By derivatizing the purely competitive CK2 inhibitor N1-(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)-propane-1,3-diamine (K137) at its 3-amino position with a peptidic fragment composed of three or four glutamic or aspartic acid residues, a new family of bisubstrate inhibitors has been generated whose ability to simultaneously interact with both the ATP and the phosphoacceptor substrate-binding sites has been probed by running mixed competition kinetics and by mutational mapping of the kinase residues implicated in substrate recognition. The most effective bisubstrate inhibitor, K137-E4, interacts with three functional regions of the kinase: the hydrophobic pocket close to the ATP-binding site, the basic residues of the p+1 loop that recognizes the acidic determinant at position n+1 and the basic residues of α-helixC that recognize the acidic determinant at position n+3. Compared with the parent inhibitor (K137), K137-E4 is severalfold more potent (IC50 25 compared with 130 nM) and more selective, failing to inhibit any other kinase as drastically as CK2 out of 140 enzymes, whereas 35 kinases are inhibited more potently than CK2 by K137. K137-E4 is unable to penetrate the cell and to inhibit endogenous CK2, its pro-apoptotic efficacy being negligible compared with cell-permeant inhibitors; however, it readily inhibits ecto-CK2 on the outer cell surface, reducing the phosphorylation of several external phosphoproteins. Inhibition of ecto-CK2 by K137-E4 is accompanied by a slower migration of cancer cells as judged by wound healing assays. On the basis of the cellular responses to K137-E4, we conclude that ecto-CK2 is implicated in cell motility, whereas its contribution to the pro-survival role of CK2 is negligible.
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Research Article|
October 16 2015
Design, validation and efficacy of bisubstrate inhibitors specifically affecting ecto-CK2 kinase activity Available to Purchase
Giorgio Cozza;
Giorgio Cozza
1
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
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Sofia Zanin;
Sofia Zanin
1
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
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Stefania Sarno;
Stefania Sarno
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
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Elena Costa;
Elena Costa
‡Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy
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Cristina Girardi;
Cristina Girardi
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
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Giovanni Ribaudo;
Giovanni Ribaudo
‡Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy
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Mauro Salvi;
Mauro Salvi
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
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Giuseppe Zagotto;
Giuseppe Zagotto
‡Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy
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Maria Ruzzene;
Maria Ruzzene
2
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
2Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Lorenzo A. Pinna
Lorenzo A. Pinna
2
*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
†CNR Institute of Neurosciences, Via Ugo Bassi 58B, 35131 Padova, Italy
2Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Publisher: Portland Press Ltd
Received:
September 11 2014
Revision Received:
August 10 2015
Accepted:
September 08 2015
Accepted Manuscript online:
September 08 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2015 Authors; published by Portland Press Limited
2015
Biochem J (2015) 471 (3): 415–430.
Article history
Received:
September 11 2014
Revision Received:
August 10 2015
Accepted:
September 08 2015
Accepted Manuscript online:
September 08 2015
Citation
Giorgio Cozza, Sofia Zanin, Stefania Sarno, Elena Costa, Cristina Girardi, Giovanni Ribaudo, Mauro Salvi, Giuseppe Zagotto, Maria Ruzzene, Lorenzo A. Pinna; Design, validation and efficacy of bisubstrate inhibitors specifically affecting ecto-CK2 kinase activity. Biochem J 1 November 2015; 471 (3): 415–430. doi: https://doi.org/10.1042/BJ20141127
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