生物活性

Sirtinol, a cell permeable class III HDAC inhibitor, inhibits both SIRT1 andSIRT2 and has been shown to have anti-tumor and anti-inflammatory properties. Sirtinol is a cell-permeable, specific inhibitor of sirtuin NAD-dependant histone deacetylases. Significantly decreases growth and viability of PCa and HEK293T cells in vitro. Sirtinol has been shown to inhibit yeast Sir2 (IC50 = 70 μM), human SIRT2 (IC50 = 40 μM), and human SIRT1 (IC50 = 131 μM), yet has shown no effect on human HDAC1. In Arabidopsis, sirtinol strongly affected root and vascular tissue development by activating the auxin signaling pathway. Sirtinol has also been shown to induce senescence-like growth arrest in human breast cancer MCF-7 and lung cancer H1299 cells by impaired activation of MAPK.

Anti-proliferation of sirtinol

Kinaseactivities of sirtinol and the enantiomers[5]

体外研究

体内研究

1% DMSO+30% polyethylene glycol+1% Tween 80, pH 4

激酶实验

Assays of Recombinant Yeast Sir2 and Human SIRT1[5]

Recombinant His-tagged yeast Sir2p,His-tagged human SIRT1, and His-tagged human SIRT2 were purified and assayedfor deacetylase activity using the HDAC fluorescent activity assay. This assaysystem allows detection of a fluorescent signal upon deacetylation of a histonesubstrate when treated with developer. Fluorescence was measured on afluorometric reader with excitation set at 360 nm and emission detection set at450 nm. Reactions consisted of either 3μg of ySir2 or1μg of SIRT1 or SIRT2, incubated with 250μM acetylatedhistone substrate, 1mM dithiothreitol, and a range of inhibitor concentrationsas described. Reactions with the yeast and human proteins were carried out at30 and 37 °C, respectively, for 60 min. Assays were performed in the presenceof 200μM NAD+ and each of the inhibitors at 0, 20, 75, 100, 150, or 300μM.

In Vitro Histone Deacetylation Assays[3]

1.5μg ofrecombinant human GST-SIRT2 (amino acids 18–340) or 0.5μg ofrecombinant yeast Sir2p were incubated for 2 h at 30 °C in 50μlof assay buffer (50mM Tris-HCl, pH 8.8, 4mM MgCl2, 0.2mM dithiothreitol), withor without 50μM NAD and acetylated HeLa histones (1000cpm), purified by acidextraction. HDAC activity was determined by scintillation counting of the ethylacetate-soluble [³H] acetic acid.

细胞实验

Sirtinol[6]

Sirtinol (2-[(2-hydroxynaphthalen-1-ylmethylene)-amino]-N-(1-phenyl-ethyl)-benzamide)was prepared in 20mM stocks in dimethylsulfoxide(DMSO) stored at −20 ℃ until use.

Cellculture and hCMV infection

Primary human embryonic lung diploidfibroblasts (WI-38) were cultured in Dulbecco’s modifiedEagle’s medium (DMEM)supplemented with 10% fetal bovineserum (FBS), 100 U/mL penicillin, and 100μg/mLstreptomycin. The cells were young at population doubling (PD) of 32 or lessand became replicative senescent at PD50 or higher. The cultured cells weresplit in ratios of 1:2 or 1:4 when the confluence of the culture was over 80%.The cumulative population doublings were calculated as log₂ (D/D0),where D and D0are defined as the density of cells at the time of harvesting andseeding, respectively. All experiments were performed using cells that werebetween 27–32 PD for hCMV infections unless indicated otherwise.  

Stocks of the Towne strain of hCMV (ATCC,VR977) were routinely prepared and viral titers were measured in MRC-5 cells.Supernatants collected from uninfected MRC-5 culture were processed the sameway as viral stock and used as mock control medium. WI-38 cells seeded at 2 ×10⁴cells/cm² in DMEM—10% FBS were cultured for 24h after whichthe cells were serum starved for 48–72h in DMEM—0.2% FBS to synchronize cellsin G0 phase of cell cycle before infection. Virus inoculum was added for 2 h at37◦C and then discarded and replaced with fresh DMEM—0.2% FBS. Mock-infectedcontrols were exposed to an equal volume of mock control medium described above.Cells were harvested at indicated time points post infection for furthertesting. For cultures in the presence of sirtinol, sirtinol was added 2 h before(−2 h) or after (+2 h) virus inoculation with its presence until cells wereharvested.

MTTassay

MTT assay was performed. Briefly, cellswere seeded in flat-bottomed 96-well microplates at the density of 3 × 10³ cells in 0.2mL per well. After 24h, cells were incubated in the culture mediumcontaining sirtinol at different concentrations for 48h. Then 20μLMTT [3-(4, 5)-dimethylthiahiazo(-z-y1)-3,5-diphenlytetrazoliumbromide] of 5mg/mLwas added to each well. After incubation for 4 h, the supernatant was discardedand 0.2mL DMSO was added to stop reactions. The absorbance values of each wellwere determined spectrophotometrically at 490nm using BIO-RAD microplate reader.

动物实验

Animal Experiments[7]

Mice were treated with sirtinol at a doseof 10mg/kg bodyweight in 100 ml volume intraperitoneal injection twice a weekfor three weeks. At 1 hour following tail vein injection, blood was collected fromthe tail vein for glucose detection. Tissues were collected rapidly fromanaesthetized mice and frozen in liquid nitrogen for further analysis.

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] Wang J, Kim TH, Ahn MY, et al. Sirtinol, a class III HDAC inhibitor, induces apoptotic and autophagic cell death in MCF-7 human breast cancer cells. Int J Oncol. 2012;41(3):1101-1109.
[2] Tsai YF, Yu HP, Chang WY, Liu FC, Huang ZC, Hwang TL. Sirtinol inhibits neutrophil elastase activity and attenuates lipopolysaccharide-mediated acute lung injury in mice. Sci Rep. 2015;5:8347.
[3] Grozinger CM, Chao ED, Blackwell HE, Moazed D, Schreiber SL. Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem. 2001;276(42):38837-38843.
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体内溶解度
约1 mg/mL

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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