SirReal2 is a potent and selective Sirt2 inhibitor with IC50 of 140 nM.

SIRT2:140 nM

SirReal2 selectively inhibits Sirt2 via a Sirt2-specific amino acid network, and induces a tubulin hyperacetylation and a significant depletion of BubR1 in HeLa cells. [1]

In vivo, SirReal2 inhibits Sirt2 activity without affecting the activity of the other Class-I sirtuins Sirt1 and Sirt3. [1]

Wee1 Mass Screening: Wee1 mass screening is performed using Amersham's p34cdc2 kinase SPA kit with some modifications. Briefly, 45–60 nM full-length Wee1 kinase is incubated with 25 μM compounds, 20 μM ATP, and 122–441 nM Cdc2/cyclin B in a final volume of 50 μl of enzyme dilution buffer [50 mM Tris (pH 8.0), 10 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mM Na3VO4]. After 30 min incubation at 30°C, 30 μl of [33P]ATP containing kinase buffer [67 mM Tris (pH 8.0), 40 mM NaCl, 13 mM MgCl2, 1 mM DTT, and 0.13 mM Na3VO4] containing 1 μM biotinylated peptide, and 0.25 μCi of [γ-33P]ATP is added to the reaction and incubated for another 30 min at 30°C. The reaction is stopped by adding 200 μl of stop buffer [50 μM ATP, 5 mM EDTA, 0.1% Triton X-100, and 1.25 mg/ml SPA beads in PBS]. After centrifugation at 2400 rpm for 15 min, the plate is counted with Wallac's Microbeta counter.

HeLa cells were plated in petri dishes (5?cm), incubated overnight to a confluency of 30–40% and then treated with SirReal2 dissolved in RPMI1640 medium supplemented with fresh 20% (v/v) FCS, 1% (v/v) penicillin, 1% (v/v) streptomycin, 1% (v/v), L-glutamine, 1% (v/v) DMSO for 5?h at various concentrations. Cells were then washed with prewarmed PBS (2?ml), lysed in SDS–PAGE sample buffer (70?μl, 50?mM Tris/HCl, 0.5?mM EDTA, 1 × Complete Protease Inhibitors, 2% (v/v) IGEPAL, 2% (w/v) SDS, 10% (v/v) glycerol, 50?mM NCA, 3.3?μM trichostatin A, 50?mM DTT, 0.01% (w/v) bromophenol blue, pH 6.8) and sonicated (5?min). Cell samples were then separated using SDS–PAGE (12.5% (w/v) polyacrylamide), transferred to an activated nitrocellulose membrane (Bio-Rad), blocked with non-fat dry milk (Roth, 5% (w/v), TBS, 0.1% (v/v) Tween 20) and probed with an anti-acetyl-α-tubulin antibody (1:1,000) and an anti-GAPDH antibody (1:2,000–1:5,000) as a loading control.