Mechanism of CK2 Inhibition by a Ruthenium-Based Polyoxometalate

Abstract

CK2 is a Ser/Thr protein kinase involved in many cellular processes such as gene expression, cell cycle progression, cell growth and differentiation, embryogenesis, and apoptosis. Aberrantly high CK2 activity is widely documented in cancer, but the enzyme is also involved in several other pathologies, such as diabetes, inflammation, neurodegeneration, and viral infections, including COVID-19. Over the last years, a large number of small-molecules able to inhibit the CK2 activity have been reported, mostly acting with an ATP-competitive mechanism. Polyoxometalates (POMs), are metal-oxide polyanionic clusters of various structures and dimensions, with unique chemical and physical properties. POMs were identified as nanomolar CK2 inhibitors, but their mechanism of inhibition and CK2 binding site remained elusive. Here, we present the biochemical and biophysical characterizing of the interaction of CK2α with a ruthenium-based polyoxometalate, [Ru4(μ-OH)2(μ-O)4(H2O)4 (γ-SiW10O36)2]10− (Ru4POM), a potent inhibitor of CK2. Using analytical Size-Exclusion Chromatography (SEC), Isothermal Titration Calorimetry (ITC), and SAXS we were able to unravel the mechanism of inhibition of Ru4POM. Ru4POM binds to the positively-charged substrate binding region of the enzyme through electrostatic interactions, triggering the dimerization of the enzyme which consequently is inactivated. Ru4POM is the first non-peptide molecule showing a substrate-competitive mechanism of inhibition for CK2. On the basis of SAXS data, a structural model of the inactivated (CK2α)2(Ru4POM)2 complex is presented.

Exploiting Interactions between Polyoxometalates and Proteins for Applications in (Bio)chemistry and Medicine

“…For example, several POMs have been shown to be highly potent nanomolar inhibitors of protein kinase CK2, which when overexpressed results in abnormally high activity that has been associated with multiple diseases ranging from cancer to diabetes and COVID-19. [113,114] The ability of POMs to inhibit the activity of CK2 was found to be roughly correlated to the size and charge of the POM, with larger and highly charged POMs displaying generally higher activity, which is likely due to stronger binding interactions. [114] Interestingly, [P 2 Mo 18 O 62 ] 6À showed remarkable selectivity for CK2 over other kinases, however the mechanism of action could not be established owing to the low stability of the POM.…”

“…Owing to their selective interactions with proteins, POMs have been shown to act as inhibitors for a wide range of enzymes, such as kinases, [113,114] proteases, [115] ATPases, [116] choline esterases, [117] etc. [67,118,119] Furthermore, POMs have been shown to act as effective inhibitors of Aβ aggregation, [19,79,95,[120][121][122] which in the brain leads to the formation of plaques and neurofibrillary tangles that are characteristic of Alzheimer's disease.…”

“…On the other hand, the more stable Ru-substituted POM ([Ru 4 (μ-OH) 2 (μ-O) 4 (H 2 O) 4 (γ-SiW 10 O 36 ) 2 ] 10À ) has been recently shown to display high inhibitory activity towards CK2 owing to the formation of a protein dimer induced by the POM, thereby preventing the binding of the substrate. [113] Similarly, several POMs have been investigated as inhibitors for Aβ aggregation, and it was determined that their inhibitory activity increased with the binding affinity of the POM, which was linked to electrostatic interactions with positively charged regions of the peptide, and with the size of the POM. [79,120] Moreover, as previously discussed, the inhibitory activity and selectivity could be enhanced through metal substitution or through functionalization of the POM.…”

“…This makes POMs promising candidates for therapeutic agents towards a wide range of diseases, such as cancer, Alzheimer's, AIDS, and COVID‐19. For example, several POMs have been shown to be highly potent nanomolar inhibitors of protein kinase CK2, which when overexpressed results in abnormally high activity that has been associated with multiple diseases ranging from cancer to diabetes and COVID‐19 [113, 114] . The ability of POMs to inhibit the activity of CK2 was found to be roughly correlated to the size and charge of the POM, with larger and highly charged POMs displaying generally higher activity, which is likely due to stronger binding interactions [114] .…”

“…Interestingly, [P 2 Mo 18 O 62 ] 6− showed remarkable selectivity for CK2 over other kinases, however the mechanism of action could not be established owing to the low stability of the POM. On the other hand, the more stable Ru‐substituted POM ([Ru 4 (μ‐OH) 2 (μ‐O) 4 (H 2 O) 4 (γ‐SiW 10 O 36 ) 2 ] 10− ) has been recently shown to display high inhibitory activity towards CK2 owing to the formation of a protein dimer induced by the POM, thereby preventing the binding of the substrate [113] . Similarly, several POMs have been investigated as inhibitors for Aβ aggregation, and it was determined that their inhibitory activity increased with the binding affinity of the POM, which was linked to electrostatic interactions with positively charged regions of the peptide, and with the size of the POM [79, 120] .…”