Inhibition of GST-SIRT1 deacetylase activity

293T cells were transiently transfected with GST-tagged human SIRT1 in the pDEST27 Gateway vector using FuGENE-6. After 48 hrs, the cells were lysed with 50 mM Tris, pH 8.0, 120 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40, supplemented with Complete Mini protease inhibitor cocktail tablets. GST-SIRT1 was purified from lysates using glutathione-Sepharose beads and washed extensively in the above buffer. The deacetylation assay was performed with approximately 30 ng of GST-SIRT1 in the presence of EX 527 (48 pM to 100 μM). Deacetylation was measured using the Fluor de Lys kit using a fluorogenic peptide encompassing residues 379 to 382 of p53, acetylated on lysine 382. The acetylated lysine residue was coupled to an aminomethylcoumarin moiety. The peptide was deacetylated by SIRT1, followed by the addition of a proteolytic developer that released the fluorescent aminomethylcoumarin. Briefly, enzyme preparations were incubated with 170 μM NAD+ and 100 μM p53 fluorogenic peptide for 45 mins at 37 °C followed by incubation in developer for 15 mins at 37 °C. Fluorescence was measured by excitation at 360 nm and emission at 460 nm and enzymatic activity was expressed in relative fluorescence units.

Cell lines

NCI-H460, MCF-7, U-2 OS and HMEC cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 °C for several months.

Reaction Conditions

1 μM; 48 or 72 hrs

Applications

In cells treated with Etoposide, inhibition of SIRT1 catalytic activity by EX 527 had no effect on cell growth, viability or p53-controlled gene expression.

Animal models

Male SD rats

Dosage form

1 to 5 ~ 10 μg in a total volume of 5 μL; intracerebroventricular injection

Applications

EX 527 (~ 10 μg) increased hypothalamic acetyl-p53 levels by inhibiting hypothalamic SIRT1 activity.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Jonathan M. Solomon, Rao Pasupuleti, Lei Xu, Thomas McDonagh, Rory Curtis, Peter S. DiStefano, L. Julie Huber. Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage. Molecular Cellular Biology January 2006 vol. 26 no. 1 28-38.

[2]. Velásquez DA, Martínez G, Romero A, Vázquez MJ, Boit KD, Dopeso-Reyes IG, López M, Vidal A, Nogueiras R, Diéguez C. The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin. Diabetes. 2011 Apr;60(4):1177-85.