Functional proteomics strategy for validation of protein kinase inhibitors reveals new targets for a TBB-derived inhibitor of protein kinase CK2.

CK2 is a constitutively active protein kinase with key regulatory roles in many cellular signaling events which has been implicated in several human diseases. To investigate its roles in biological events and potential as a therapeutic target, several potent CK2 inhibitors have been developed including TBB and its derivatives that have been employed in many studies. Despite the utility of these compounds, a precise understanding of their mode of action within cells remains incomplete. In fact, cells are typically treated with inhibitor concentrations (>5 μM) that are orders of magnitude higher than their in vitro inhibitory constants (<0.05 μM). Accordingly, we hypothesized that CK2 inhibitors could have off-target effects that are not recognized when inhibitors are profiled using panels of recombinant protein kinases. To address this issue, we combined structural modeling with inhibitor-affinity purification and proteomics to test the specificity of derivatives of TBB using whole cell lysates of HeLa cells. While these studies confirmed that CK2 does bind to the immobilized inhibitor, several other abundant ATP/GTP-binding proteins were also identified and confirmed. These results suggest that highly abundant nucleotide binding proteins may limit the bioavailability of the free inhibitor and interactions with CK2 in the cellular environment. This article is part of a Special Issue entitled: From protein structures to clinical applications.