Inauhzin (INZ)(INZ) is a novel small molecule that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human Y cells without causing apparently genotoxic stress(IC50=3 uM, in A549 cell).

SIRT1:3 μM

Kinase Profile Assay: Assays for serine/threonine kinases using radio labeled [γ-33P] ATP are performed in 96 well plates. BRAF and c-RAF are expressed as N-terminal FLAG-tagged protein using a baculovirus expression system. The reaction conditions are optimized for each kinase: BRAF (25 ng/well of enzyme, 1 μg/well of GST-MEK1(K96R), 0.1 μCi/well of [γ-32P] ATP, room temperature, 20 min reaction); c-RAF (25 ng/well of enzyme, 1 μg/well of GST-MEK1 (K96R), 0.1 μCi/well of [γ-32P] ATP, room temperature, 20 min reaction). Enzyme reactions are performed in 25 mM HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol and 0.5 μM ATP containing optimized concentration of enzyme, substrate and radiolabeled ATP as described above in a total volume of 50 μL. Prior to the kinase reaction, compound and enzyme are incubated for 5 min at reaction temperature as described above. The kinase reactions are initiated by adding ATP. After the reaction period as described above, the reactions are terminated by the addition of 10% (final concentration) trichloroacetic acid. The [γ-33P] or [γ-32P]-phosphorylated proteins are filtered in GFC filter plates with a Cell Harvester and then the plates are washed out with 3% phosphoric acid. The plates are dried, followed by the addition of 40 μL of MicroScint0. The radioactivity is counted by a TopCount scintillation counter.