HIPKs in general and HIPK2 in particular, and they highlight the striking superiority of this new compound over both TBI and SB203580. To note that in our hands SB203580 is not appreciably affecting HIPK2 activity up to 40 mM concentration consistent with previous reports. In contrast the IC50 values with TBI was only slightly higher than that with TBID, the latter however being much more selective as also highlighted by the observation that the number of kinases inhibited.90% by either 10 mM TBID or TBI in the same panel is 1 and 10 respectively. From the selectivity data of Figure 4 it was possible to draw a Lorenz curve allowing to calculate a Gini coefficient whose value denotes a remarkable selectivity, especially if compared to that of TBI. The difference in selectivity between TBID and TBI is also striking if their hit rates are compared. Dealing with protein kinase inhibitors, a crucial issue is their cell permeability which is essential to make these reagents useful for in vivo studies. Cell permeability of TBID was 1000413-72-8 chemical information firstly assessed by treating HepG2 cells with increasing concentrations of either TBID or its very close analog 5e almost devoid of inhibitory efficacy and measuring HIPK2 activity in the cell 870281-82-6 lysate : HIPK2 was immunoprecipitated and then assayed for its activity using a specific peptide substrate. As shown in Figure 6A endogenous HIPK2 activity is reduced in a dose dependent manner upon cell treatment with TBID, but not with its inactive analog 5e, providing the evidence that TBID is cell permeant. Incidentally this outcome places TBID in that category of protein kinase inhibitors whose efficacy persists after the kinase has been isolated from the treated cells. Such a behaviour is typical of many CK2 inhibitors, TBB and TBI included, but it has also been reported in the case of other kinases, e.g. PIM-1. The molecular features underlying persistent inhibition, suggestive of a very low Koff rate, are presently unclear, but it is plausible to assume that these compounds, once entrapped in the hydrophobic pocket of the kinase, undergo a thermodynamic advantage, hindering their release into the surrounding aqueous medium. We also considered the possibility that intracellular TBID could irreversibly inactivate HIPK-2 by preventing the
