generally tend to be susceptible to CK2 inhibitors, notably CK2 itself, DYRK1A and PIM1. To gain more information about the selectivity of TBID the compound was profiled at 1 instead of 10 mM concentration on a larger panel of 125 GNF-7 protein kinases, implemented with other members of the HIPK sub-family and many protein tyrosine kinases which were scarcely represented in the smaller panel. The data, shown in File S1, corroborate the concept that HIPK-2 is the kinase most susceptible to TBID. HIPK1 and HIPK3 however are also significantly inhibited with residual activities of 39% and 53%, respectively. In EW-7197 contrast none of the protein tyrosine kinases tested is appreciably affected by TBID with the only possible exception of IGF-IR. This together with CAMK1 and CAMKKb are the only kinases inhibited more than 20% a part from the HIPKs. Collectively taken these data denote TBID as a very selective inhibitor of 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 firstly assessed by treating HepG2 cells
