MN 64

Kinase experiment:

Inhibitory potency of compounds on Tankyrase-1 enzymatic activity is evaluated using a Scintillation Proximity Assay (SPA). The assay is designed to measure compound inhibition of Tankyrase-1 autoPARsylation (Tankyrase-1 is both enzyme and substrate in this assay). Truncated recombinant human Tankyrase-1 protein (amino acids E1023-T1327) is purified from SF9 cells. The assay is conducted using 0.11 μM of Tankyrase-1 protein and 3 μM nicotinamide adenine dinucleotide (NAD+, 2.12 μM 3H-NAD+ with a specific radioactivity of 1690 Ci/mol, 0.88 μM biotin- NAD+), in pH 7.5 Tris buffer (60 mM Tris, 1 mM DTT, 0.01% (v/v) Tween-20®, 2.5 mM MgCl2, 0.3 mg/mL BSA). For IC50 determination, 10 mM DMSO stock solution of a compound (MN-64) is sequentially diluted by two-fold in DMSO, and aliquots of the diluted solutions are transferred to 384-well assay plates and mixed with Tankyrase-1 solution[1].

References:

[1]. Narwal M, et al. Discovery of tankyrase inhibiting flavones with increased potency and isoenzyme selectivity. J Med Chem. 2013 Oct 24;56(20):7880-9.