DataSheet1_Mechanism of CK2 Inhibition by a Ruthenium-Based Polyoxometalate.PDF

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.

酪蛋白激酶2（CK2）是一种丝氨酸/苏氨酸蛋白激酶，参与诸多细胞生物学过程，包括基因表达、细胞周期进程、细胞生长与分化、胚胎发生以及细胞凋亡。CK2活性异常升高的情况在多种癌症中已有广泛报道，同时该酶还参与多种其他病理过程，如糖尿病、炎症、神经退行性疾病以及包括新型冠状病毒肺炎（COVID-19）在内的病毒感染。近年来已有大量能够抑制CK2活性的小分子化合物被报道，其中多数以ATP竞争性机制发挥作用。多金属氧酸盐（Polyoxometalates, POMs）是一类具有不同结构与维度的金属氧簇阴离子，具备独特的理化性质。POMs曾被鉴定为纳摩尔级的CK2抑制剂，但其抑制机制与CK2结合位点仍未明确。本研究针对CK2α与钌基多金属氧酸盐[Ru4(μ-OH)2(μ-O)4(H2O)4(γ-SiW10O36)2]10−（Ru4POM）——一种强效CK2抑制剂——之间的相互作用，开展了生化与生物物理表征分析。通过分析型尺寸排阻色谱（SEC）、等温滴定量热法（ITC）以及小角X射线散射（SAXS），我们阐明了Ru4POM的抑制机制：Ru4POM通过静电相互作用结合于酶的带正电底物结合区域，触发酶的二聚化，进而使其失活。Ru4POM是首个被发现可通过底物竞争性机制抑制CK2的非肽类分子。基于SAXS实验数据，本研究构建了失活状态的(CK2α)2(Ru4POM)2复合物的结构模型。