Inhibition of Protein Kinase CK2 by Flavonoids and Tyrphostins. A Structural Insight Graziano Lolli, ⊥,∥,§ Giorgio Cozza, ⊥,† Marco Mazzorana, #,∥ Elena Tibaldi, ‡ Luca Cesaro, † Arianna Donella-Deana, † Flavio Meggio, † Andrea Venerando, † Cinzia Franchin, ∥ Stefania Sarno, † Roberto Battistutta,* ,∥,§ and Lorenzo A. Pinna* ,†,∥ ∥ Venetian Institute of Molecular Medicine (VIMM), Padova, Italy, Via G. Orus 2 35129 Padova, Italy § Department of Chemical Sciences, University of Padova, Via Marzolo 1 35131 Padova, Italy † Department of Biomedical Sciences, University of Padova, Viale G. Colombo 35131 Padova, Italy ‡ Department of Molecular Medicine, University of Padova, Viale G. Colombo 35131 Padova, Italy * S Supporting Information ABSTRACT: Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 μM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC 50 values < 1 μM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4′. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site. F lavonoids belong to a group of natural substances and are found in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine. Flavonoids are the most important plant pigments for flower coloration and the most common group of polyphenolic compounds in the human diet. 1 Over 9000 naturally occurring flavonoids have been characterized and their aglyconated forms classified into four main groups, flavones, flavanols, flavanones, and flavanonols, based on differences in molecular backbone structure as outlined in Table 1. 2 Moreover, the position of the benzenoid substituent splits flavonoids into two subclasses: flavonoids (2-position) and isoflavonoids (3-position). Because of the variety of pharmacological activities in the mammalian body, flavonoids are intensively studied for their potentials as therapeutic agents. The best known property of almost every group of flavonoids is their ability to act as antioxidants and metal chelators, protecting against reactive oxygen species (ROS). Free radicals and ROS have been implicated in a large number of human diseases, and many flavonoids, notably, quercetin, kaempferol, myricetin, have been reported to display beneficial effects as anti-inflammatory, anticancer, and cardioprotective com- pounds. 3−5 This seems to account for the decreased incidence of cardiovascular disease in the Mediterranean population associated with red wine consumption (known as the “French Paradox”, i.e., the observation that French people suffer a telatively low incidence of coronary heart disease despite having a diet rich in saturated fats, possibly balanced by red wine rich in flavonoids 6 ). Moreover, several other beneficial effects of flavonoids have been reported; for example, flavonoids may display anti-inflammatory and antiulcer activity by inhibiting lipooxygenase (LO) and cyclo-oxygenase (COX). 7,8 An antidiabetic effect has been also reported due to the stimulation by flavonoids of insulin release. 9 Finally antibacterial, antiviral, Received: February 10, 2012 Revised: July 13, 2012 Published: July 14, 2012 Article pubs.acs.org/biochemistry © 2012 American Chemical Society 6097 dx.doi.org/10.1021/bi300531c | Biochemistry 2012, 51, 6097−6107