Proposed Allosteric Inhibitors Bind to the ATP Site of CK2α

Abstract

CK2α is a ubiquitous, well-studied kinase that is a target for small-molecule inhibition, for treatment of cancers. While many different classes of adenosine 5′-triphosphate (ATP)-competitive inhibitors have been described for CK2α, they tend to suffer from significant off-target activity and new approaches are needed. A series of inhibitors of CK2α has recently been described as allosteric, acting at a previously unidentified binding site. Given the similarity of these inhibitors to known ATP-competitive inhibitors, we have investigated them further. In our thorough structural and biophysical analyses, we have found no evidence that these inhibitors bind to the proposed allosteric site. Rather, we report crystal structures, competitive isothermal titration calorimetry (ITC) and NMR, hydrogen–deuterium exchange (HDX) mass spectrometry, and chemoinformatic analyses that all point to these compounds binding in the ATP pocket. Comparisons of our results and experimental approach with the data presented in the original report suggest that the primary reason for the disparity is nonspecific inhibition by aggregation.

Structure- and Similarity-Based Survey of Allosteric Kinase Inhibitors, Activators, and Closely Related Compounds

“…However, the discovery of allosteric kinase inhibitors is far from being routine and has often been serendipitous. Moreover, confirming allosteric mechanisms of action is prone to complications …”

Crystal structure of the Rho-associated coiled-coil kinase 2 inhibitor belumosudil bound to CK2α

“…The CK2_KA mutant contains the mutations K74A, K75A, K76A and R21S. These mutations do not affect binding in the ATP site or the conformation of the ATP site (Brear et al, 2020;De Fusco et al, 2017;Iegre et al, 2018;Brear et al, 2016). These crystals were then soaked overnight with 10 mM belumosudil (MedChemExpress) in mother liquor with 10% DMSO.…”

Chemical probes targeting the kinase CK2: a journey outside the catalytic box

“…100,101 However, Brear et al used a combination of crystal structures, competitive ITC and NMR, hydrogen-deuterium exchange (HDX) mass spectrometry, and computational analyses of the amino thiazole compounds to confirm that these molecules were binding to the ATP binding site of the kinase, and hence acted as type II inhibitors. 104 Independently, Lindenblatt et al also obtained co-crystal structures of these compounds with CK2 showing binding to the ATP site and used kinase enzyme assay to demonstrate that these inhibitors do indeed act via an ATP-competitive mechanism. 105 Modulators of the CK2β subunit Lastly, another approach to inhibit CK2 outside its catalytic pocket is to interact with the CK2β subunit.…”