with increasing concentrations of either TBID or its very close analog 5e almost devoid of inhibitory efficacy and measuring HIPK2 activity in the cell 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 325970-71-6 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 phosphorylation of its up-regulatory Solvent Yellow 14 structure tyrosine, an event occurring only during translation. In our cell model, however, we couldn��t detect any phospho-Tyr signal in HIPK-2 immunoprecipitated from either untreated or treated cells. To reinforce the view that endogenous HIPK-2 is inhibited upon cell treatment with TBID, advantage has been also taken of p53 Ser46, a known target of the kinase. As shown in Figure 6B, TBID treatment markedly reduces the phosphorylation level of this residue, without affecting the amount of p53, under conditions devoid of cell toxicity. To note that, although p53 Ser46 is not targeted exclusively by HIPK2, other putative phosphorylating agents of this residue, notably DYRK2 and PKC, are nearly unaffected by the inhibitor under conditions where HIPK2
