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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CAS No. : 1808714-73-9

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10 mM * 1 mL in DMSO
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Based on 5 publication(s) in Google Scholar

EN6 Related Antibodies

Top Publications Citing Use of Products

    EN6 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2024 May 13:e2400790.  [Abstract]

    EN6 (50 mg/kg; every other day for 14 d) significantly impaired the level of extracellular matrix calcification of tendon fibroblasts.

    EN6 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2024 May 13:e2400790.  [Abstract]

    EN6 (50 mg/kg; every other day for 14 d) reduced extracellular vesicles specific protein CD9 and autolysosomes associated LC3 and LAMP1 in tendon fibroblasts treated with IL‐1β+TNF‐α.

    EN6 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2024 May 13:e2400790.  [Abstract]

    EN6 (50 mg/kg; every other day) decreased the green fluorescent signal in the Achilles tendon of C57BL/6J mice, suggesting that autophagosomes were hydrolyzed after binding to lysosomes and autophagic flux was restored. Impaired autophagic flux (yellow arrows) and normal autophagic flux (red arrows).

    EN6 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2024 May 13:e2400790.  [Abstract]

    EN6 (50 mg/kg; every other day) significantly decreased the expression level of LC3B and P62 in the Achilles tendons of C57BL/6J mice, suggesting restoration of autophagic flux.

    EN6 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2024 May 13:e2400790.  [Abstract]

    EN6 (50 mg/kg; every other day) significantly reduced heterotopic ossification (HO) in tendons of C57BL/6J mice at 1 and 3 weeks after surgery.

    • Biological Activity

    • Purity & Documentation

    • References

    • Customer Review

    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.

    • Molarity Calculator

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

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    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.
    Calculation results:
    Working solution concentration: mg/mL
    Purity & Documentation

    Purity: 99.42%

    SDS (396 KB)

    COA (247 KB) LCMS (102 KB)

    Handling Instructions (2659 KB)
    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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