Bivalent CK2 Inhibition as a Potent Strategy Against SARS-CoV-2 and Other Coronaviruses

Protein phosphorylation is a critical host-signaling pathway hijacked by viruses to facilitate their replication. Casein Kinase 2 (CK2), a ubiquitously expressed serine/threonine kinase, has emerged as a key host factor exploited by several viruses, including SARS-CoV-2. Here, we investigate the role of CK2 subunits (CK2α, CK2α’, and CK2β) in SARS-CoV-2 replication and evaluate the antiviral potential and broad-spectrum anti-coronavirus activity of two CK2 inhibitors: the previously described ATP-competitive inhibitor CX4945 and a newly developed, highly selective bivalent inhibitor, AB668.

SiRNA-mediated knockdown of CK2α, CK2α’, and CK2β significantly reduced SARS-CoV-2 RNA production, nucleoprotein expression, and viral titers, highlighting the essential role of CK2 in viral replication. Co-immunoprecipitation assays revealed that the SARS-CoV-2 nucleoprotein preferentially interacted with CK2α and CK2β rather than CK2α’. Furthermore, bio-layer interferometry assays demonstrated that the nucleoprotein specifically binds to the tetrameric CK2 holoenzyme (CK2α2β2) but not to individual CK2α or CK2β subunits.

Pharmacological inhibition of CK2 with AB668 exhibited potent antiviral activity, significantly reducing viral replication. Time-of-addition experiments indicated that AB668 targets a pre-replicative or RNA-replicative step of the viral life cycle. Importantly, AB668 displayed broad-spectrum antiviral activity, effectively inhibiting both HCoV-OC43 (β-CoV) and HCoV-229E (α-CoV).

In summary, our findings establish CK2 as a critical host factor for SARS-CoV-2 replication and identify AB668 as a promising candidate for broad-spectrum antiviral therapy. With its high selectivity, minimized off-target effects, and efficacy against multiple coronaviruses, AB668 represents a valuable chemobiological and therapeutic tool.