteosarcoma cellmTORC1LC3 punctaTDP-43frontotemporal dementiaInhibitorinhibitorinhibit

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