生物活性

MHY1485 is a mTOR activator which inhibits the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. Treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy.

体外研究

体内研究

激酶实验

细胞实验

Cell Culture System[1]

Rat hepatocytes, Ac2F cells were grown inDulbecco’s modified eagle medium (DMEM) containing 2 mM L-glutamine, 100units/ml penicillin, 100μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum(FBS). Cells were maintained at 37^oC in a humidified atmospherecontaining 5% CO₂/95% air. Under starvation conditions, cells weretreated with Hank’s Balanced Salt Solution (HBSS). Compound was treated underthe same conditions as the growth medium to exclude the effect of serumreduction to induce autophagy. For assessment of autophagic flux, thelysosomotropic agents bafilomycin and chloroquine were applied 1 h before the cellharvest at concentrations of 10 nM and 100 mM, respectively.

WesternBlotting

Cells were washed with cold PBS andharvested. Cell lysates were prepared using RIPA buffer containing 50 mM Tris-HCl(pH), 150 mM NaCl, 1% NP-40, 1 mM DTT, 0.1 mM NaF, 1 mM PMSF, 1 μg/mlpepstatin, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Proteinconcentration was determined by the bicinchoninic acid (BCA) method usingbovine serum albumin (BSA) as a standard. Equal amounts of protein wereseparated on 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDSPAGE) gels. The gels were subsequently transferred onto a polyvinylidenedifluoride membrane by electroblotting for 2 h at 60–75 V. The membranes wereblocked in a 5% nonfat milk solution in Trisbuffered saline (TBS) with 0.5%Tween-20, and incubated with primary antibodies as indicated. Pre-stainedprotein markers were used for molecular-weight determination.

Stainingof Autophagosomes with GFP-LC3 and Confocal Microscopy

Approximately 1x10 ^ˆ5 cells were seeded in coverglass-bottom-dish, incubated overnight,and then transfected with the adenovirus encoding green fluorescentprotein-microtubule-associated protein 1 light chain 3 (AdGFP-LC3) at a concentrationof 1,000 virus particles/ cell in DMEM. After incubation for 24 h, cells weretreated with compounds or starved. For visualization of lysosomes, cells were incubatedwith LysoTrackerH at a concentration of 60 nM for 1 h. Confocal images were obtainedwith FV10i FLUOVIEW Confocal Microscope.

动物实验

Animals[2]

CD-1 and B6D2F1 mice were housed 12hlight/dark with free access to water and food.

Ovariantissue grafting

Paired ovaries from day 10 mice werecultured on plate culture inserts in MEMα medium containing 3mg/ml BSA, 0.23mMsodium pyruvate, 50μg/ml vitamin C, 30mIU/ml FSH, 50mg/L streptomycin sulfateand 75 mg/L penicillin G. Ovaries were treated with 3–20μM MHY1485 for 48h withmedium changes after 24h of culture. Paired ovaries (without or with MHY1485 treatment)from the same donor were grafted under kidney capsules of the same adult ovariectomizedhosts (9–10 weeks of age) for 5 days with daily FSH injections (1 IU/animal).At the end of transplantation, grafts were collected for weight determinationand histological analysis.

To test the combined effects of mTOR activatorand AKT stimulators, some ovaries from day 10 mice were treated with IVA drugs(PTEN inhibitor: (bpv(hopic) at 30 μM for the first day and an activator forphosphoinositol-3-kinase740YP at 150μg/mL for two days) , together with orwithout mTOR activator before grafting.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Choi YJ, Park YJ, Park JY, et al. Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion. PLoS One. 2012;7(8):e43418.
[2] Cheng Y, Kim J, Li XX, Hsueh AJ. Promotion of ovarian follicle growth following mTOR activation: synergistic effects of AKT stimulators. PLoS One. 2015;10(2):e0117769.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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