In order to validate the ATP competitive
In order to validate the ATP competitive mode of inhibition, compound 12 was selected as a representative inhibitor of this series, and the IC50 values were determined at eight different concentrations of compound 12 ranging from 0.001 to 100 μM, and repeated four times using different ATP concentrations each time. The IC50 values were observed to increase linearly with the ATP concentration, indicating the ATP competitive mode of CK2 inhibition (Fig. 6). The Ki value of CK2 inhibitory by compound 12 was determined from the IC50 values obtained at various ATP concentrations. The regression line showed a Y-axis intercept at an IC50 value of 0.39 μM (Fig. 6) and thereby defined the Ki value of compound 12 to be at this concentration.
Conclusion From the screening on CK2 activity of a set of 60 structurally diverse natural and synthetic quinones, an embelin derivative (1) with an IC50 = 0.90 ± 0.052 μM was identified. The SAR of the compound class was investigated by synthesizing structural variations and by determination of their CK2 inhibitory activities. It was possible to enhance the activity of compound (1) and an IC50 value of 0.22 ± 0.04 μM was achieved for the most active compound. The cell viability for some compounds was determined, and the binding pattern of these compounds into the ATP binding site of CK2 was analysed by molecular docking, and it also was corroborated by ATP competitive assay. This family of inhibitors is a promising start point for further chemical modifications focused on the nature of the heterocyclic ring fused to the benzoquinone core to obtain more efficient CK2 inhibitors.
Acknowledgment We gratefully acknowledge the financial support from the Spanish MINECO SAF2015-65113-C2-1-R to A.E.B. This project is also co-funded by the European Regional Development Fund (FEDER). PMA thanks to ACIISI for a predoctoral grant. We also thank to Prof. Alejandro Tapia and Gabriela Feresin for providing the natural embeline.
Introduction By 1979, when Beavo and Krebs published a set of guidelines for the establishment of kinase–substrate interactions, just over twenty proteins had been identified as regulated through reversible phosphorylation . With no way of knowing how widespread, and crucial to cellular signaling, phosphorylation would prove to be, the authors optimistically speculated that “… if the present trend continues, another 10–15 [substrates] could be added to the group within the next five years.” . Several decades of work later, we possess a huge body of knowledge regarding kinase specificity, kinase–substrate reactions, and their individual functional effects on the cell. It is estimated that one third of intracellular proteins are phosphorylated, many on several distinct sites , and large scale phosphoproteomic screens have contributed large datasets of potential phosphorylation sites (for example, as of June 2014, the PhosphoSitePlus database  lists almost 120,000 human phosphorylation sites). Interestingly, global analysis of known phosphorylation sites in humans and mice demonstrates significant clustering of phosphorylation sites into specific regions, often showing concurrent phosphorylation . Strikingly, in a study of 70,000 in vivo phosphorylation sites, 54% of phosphoserine (pS) and phosphothreonine (pT) residues were located no further than four Estradiol valerate from another pS/pT residue . While it is apparent that the regulated phosphorylation of proteins is intricately involved in every fundamental cellular process , our understanding of the interplaybetween distinct phosphorylation sites has lagged considerably. Hierarchical protein phosphorylation is a phenomenon in which a kinase phosphorylates a substrate based on its unique sequence determinants, and the addition of phosphate creates adequate sequence determinants for nearby phosphorylation events to occur. These events can result in processive phosphorylation events catalyzed by one kinase, or can involve two or more distinct kinases. Protein kinase families such as GSK3 or CK1 catalyze primed phosphorylation events almost exclusively, as phosphorylation by these kinases usually requires prior phosphorylation of a nearby residue , . By comparison, protein kinase CK2 seems to be more distinct in its ability to use either non-phosphorylated or phosphorylated determinants for phosphorylation . Interestingly, CK2 may therefore be capable of generating clusters of phosphorylation sites both independently and in concert with other kinases. CK2 is a ubiquitously expressed serine/threonine kinase with a multitude of substrates. It participates in a variety of cellular processes, including proliferation, apoptosis, transcription, and translation . In fact, CK2 phosphorylation is so widespread that an estimated 20% of the phosphoproteome can be attributed to CK2 on the basis of phosphopeptides that conform to the minimal consensus recognition sequence for CK2 . CK2 is an acidophilic kinase, with canonical (non-hierarchical) CK2 phosphorylation requiring one or more acidic residues C-terminal to the phosphoacceptor site. Accordingly, the minimal consensus sequence for CK2 phosphorylation is S/T–X–X–D/E. While X can be any amino acid, studies have shown that proline, lysine, or arginine at the +1 position are unfavorable , . In some instances, phosphorylation by CK2 is enabled by an acidic determinant in the +1 position instead of the +3 position. Multiple aspartic or glutamic acid residues seem to have an additive effect, and due to this, many known CK2 sites consist of a serine/threonine residue followed by a string of acidic residues . It has long been recognized that phosphoserine (pS) can substitute for the acidic determinant at the +3 position, enabling CK2 to participate in hierarchical signaling events , , . However, the precise consensus requirements for these events, as well as their impact on cellular signaling, have not been thoroughly investigated. In this study, we use a peptide-based approach to outline favorable consensus requirements for hierarchical phosphorylation by CK2. The results indicate that efficient hierarchical phosphorylation by CK2 requires either multiple phosphoserine residues, or a mix of canonical and hierarchical determinants, with precise spacing. Kinetic analysis indicates that these reactions may be as enzymatically favorable as canonical phosphorylation. Using these determinants, a search of the human proteome for CK2 hierarchical consensus sequences revealed over 1600 proteins that contain at least one motif for CK2 hierarchical phosphorylation, with significant enrichment for proteins involved in transcriptional regulation, development, differentiation, and other fundamental processes. Notably, a number of these sites are previously reported in vivo phosphorylation sites. These results provide compelling in vitro evidence that hierarchical phosphorylation by CK2 has the potential to regulate several crucial cellular processes, demonstrating the impact that hierarchical phosphorylation could have on signal transduction.