Proposed Allosteric Inhibitors Bind to the ATP Site of CK2α

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.

CK2α是一种广泛分布且研究充分的激酶，可作为小分子抑制剂的靶点用于癌症治疗。尽管目前已报道了多种不同结构类别的腺苷5'-三磷酸（ATP）竞争性CK2α抑制剂，但这类抑制剂普遍存在显著的脱靶活性，因此亟需开发全新的抑制策略。近期有研究报道了一系列别构CK2α抑制剂，其作用位点为此前未被鉴定的结合口袋。鉴于这类抑制剂与已知的ATP竞争性抑制剂结构相似，我们对其展开了进一步研究。在全面的结构与生物物理分析中，我们未发现任何证据表明这些抑制剂结合于所提出的别构位点。相反，我们通过晶体结构解析、竞争性等温滴定量热法（ITC）、核磁共振波谱（NMR）、氢氘交换（HDX）质谱法以及化学信息学分析，证实这类化合物均结合于ATP结合口袋中。将本研究的结果与实验方法同原报道中的数据进行对比后发现，两项研究结论出现差异的核心原因在于原研究中存在由蛋白聚集引发的非特异性抑制作用。