Associate editor: D. Shugar Toward the rational design of protein kinase casein kinase-2 inhibitors Stefania Sarno a , Stefano Moro b , Flavio Meggio a , Giuseppe Zagotto b , Diego Dal Ben b , Paola Ghisellini c , Roberto Battistutta d , Giuseppe Zanotti d , Lorenzo A. Pinna a, * a Department of Biological Chemistry, CNR Biomembrane Research Center, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy b Department of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy c DISTBIMO, University of Genova, Genova, Italy d Department of Organic Chemistry, CNR Biopolymer Research Center, University of Padova, Via F. Marzolo 1, 35131 Padova, Italy Abstract Casein kinase-2 (CK2) probably is the most pleiotropic member of the protein kinase family, with more than 200 substrates known to date. Unlike the great majority of protein kinases, which are tightly regulated enzymes, CK2 is endowed with high constitutive activity, a feature that is suspected to underlie its oncogenic potential and possible implication in viral infections. This makes CK2 an attractive target for anti-neoplastic and antiviral drugs. Here, we present an overview of our present knowledge about CK2 inhibitors, with special reference to the information drawn from two recently solved crystal structures of CK2a in complex with emodin and with 4,5,6,7-tetrabromo-2-azaben- zimidazole (TBB), this latter being the most specific CK2 inhibitor known to date. A comparison with a series of anthraquinone and xanthenone derivatives highlights the crucial relevance of the hydroxyl group at position 3 for inhibition by emodin, and discloses the possibility of increasing the inhibitory potency by placing an electron withdrawing group at position 5. We also present mutational data corroborating the relevance of two hydrophobic residues unique to CK2, Val66 and Ile174, for the interactions with emodin and TBB, but not with the flavonoid inhibitors quercetin and fisetin. In particular, the CK2a mutant V66A displays 27- and 11-fold higher IC 50 values with emodin and TBB, respectively, as compared with the wild-type, while the IC 50 value with quercetin is unchanged. The data presented pave the road toward the rational design of more potent and selective inhibitors of CK2 and the generation of CK2 mutants refractory to inhibition, useful to probe the implication of CK2 in specific cellular functions. D 2002 Elsevier Science Inc. All rights reserved. Keywords: CK2; Inhibitors; Protein kinase; Emodin; Tetrabromobenzotriazole; Flavonoids Abbreviations: CK2, casein kinase-2; DRB, 5,6-dichloro-1-(b-D-ribofuranosyl)benzimidazole; TBB, 4,5,6,7-tetrabromo-2-azabenzimidazole. Contents 1. Introduction ............................................ 160 2. Experimental procedures ..................................... 160 2.1. Phosphorylation assay ................................... 160 2.2. Computer-assisted analysis ................................ 160 3. Looking for casein kinase-2 Achilles’ heel ............................ 161 4. ATP site-directed inhibitors .................................... 162 4.1. Emodin and related compounds .............................. 162 4.2. Flavonoids ......................................... 164 4.3. Halogenated (aza)benzimidazole derivatives ........................ 164 5. Conclusions and perspectives ................................... 166 Acknowledgments ........................................... 167 References ............................................... 167 0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII:S0163-7258(02)00185-7 * Corresponding author. Tel.: +39-049-8276108; fax: +39-049-8073310. E-mail address: pinna@civ.bio.unipd.it (L.A. Pinna). Pharmacology & Therapeutics 93 (2002) 159 – 168