SGC-CK2-1 A chemical probe for CK2

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Overview

SGC-CK2-1

SGC-CK2-1N

Based upon a pyrazolopyrimidine scaffold with potent CK2 inhibition exemplified by AstraZeneca, the SGC has developed a high-quality chemical probe and its negative control for protein kinase CK2.1 This compound shows equal potency in binding to both catalytic subunits of CK2: CK2α (CSNK2A1) and CK2α' (CSNK2A2). CK2 is a serine/threonine kinase that is part of the larger CMGC family, which is named after the initials of its subfamily members including cyclin-dependent kinases (CDK), mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK), and CDC-like kinase (CLK). A plethora of biological functions have been ascribed to CK2 ranging from cell survival and proliferation to inflammation. CK2 phosphorylates more than 300 proteins and, via genetic and biochemical studies in a variety of experimental models, has been found to be both constitutively active and ubiquitously expressed.2-5 While CK2 inhibition has been widely studied in the context of cancer, pervasive use of non-selective CK2 inhibitors has confounded interpretation of results in many oncological studies.

Figure 1: Phylogenetic kinase tree with CK2α and CK2α' highlighted with red circles. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com)

Key profiling data:

Enzymatic assays (Eurofins): CK2α IC₅₀ = 4.2 nM; CK2α' IC₅₀ = 2.3 nM at 10µM ATP
Cellular data (nanoBRET): CK2α IC₅₀ = 36 nM; CK2α' IC₅₀ = 16 nM
Only 11/403 kinases with PoC <35 when screened at 1 μM

Properties

SMILES:
CC1=CC=C(NC2=NC3=C(C#N)C=NN3C(NC4CC4)=C2)C=C1NC(CC)=O
InChI:
InChI=1S/C20H21N7O/c1-3-19(28)25-16-8-15(5-4-12(16)2)23-17-9-18(24-14-6-7-14)27-20(26-17)13(10-21)11-22-27/h4-5,8-9,11,14,24H,3,6-7H2,1-2H3,(H,23,26)(H,25,28)
InChIKey:YKDZIFFKQUNVHH-UHFFFAOYSA-N

Physical and chemical properties
Molecular weight	375.44
Molecular formula	C20 H21 N7 O
IUPAC name	N-(5-((3-cyano-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-2-methylphenyl)propionamide
clogP	1.94
PSA	104.91
No. of chiral centres	0
No. of rotatable bonds	7
No. of hydrogen bond acceptors	4
No. of hydrogen bond donors	3
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20^oC
Dissolution	Soluble up to 10mM in DMSO

SMILES:
CC1=CC=C(N(C)C2=NC3=C(C#N)C=NN3C(NC4CC4)=C2)C=C1N(C)C(C5CC5)=O
InChI:
InChI=1S/C23H25N7O/c1-14-4-9-18(10-19(14)29(3)23(31)15-5-6-15)28(2)20-11-21(26-17-7-8-17)30-22(27-20)16(12-24)13-25-30/h4,9-11,13,15,17,26H,5-8H2,1-3H3
InChIKey:
IWJQIDLFOBMRNF-UHFFFAOYSA-N

Physical and chemical properties
Molecular weight	415.5
Molecular formula	C23 H25 N7 O
IUPAC name	N-(5-((3-cyano-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-5-yl)(methyl)amino)-2-methylphenyl)-N-methylcyclopropanecarboxamide
clogP	3.04
PSA	87.33
No. of chiral centres	0
No. of rotatable bonds	7
No. of hydrogen bond acceptors	4
No. of hydrogen bond donors	1
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20^oC
Dissolution	Soluble up to 10mM in DMSO

Selectivity Profile

SGC-CK2-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM. Only 11 kinases showed PoC <35 giving an S(35) at 1 μM = 0.027. Potential off-targets were tested in the nanoBRET target engagement assay (DYRK2) and via Eurofins radiometric and LANCE kinase assays. Data corresponding with off-target kinase activity is shown in the table below.

Kinase	% Control at 1µM	Enzymatic IC50 (nM)	NanoBRET IC50 (nM)
Kinase	% Control at 1µM	Enzymatic IC50 (nM)	NanoBRET IC50 (nM)
CK2α'/CSNK2A2	0	2.3	16
DRAK1	0	>10000	NT
CK2α/CSNK2A1	0.5	4.2	36
DYRK2	14	440	3700
PLK4	23	>10000	NT
HIPK2	26	3400	NT
MEK5	28	0% AT 1µM	NT
HIPK1	32	3700	NT
HIPK3	34	8100	NT
RIOK2	32	NT	NT
SGK3	34	>10000	NT

Figure 1: SGC-CK2-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and off-target kinases inhibited PoC <35 were tested in an orthogonal assay.

SGC-CK2-1N was also tested in the DiscoverX panel and 1 kinase had a PoC <35. The negative control was sent to Eurofins for testing in enzyme assays for CSNK2A1 and CSNK2A2. The results are in the table below.

Kinase	% Control at 1µM	Enzymatic IC50 (nM)	NanoBRET IC50 (nM)
Kinase	% Control at 1µM	Enzymatic IC50 (nM)	NanoBRET IC50 (nM)
RIOK1	23	NT	NT
RET	35	NT	NT
FLT3	40	NT	NT
BRAF	55	NT	NT
IKK-alpha	58	NT	NT
MKNK1	58	NT	NT
STK33	61	NT	NT
TNK1	62	NT	NT
EPHA2	63	NT	NT
CK2α'/CSNK2A2	65	>10000	>10000
CK2α/CSNK2A1	100	>10000	NT

Figure 2: SGC-CK2-1N was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and follow-up CK2α and CK2α' enzymatic assays were done to confirm no activity.

Cell-based Assay Data

A NanoBRET assay was utilized to assess the binding affinity of SGC-CK2-1 to CK2α and CK2α'. The negative control shows no binding affinity for CK2α'.

Figure 1: SGC-CK2-1 and SGC-CK2-1N were profiled in the CK2 NanoBRET assays.

References

Wells, C., Drewry, D. H., Pickett, J. E. & Axtman, A. D. SGC-CK2-1: the first selective chemical probe for the pleiotropic kinase CK2. ChemRxiv, 10.26434/chemrxiv.12296180.v12296181 (2020).
Rabalski, A. J., Gyenis, L. & Litchfield, D. W. Molecular Pathways: Emergence of Protein Kinase CK2 (CSNK2) as a Potential Target to Inhibit Survival and DNA Damage Response and Repair Pathways in Cancer Cells. Clinical Cancer Research 22, 2840-2847, doi:10.1158/1078-0432.Ccr-15-1314 (2016).
Meggio, F. & Pinna, L. A. One-thousand-and-one substrates of protein kinase CK2? FASEB journal : official publication of the Federation of American Societies for Experimental Biology 17, 349-368, doi:10.1096/fj.02-0473rev (2003).
Nuñez de Villavicencio-Diaz, T., Rabalski, A. J. & Litchfield, D. W. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks. Pharmaceuticals 10, 27 DOI: 10.3390/ph10010027 (2017).
Ahmed, K., Gerber, D. A. & Cochet, C. Joining the cell survival squad: an emerging role for protein kinase CK2. Trends in Cell Biology 12, 226-230, doi:10.1016/S0962-8924(02)02279-1 (2002).