Unveiling the significance of His160 residue in CK2α-CX-4945 interaction with implications for innovative diabetes treatment

Diabetes mellitus is a chronic metabolic disorder characterized by disturbances in the body's sugar metabolism due to inadequate insulin production or utilization. Current therapeutic approaches focus on management rather than a cure necessitating the pursuit of novel treatments. Understanding the molecular mechanisms of the disease may aid innovative therapy development. The “Diabetic Kinome” encompasses protein kinases including DYRK1A, GSK3β, and CK2 responsible for regulating functions involved in diabetes. Targeted inhibition of these kinases shows potential as an alternative treatment, although this approach is still under investigation. Challenges arise due to the significant similarity in the kinase domains of diabetic kinome members, which requires meticulous design of highly specific inhibitors. Precision in this regard is crucial for minimizing off-target effects and promoting the advancement of effective anti-diabetic treatment. CK2, also known as Casein Kinase 2, is a tetrameric enzyme consisting of two catalytic subunits (CK2α and CK2α') and two regulatory subunits (CK2β). It is a key regulator of various cellular processes. Additionally, the recent studies have indicated an involvement of this particular kinase in the endocrine functions of pancreatic β-cells. Therefore, CK2 becomes one of the molecular targets in the treatment of diabetes. One of the most renowned CK2 inhibitors is CX-4945, a potent ATP-competitive inhibitor. However, this inhibitor also binds to other kinases within the diabetic kinome such as DYRK1A, making it challenging to attribute the observed effect to a specific kinase. Further investigation is essential to elucidate the intricate interaction mechanism between the inhibitor and the kinases. The analysis of the available CK2α-CX-4945 and DYRK1A-CX-4945 structures indicated a crucial role of the His160 residue in CK2α, in facilitating the unique inhibitor-kinase interaction. The exact role of His160 was further examined in the presented thesis. Two mutants of His160 (Ala and Glu) were created using site-directed mutagenesis, expressed in E. coli, and subsequently purified. The thermal shift assay analysis confirmed that introduced changes did not affect the stability of the protein. Moreover, these alterations did not affect the enzymatic activity, and the analysed CK2α variants remained equally active, as demonstrated in the ADP-GLO assay. Substitution of His160 by Ala or Glu affected the kinase-CX-4945 interaction. The strongest inhibition was observed for the wild-type protein, while both mutants (H160A and H160E) displayed higher IC50 values. Then, crystallization trials were conducted, yielding crystals of three CK2α kinase variants (WT, H160A, and H160E) in complex with the CX-4945 inhibitor. This will enable the explanation of the observed differences in the interaction between CK2α variants and CX-4945 at the molecular level. These results emphasize His160 residue role in CK2α-CX-4945 interaction, guiding the design of potent and selective inhibitors. These findings hold promise for design innovative ATP-competitive inhibitors tailored for CK2α, advancing potential therapies for diabetes treatment.