Design, validation and efficacy of bisubstrate inhibitors specifically affecting ecto-CK2 kinase activity.
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.
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.
Giorgio
G
Cozza
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.
Sofia
S
Zanin
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.
Stefania
S
Sarno
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy.
Elena
E
Costa
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.
Cristina
C
Girardi
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy.
Giovanni
G
Ribaudo
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.
Mauro
M
Salvi
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy.
Giuseppe
G
Zagotto
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 maria.ruzzene@unipd.it lorenzo.pinna@unipd.it.
Maria
M
Ruzzene
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 maria.ruzzene@unipd.it lorenzo.pinna@unipd.it.
Lorenzo A
LA
Pinna
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001562:Benzimidazoles / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000187:drug effects*
D001562:Benzimidazoles; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C034454:ectoprotein kinase
CK2; bisubstrate inhibitor; cell migration; ecto-kinase; protein kinase inhibitor; protein phosphorylation; signal transduction