EN6

Autophagy; ATP6V1A subunit of the lysosomal v-ATPase

EN6（50 μM；1、4、8 小时）会触发 HEK293A 细胞中时间和剂量依赖性 LC3 的产生，并增加 LC3BII 水平 [1]。在 HEK293A 细胞中，EN6（25 μM；1 小时）抑制 mTORC1 溶酶体定位和激活，导致自噬体和自噬 [1]。在 GFP-TDP43 U2OS 骨肉瘤细胞系模型中，EN6（50 μM；4 小时）刺激 HEK293A 细胞中的 v-EN6（25 μM；7 小时），以支持 IPTG 诱导的蛋白质聚集体自噬清除 [1]。

在体内，EN6（50 mg/kg；腹腔注射；单次）抑制 mTORC1 并促进自噬 [1]。

体外v-ATP酶测定[1]
简言之，将两个15cm汇合的HEK293T细胞与30μg/ml与Oregon Green 514（Dx-OG514，Invitrogen）缀合的70 kDa葡聚糖孵育过夜。第二天，洗涤细胞并在无血清DMEM中追逐2小时，以允许Dx-OG514的溶酶体积累。裂解前15分钟，加入1μM FCCP以消散溶酶体pH梯度。然后收获细胞，并使用23G针在部分缓冲液中机械破碎：140mM KCl、1mM EGTA、20mM HEPES、50mM蔗糖（pH 7.4），补充有5mM葡萄糖、蛋白酶抑制剂和1μM FCCP。将溶解的细胞在4°C下以1700rpm离心10分钟，并收集上清液。将得到的核后上清液（PNS）在4°C下以最大速度旋转，得到含有器官部分的沉淀。将每个级分重悬于180μl不含FCCP的分馏缓冲液中，并转移到96多孔（黑色）中。在SpectraMax i3中以30秒的间隔在490nm激发后，在530nm处收集基线荧光，持续5分钟。将各种化合物加入每个孔中，然后加入5mM ATP和MgCl2，并在45分钟内恢复荧光读数。由于v-ATP酶的溶酶体再酸化，Dx-OG514的荧光发射随时间呈指数衰减。

蛋白质印迹分析[1]
细胞类型： HEK293A 细胞
测试浓度： 50 μM
孵育时间： 1 、4、8 小时
实验结果：时间和剂量依赖性触发 LC3 斑点形成并增加 LC3BII 水平。

---

蛋白质印迹分析[1]
细胞类型： HEK293A 细胞
测试浓度： 25 μM
孵育时间： 1 小时
实验结果： 导致 mTORC1 信号传导完全失活，例如经典底物、S6 激酶 1 (S6K1)、4EBP1 和 ULK1 的磷酸化。

---

免疫荧光 [1]
细胞类型： HEK293A 细胞
测试浓度： 50 μM
孵育时间： 4 h
实验结果：导致HEK293A细胞中溶酶体酸化显着增加，而这种增强的酸化被BafA1阻断。

---

免疫荧光[1]
细胞类型： IPTG诱导的 GFP-TDP43 U2OS 骨肉瘤细胞系模型
测试浓度： 25 μM
孵育持续时间：7小时
实验结果：IPTG诱导的TDP43聚集体减少了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.

---

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

C₁₉H₁₄F₂N₄O₂

368.34

368.11

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

1808714-73-9

White to off-white solid

2.7

76Ų

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

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)

配方 1 中的溶解度: 10 mg/mL (27.15 mM) in 50% PEG300 +50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。