br Acknowledgement br Introduction CK kinases

Acknowledgement
Introduction CK2 kinases are ubiquitous highly conserved pleiotropic protein kinases (PKs) that have roles in various essential and pathological biological processes.1, 2, 3, 4, 5, 6, 7 In mammals the CK2 serine/threonine kinase family is composed of 2 enzymes, CK2α and CK2α’. CK2 kinases can function as monomeric kinases and within a tetrameric complex. The latter is composed of two CK2 catalytic units (CK2α and/or CK2α’) and two regulatory units (CK2β). In many types of cancer CK2 is overexpressed, it favors rapid proliferation and survival of cancer Pam2CSK4 and supports angiogenesis.8, 9, 10, 11, 12, 13, 14, 15 In turn, pharmacological inhibition or downregulation of CK2 by gene silencing has been shown to suppress angiogenesis and induce apoptosis.10, 16, 17, 18, 19, 20, 21 Within the past decade, two inhibitors of CK2-catalyzed protein phosphorylation, CX-4945 (Silmitasertib) and CIGB-300, entered clinical trials (www.clinicaltrials.gov identifiers: NCT02128282 and NCT01639625), both as anticancer agents. CX-4945 is an ATP-competitive CK2 inhibitor, whereas CIGB-300 is a peptidic inhibitor of CK2-catalyzed reaction that binds to the phospho-acceptor domain of CK2 substrates, thus impairing the correct phosphorylation by the enzyme. In addition, the overexpression of CK2 often correlates with the diagnosis of cancer, suggesting that the PK is not only a potential drug target but may also serve as a biomarker for certain types of malignancy.8, 11, 12, 18, 22 The implication of CK2 in malaria, neurodegenerative and several other human diseases23, 24, 25 has been described that increases the pharmacological potential of inhibition of CK2. A large number of inhibitors of CK2 has been developed. Most of them are rigid small molecules that contain one or more aromatic carbo- or heterocycles; these compounds target the ATP-binding pocket of the enzyme. CK2 is an acidophilic PK that has the preference to phosphorylate serine/threonine residues in negatively charged regions of proteins. Accordingly, CK2 is also inhibited by negatively charged oligomers such as heparin28, 29 and bisubstrate inhibitors comprising an anionic peptide or peptoid fragment.30, 31, 32, 33 The activity of CK2 and inhibitory potential of compounds towards CK2 have been mostly assessed by monitoring the CK2-catalyzed phosphorylation of synthetic peptide substrates with radioactively labeled ATP, the handling of which requires special precautions. Therefore, photoluminescence-based binding and displacement assays could be valuable alternatives for quantification of catalytically active PKs and for screening and characterization of inhibitors.30, 34 Previously, we have shown that complexes of protein PKs with inhibitors that comprised sulfur or selenium atoms in aromatic structures were phosphorescent at room temperature, while the long-lifetime signal emitted by the free inhibitor was negligible.34, 35, 36 This phenomenon was caused by stabilization of the triplet excited state of the aromatic structure of the inhibitor inside the ATP-binding pocket of PK that slowed down competing non-radiative relaxation pathways (e.g., quenching by dissolved oxygen). Moreover, significant enhancement of kinase binding-responsive long lifetime photoluminescence was achieved when the inhibitor was covalently labeled with a bright fluorescent dye. This enhancement resulted from efficient radiationless intramolecular Förster-type resonant energy transfer (RET) from the donor phosphor in the excited triplet state to the non-excited acceptor fluorophore, leading it to the excited singlet state of the latter and emission of light.35, 38