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

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

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%.

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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Assessing mTORC1 inhibition in vivo and pharmacokinetics of EN6 in mice [1]
6-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]
For 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.

[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 eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling. [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.)