A-196 is a potent and selective inhibitor of SUV420 h1 and SUV420 h2 with IC50 values of 0.025 and 0.144 μM, respectively; more than 100-fold selective over other histone methyltransferases and non-epigenetic targets.

SUV420H1:25 nM, SUV420H2:144 nM

A-196 在1和10μM浓度下强效抑制SUV420 h1和SUV420 h2，但对其它PKMTs（包括另一种H4K20修饰酶PR-SET7以及以H3K4、H3K9、H3K27和H3K79为底物的酶）无活性。在细胞内，A-196引起全局性H4K20me2和H4K20me3的下降，同时使H4K20me1上升，但对其它组蛋白修饰无影响。A-196抑制电离辐射后53BP1焦点的形成，并减少NHEJ介导的DNA断裂修复，但不影响同源指导修复。

Briefly, Hsp90 from porcine spleen extract is isolated by affinity capture on a purine-affinity media. The Hsp90 loaded media is then challenged with test compound (PF-04929113) at a given concentration, ranging from 0.8 to 500 μM, and the amount of Hsp90 liberated at each concentration is determined. The resulting IC50 values are corrected for the ATP ligand concentration and presented as apparent Kd values.

U2OS cells are seeded on 6-well plates with 3 μM A-196 or DMSO as a control, and incubated for 48 h. The cells are washed once in 1 X PBS and then lysis buffer (20 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, PMSF, protease inhibitors, benzonase) is added to half the cells to create whole cell extract (WCE). The remaining cells are subjected to sequential cellular fractionation. First the cell pellet is resuspended in hypotonic buffer A (10 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.3 M sucrose, 1 mM DTT and protease inhibitors) and then 0.1% triton X-100 is added. The cells are incubated for 15 min on ice and pelleted by centrifugation at 1,500 g. The supernatant is clarified by centrifugation at max speed and saved as the cytoplasmic fraction. The pellet is resuspended in buffer B (3 mM EDTA, 3 mM EGTA, 1 mM DTT and protease inhibitors) and incubated on ice for 40 min and then centrifuged at 1,500 g for 5 min. The supernatant is clarified and saved as the nucleoplasmic fraction. The pellet is resuspended in lysis buffer and incubated for 5 min at room temperature before being resuspended in 4× loading dye. The final lysate contains the solubilized chromatin fraction.