Autophagy; ATP6V1A subunit of the lysosomal v-ATPase
Time- and dose-dependent LC3 production is triggered and LC3BII levels are increased in HEK293A cells by EN6 (50 μM; 1, 4, 8 h) [1]. In HEK293A cells, EN6 (25 μM; 1 h) inhibits mTORC1 lysosomal localization and activation, leading to autophagosomes and autophagy [1]. In the GFP-TDP43 U2OS osteosarcoma cell line model, EN6 (50 μM; 4 h) stimulates v-EN6 (25 μM; 7 h) in HEK293A cells to support IPTG-induced autophagic clearance of protein aggregates [1].Cell

Time- and dose-dependent LC3 production is triggered and LC3BII levels are increased in HEK293A cells by EN6 (50 μM; 1, 4, 8 h) [1]. In HEK293A cells, EN6 (25 μM; 1 h) inhibits mTORC1 lysosomal localization and activation, leading to autophagosomes and autophagy [1]. In the GFP-TDP43 U2OS osteosarcoma cell line model, EN6 (50 μM; 4 h) stimulates v-EN6 (25 μM; 7 h) in HEK293A cells to support IPTG-induced autophagic clearance of protein aggregates [1].Cell

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Treatment of HEK293A cells with EN6 (50 µM) increased the levels of processed LC3BII and triggered the formation of LC3 puncta in a time- and dose-dependent manner, comparable to the mTORC1 inhibitor Torin1. [1]
In HEK293A cells, EN6 treatment led to a significant increase in the number of autophagosomes and autolysosomes as observed by transmission electron microscopy. [1]
EN6 treatment (25 µM, 1 h) in HEK293A cells completely inactivated mTORC1 signaling, as shown by reduced phosphorylation of canonical substrates S6 kinase 1 (S6K1), 4EBP1, and ULK1 in a dose-responsive and time-dependent manner. Unlike the ATP-competitive mTOR inhibitor Torin1, EN6 had no effect on mTORC2-dependent AKT phosphorylation. [1]
The inhibition of mTORC1 signaling and activation of autophagy (LC3BII accumulation, p62 degradation) by EN6 was strictly dependent on covalent modification of C277 of ATP6V1A, as demonstrated using ATP6V1A-knockdown cells rescued with non-modifiable C277A or C277S mutants. [1]
EN6 treatment (25 µM, 1 h) abrogated amino acid-induced recruitment of mTORC1 to LAMP2-positive lysosomes in HEK293T cells. This effect was prevented by expressing a constitutively active, GTP-locked RagB mutant (RagB^Q99L). [1]
EN6 decreased the overall binding between the v-ATPase and the Ragulator complex and rendered this interaction insensitive to amino acid levels, as shown by co-immunoprecipitation experiments. [1]
In HEK293A cells, EN6 treatment (25 µM, 4 h) induced nuclear translocation of TFEB and significantly increased mRNA levels of several TFEB target lysosomal genes. [1]
EN6 treatment (50 µM, 4 h) significantly increased lysosomal acidification in HEK293A cells, as measured by ratiometric LysoSensor DND-160 staining. This acidification was blocked by the v-ATPase inhibitor bafilomycin A1 (BafA1). [1]
In an in vitro assay using isolated lysosomes loaded with FITC-dextran, EN6 (25 µM) increased the catalytic activity of the v-ATPase, accelerating lysosomal re-acidification, whereas BafA1 inhibited it. [1]
In a U2OS cell model with IPTG-inducible GFP-TDP-43 aggregates, co-treatment with EN6 (25 µM) for 7 hours reduced TDP-43 aggregate formation by 75%. This clearance was lysosome- and v-ATPase-dependent, as it was prevented by co-treatment with BafA1. Clearance of pre-formed aggregates was also observed within 4 hours of EN6 treatment. [1]
EN6 was ineffective at blocking mTORC1 signaling in cells expressing a constitutively active RagB^Q99L mutant or in cells with knockout of the GATOR1 complex protein NPRL3. [1]
A cysteine non-reactive analog of EN6, CC2-49, did not inhibit mTORC1 signaling or elevate LC3BII levels in HEK293A cells. [1]

In vivo, EN6 (50 mg/kg; ip; single) suppresses mTORC1 and promotes autophagy [1].

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Intraperitoneal (i.p.) administration of EN6 (50 mg/kg) to C57BL/6 mice significantly inhibited mTORC1 signaling in both skeletal muscle and heart tissue after 4 hours, as demonstrated by reduced phosphorylation of S6, 4EBP1, and ULK1. Autophagy was strongly activated, shown by increased LC3BII levels and decreased p62 levels. [1]
EN6 was more effective than rapamycin (10 mg/kg, i.p.) at inhibiting phosphorylation of 4EBP1 and ULK1, as well as inducing LC3B cleavage and p62 degradation in mouse heart and skeletal muscle. [1]
EN6 treatment did not inhibit mTORC2-dependent AKT phosphorylation in mouse heart or skeletal muscle. [1]

In vitro v-ATPase assays [1]
In brief, two 15cm confluent HEK293T cells were incubated overnight with 30 μg/ml 70 kDa Dextran conjugated to Oregon Green 514 (Dx-OG514, Invitrogen). The next day, cells were washed and chased for 2 hours in serum free DMEM to allow lysosomal accumulation of Dx-OG514. 15 minutes prior to lysis, 1 μM FCCP was added to dissipate the lysosomal pH gradient. Cell were then harvested and mechanically broken using a 23 G needle in fractional buffer: 140 mM KCl, 1 mM EGTA, 20 mM HEPES, 50 mM Sucrose (pH 7.4), supplemented with 5 mM Glucose, protease inhibitor and 1 μM FCCP. Lysed cells were spun down at 1700 rpm for 10 min at 4°C and the supernatant was collected. The resulting post-nuclear supernatant (PNS) was spun down at max speed at 4°C, yielding a pellet containing the organellar fraction. Each fraction was resuspended in 180 μl of fractionation buffer devoid of FCCP and transferred to a 96-multiwell (black). Baseline fluorescence was collected at 530 nm upon 490 nm excitation in SpectraMax i3 at 30sec intervals for 5 min. Various compounds were added to each well followed by 5 mM ATP and MgCl2, and fluorescence reading was resumed for further 45 min. The fluorescence emission of Dx-OG514 decayed exponentially over time due to the lysosomal reacidification of the v-ATPase.

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A gel-based activity-based protein profiling (ABPP) assay was used to assess the interaction between EN6 and recombinant ATP6V1A. Purified recombinant human ATP6V1A protein was pre-incubated with vehicle or varying concentrations of EN6 for 1 hour at 37°C in PBS buffer. The mixtures were then treated with a rhodamine-functionalized iodoacetamide probe (IA-rhodamine) at a final concentration of 1 µM for 1 hour at room temperature. The reactions were quenched with SDS loading buffer, heated, and separated by SDS-PAGE. Probe-labeled proteins were visualized and quantified using in-gel fluorescence scanning and densitometry. [1]
An in vitro v-ATPase activity assay was performed. Lysosomes loaded with fluorescein-conjugated dextran (FITC-dextran or Oregon Green 514-conjugated dextran) were isolated from HEK293T cells via subcellular fractionation. The organellar fraction was resuspended in assay buffer. Baseline fluorescence was recorded. Vehicle, EN6 (25 µM), or bafilomycin A1 (200 nM) was added along with ATP (5 mM) and MgCl₂ to initiate v-ATPase-driven proton pumping and lysosomal re-acidification. The quenching of fluorescence (due to the pH sensitivity of the dextran conjugate) was monitored over time using a plate reader. The rate of fluorescence decay reflects v-ATPase activity. [1]

Western Blot Analysis[1]
Cell Types: HEK293A Cell
Tested Concentrations: 50 μM
Incubation Duration: 1, 4, 8 hrs (hours)
Experimental Results: Time- and dose-dependent triggering of LC3 puncta formation and increased LC3BII levels.

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Western Blot Analysis[1]
Cell Types: HEK293A Cell
Tested Concentrations: 25 μM
Incubation Duration: 1 hour
Experimental Results: Result in complete inactivation of mTORC1 signaling, such as phosphorylation of classical substrates, S6 Kinase 1 (S6K1), 4EBP1 and ULK1.

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Immunofluorescence [1]
Cell Types: HEK293A Cell
Tested Concentrations: 50 μM
Incubation Duration: 4 h
Experimental Results: It resulted in a significant increase in acidification of lysosomes in HEK293A cells, and this enhanced acidification was blocked by BafA1.

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Immunofluorescence[1]
Cell Types: IPTG-induced GFP-TDP43 U2OS osteosarcoma cell line model
Tested Concentrations: 25 μM
Incubation Duration: 7 hrs (hours)
Experimental Results: IPTG-induced TDP43 aggregates were diminished by 75%.

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A cell-based autophagic flux screen was conducted using MEF or HEK293A cells stably expressing a dual-color, cleavable GFP-LC3-RFP-LC3ΔG reporter. Cells were plated in 96-well plates, allowed to adhere overnight, and then treated with vehicle, covalent ligands (e.g., 50 µM EN6), rapamycin (100 nM), or Torin1 (250 nM) for 24 hours in complete medium. Cells were fixed, and the GFP/RFP fluorescence ratio was measured using a plate reader. A decrease in the GFP/RFP ratio indicates increased LC3 processing and autophagic flux. [1]
For immunoblotting analysis of signaling and autophagy markers, cells (e.g., HEK293A, HeLa) were treated with compounds as indicated. After treatment, cells were washed with ice-cold PBS and lysed in a buffer containing Triton X-100, phosphatase inhibitors, and protease inhibitors. Cleared lysates were mixed with SDS sample buffer, heated, resolved by SDS-PAGE, and transferred to membranes. Membranes were blocked, incubated with primary antibodies (e.g., against phospho-S6K1, LC3B, p62, ATP6V1A), followed by appropriate secondary antibodies, and detected using chemiluminescence or fluorescence. [1]
For amino acid starvation and stimulation experiments, sub-confluent cells were rinsed and incubated in amino acid-free medium supplemented with dialyzed serum for 50 minutes. Where applicable, drugs were included during this starvation period. Cells were then stimulated for 10 minutes with a complete amino acid mixture (or kept in starvation medium). Cells were immediately lysed for immunoblotting analysis. [1]
For imaging LC3B puncta, HEK293A cells plated on chambered slides were treated with compounds, fixed, permeabilized, and blocked. Cells were incubated overnight with an anti-LC3B primary antibody at 4°C, followed by incubation with a fluorescent dye-conjugated secondary antibody. Nuclei were stained with Hoechst 33342. Images were acquired by confocal fluorescence microscopy, and LC3B puncta per cell were quantified using image analysis software. [1]
For assessing mTORC1 lysosomal localization by immunofluorescence, cells plated on coverslips were subjected to amino acid starvation and stimulation protocols with or without drug treatment. Cells were fixed, permeabilized, and incubated with primary antibodies against mTOR and the lysosomal marker LAMP2, followed by fluorescent secondary antibodies. Images were acquired by confocal microscopy, and colocalization was analyzed. [1]
For measuring lysosomal pH, HEK293A cells were treated with compounds, then stained with the ratiometric pH-sensitive dye LysoSensor Yellow/Blue DND-160. Live cells were imaged using two-photon microscopy to collect fluorescence emission at two wavelength ranges (blue and yellow). The ratio of yellow to blue fluorescence was calculated and converted to pH values using a calibration curve generated with buffers of known pH in the presence of ionophores. [1]
For the TDP-43 aggregate clearance assay, U2OS cells stably expressing an IPTG-inducible GFP-TDP-43 were plated. For co-treatment experiments, cells were treated with IPTG to induce aggregates along with vehicle or EN6 with or without BafA1 for several hours. For pre-stimulation experiments, cells were first treated with IPTG to form aggregates, then treated with vehicle or EN6. Cells were fixed, stained with Hoechst, and imaged by confocal microscopy. GFP-TDP-43 aggregates per cell were quantified using image analysis software. [1]
For generating stable cell lines, ATP6V1A was knocked down in HeLa cells using lentivirus expressing a specific shRNA. The knockdown cells were then rescued by lentiviral transduction with vectors expressing shRNA-resistant, Flag-tagged wild-type or mutant (C277A, C277S) ATP6V1A. Cells were selected with puromycin. [1]

Animal/Disease Models: Sixweeks old male C57BL/6 mice [1].
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; Single
Experimental Results:Significant inhibition of mTORC1 signaling in skeletal muscle and heart, as evidenced by diminished phosphorylation of S6, 4EBP1, and ULK1. Increased LC3BII levels and diminished p62 levels indicate strong activation of autophagy.

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\nAssessing mTORC1 inhibition in vivo and pharmacokinetics of EN6 in mice [1]
\n6-week-old male C57BL/6 mice (Jackson Laboratory) were injected intraperitoneally with solvent control, EN6 (50 mg/kg) or rapamycin (10 mg/kg) in saline/ethanol/PEG-40 (v/v/v = 18:1:1). After 4 h, mice were euthanized, and tissues were harvested and lysed in lysis buffer (1% Triton X-100, 10 mM β-glycerol phosphate, 10 mM sodium pyrophosphate, 4 mM EDTA, 40 mM HEPES at pH 7.4, and 1 tablet of EDTA-free protease inhibitors per 50 ml) at 4 °C for 30 min. The lysates were cleared by centrifugation in a microcentrifuge at 21,130 g for 10 minutes at 4 °C and protein concentration of supernatant was determined by BCA assay. The lysates were then diluted to 1.5 mg/mL, mixed with 4× sample buffer, heated at 95 °C for 5 minutes, resolved by precast 4–20% TGX gels, and analyzed by immunoblotting. Antibodies were obtained from various commercial sources and dilutions were prepared per recommended manufacturers’ procedures.[1]
\nFor pharmacokinetics studies, mice were injected intraperitoneally with solvent control or EN6 at indicated doses. At indicated time intervals, mice were euthanized, and tissues were harvested. The tissues were then weighed and homogenized, and EN6 was extracted from chloroform:methanol:PBS solution mixture (2:1:1, v/v/v; 4 mL total) with dodecylglycerol (10 nmol) as internal standard. The organic layer was collected, evaporated under stream of N2, re-dispersed in chloroform and analyzed by multiple-reaction monitoring (MRM)-based targeted LC-MS/MS on an Agilent 6430 QQQ using a Luna reverse phase C5 column (50 mm × 4.6 mm with 5 mm diameter particles, Phenomenex). Mobile phases: Buffer A, 95:5 water / methanol; Buffer B: 60:35:5 2-propanol / methanol / water, both with 0.1 % formic acid and 50 mM ammonium formate additives. Flow rate began at 0.2 mL/min for 2 min, followed by a gradient starting at 0 % B and increasing linearly to 100 % B over the course of 23 min with a flow rate of 0.4 mL/min, followed by an isocratic gradient of 100 % B for 5 min with a flow rate increasing linearly from 0.04 mL/min to 0.4 mL/min. MS analysis was performed using electrospray ionization (ESI) with a drying gas temperature of 350 °C, drying gas flow rate of 10 L/min, nebulizer pressure 35 psi, capillary voltage 3.0 kV, and fragmentor voltage 100 V. Parent/daughter ion MRM transitions used to determine EN6 levels were 369.34/163.2 and 369.34/189 with fragmentor voltage of 10 and 20, and 30 and 40, respectively. Peak area of EN6 to that of dodecylglycerol was calibrated to amount of EN6 per gram of tissues by LC-MS/MS analysis of a set of solution mixtures containing dodecylglycerol (10 nmol) and known concentrations of EN6 (0.01, 0.1, 1, 10 and 30 nmol). Data was analyzed using Agilent Qualitative Analysis software by calculating area under the curve.

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\nTo assess mTORC1 inhibition in vivo, 6-week-old male C57BL/6 mice were injected intraperitoneally (i.p.) with a single dose of vehicle control (saline/ethanol/PEG-40, 18:1:1 v/v/v), EN6 (50 mg/kg), or rapamycin (10 mg/kg). After 4 hours, mice were euthanized. Heart and skeletal muscle tissues were harvested, homogenized in lysis buffer, and cleared by centrifugation. Protein concentrations were determined, and lysates were analyzed by immunoblotting for mTORC1 signaling and autophagy markers. [1]
\nFor pharmacokinetics studies, mice were injected intraperitoneally with vehicle or EN6 at indicated doses (e.g., 10, 25, 50 mg/kg). At specified time points (e.g., 0.5, 1, 2, 4, 8 hours), mice were euthanized, and tissues (e.g., skeletal muscle, heart) were harvested. Tissues were weighed, homogenized, and EN6 was extracted using a chloroform:methanol:PBS mixture with an internal standard. The organic layer was collected, dried, reconstituted, and analyzed by targeted LC-MS/MS using multiple-reaction monitoring (MRM) to quantify EN6 levels per gram of tissue. [1]

Following a single intraperitoneal injection of 50 mg/kg EN6 into mice, EN6 was detectable in skeletal muscle and cardiac tissues within 1 hour. [1] Dose-response and time-course studies showed that EN6 was detectable in mouse tissues (skeletal muscle and heart) after intraperitoneal injection. [1]

The study indicated that EN6 does not inhibit the mTORC2-AKT signaling pathway in cells or mice, while ATP-competitive mTOR inhibitors can inhibit this important survival signal. [1]
The authors pointed out that although isoTOP-ABPP identified C277 of ATP6V1A as the main target, EN6 may have other non-target sites with lower binding affinity or targets that react with other amino acids. The contribution of these non-target sites to its biological effects needs to be further determined. [1]

[1]. Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition. Nat Chem Biol. 2019 Aug;15(8):776-785.

Autophagy is a lysosomal degradation pathway that clears aggregated proteins and damaged organelles, thereby maintaining cellular homeostasis. One of the main pathways activating autophagy is the inhibition of mTORC1 kinase, but current compounds targeting mTORC1 cannot achieve complete and selective mTORC1 blockade. This study, combining covalent ligand library screening and activity-based proteomic analysis, discovered a small-molecule in vivo autophagy activator called EN6. EN6 covalently targets cysteine 277 in the ATP6V1A subunit of lysosomal v-ATPase, which activates mTORC1 via Rag guanosine triphosphatase. EN6-mediated ATP6V1A modification uncouples v-ATPase from Rag, thereby inhibiting the mTORC1 signaling pathway, increasing lysosomal acidification, and activating autophagy. EN6 can sustainably clear TDP-43 aggregates (a pathogenic factor in frontotemporal dementia) in a lysosomal-dependent manner. Our findings reveal how v-ATPase regulates mTORC1 and propose a unique approach to enhance cellular clearance based on covalent inhibition of the lysosomal mTORC1 signaling pathway. [1]

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EN6 is a small cysteine-reactive covalent ligand that was discovered by screening a library of covalent compounds and combining it with chemical proteomics target unconvolution (isoTOP-ABPP). [1]
Its mechanism of action involves covalent modification of the C277 site on the ATP6V1A subunit of v-ATPase. This modification uncouples v-ATPase from the Ragulator-Rag GTPase complex, inhibiting the nutrient-dependent recruitment and activation of mTORC1 on lysosomes, thereby inducing autophagy. In addition, this modification enhances the proton pump activity and lysosomal acidification of v-ATPase. [1]
EN6 represents an autophagy activation approach distinct from rapamycin (an incomplete mTORC1 inhibitor) or ATP-competitive mTOR inhibitors (which also inhibit mTORC2/AKT). It achieves complete inhibition of mTORC1 without affecting the mTORC2-AKT signaling pathway. [1]
EN6 promotes the clearance of TDP-43 protein aggregates in a lysosome-dependent manner, which are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). [1]
This study proposes a model in which v-ATPase provides energy or conformational signaling for mTORC1 activation through physical coupling with the Ragulator-Rag complex. EN6 disrupts this coupling. [1]
The authors note that EN6 is an early-stage compound requiring further medicinal chemistry optimization to improve its potency, selectivity, in vivo efficacy, and brain penetration. The duration of its inhibition of mTORC1 in vivo and its potential long-term effects on the mTORC2 signaling pathway remain to be determined. [1]
Another covalent ligand, called CZ, targets C138 on ATP6V1A, inhibiting v-ATPase activity and also inhibiting the mTORC1 signaling pathway, but does not disrupt the lysosomal localization of mTORC1 or clear TDP-43 aggregates like EN6, highlighting the functional differences in targeting different cysteine residues. [1]

C19H14F2N4O2

368.34

368.11

C, 61.96; H, 3.83; F, 10.32; N, 15.21; O, 8.69

1808714-73-9

99640033

White to off-white solid

2.7

2

5

5

27

562

0

SUSXQEYPNDORDQ-UHFFFAOYSA-N

InChI=1S/C19H14F2N4O2/c1-2-18(26)24-16-9-13(7-8-14(16)20)23-19(27)12-10-22-25(11-12)17-6-4-3-5-15(17)21/h2-11H,1H2,(H,23,27)(H,24,26)

N-(3-Acrylamido-4-fluorophenyl)-1-(2-fluorophenyl)-1H-pyrazole-4-carboxamide

EN6 EN-6 EN 6

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 : ~5 mg/mL (~13.57 mM)
Ethanol : ~1.11 mg/mL (~3.01 mM)

Solubility in Formulation 1: 10 mg/mL (27.15 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.)