Discovery of holoenzyme-disrupting chemicals as substrate-selective CK2 inhibitors

Abstract

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2β subunits is essential for substrate selectivity. The CK2α/CK2β interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2β interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.

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

“…The highest affinity reported so far for a small molecule binding at the interface site is 30 μM (ref. 82) and higher affinity inhibitors for this site are peptides or stapled peptides. This is not surprising if we consider that bigger molecules like peptides better interact with the big and shallow pocket found at the interface.…”

“…9. 78,82,85,86 Fig. 9 CK2 inhibitors reported in chronological order and acting at the protein interface between the catalytic and regulatory subunit with the cocrystal structure of CK2α and CAM7117 shown on the left (PDB: 6Q4Q).…”

“…82 Compound 6 (in Kufareva et al) successfully inhibited the phosphorylation of CK2β-dependent substrates, suppressed MDA-MB231 triplenegative breast cancer cell growth and induced apoptosis in the μM range. 82 As such, compound 6 is the first example of a small molecule inhibitor of the CK2α/β interaction which has been shown to be effective in cells. Alongside CAM187, compound 6 represents an excellent starting point for the development of selective and potent small molecule therapeutics for the inhibition of CK2 via the perturbation of the CK2 PPI.…”

Proposed allosteric inhibitors bind to the ATP site of CK2α

“…It is also a highly promiscuous kinase with hundreds of recorded cellular substrates. 19 Inhibition of CK2α has been achieved mostly through an ATPcompetitive mechanism and a number of high affinity inhibitors of distinctly different chemotypes are known and characterized structurally 17,[20][21][22][23][24][25][26][27] . Despite significant effort, most inhibitors have poor selectivity, although claims are often made to the contrary [28][29][30][31][32] .…”

“…In addition to active site targeting, another approach is the inhibition of CK2α binding to its scaffolding partner CK2β. This method has the potential to generate specific inhibitors that affect only a subset of CK2 substrates 24,33 and has led to some initial encouraging success that requires further development 24,33,34 .…”