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SGC-CK2-1 A chemical probe for CK2This probe is available from Tocris and Sigma This control is available from Sigma For any inquiries please contact proberequests@thesgc.org.group newOverview 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 -20oC 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 -20oC Dissolution Soluble up to 10mM in DMSO Selectivity ProfileSGC-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) 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) 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 DataA 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). Related Links: https://www.tocris.com/products/sgc-ck2-1_7450#product-literature