Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like β-Coronaviruses

Abstract

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human, bat, and murine β-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in β-coronavirus replication. Spike protein endocytosis was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for the development of anti-SARS-like β-coronavirus drugs.

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of β-Coronaviruses

“…When intercellular spike protein was visualized and quantified, vehicle-treated cells showed efficient uptake of the His6-tagged spike protein. In accordance with previous studies, treatment of the cells with 1 μM SGC-CK2-1 efficiently decreased spike protein uptake . Similarly, treatment of the cells with 1 μM PIKfyve inhibitors from our series ( 8 and 17 ) or apilimod also significantly reduced spike protein uptake versus the vehicle-treated control cells.…”

“…MHV belongs to the β-coronavirae genus along with SARS-CoV-2. While MHV is a common mouse pathogen, it is not infectious to humans and it has been widely used as a model to study the virulence of SARS-CoV-2. , Furthermore, there is 94% identity between human and mouse PIKfyve. The optimal titer and time point to analyze viral replication were determined via inoculation of mouse derived-from-brain-tumor (DBT) cells by MHV-NLuc .…”

“…While MHV is a common mouse pathogen, it is not infectious to humans and it has been widely used as a model to study the virulence of SARS-CoV-2. , Furthermore, there is 94% identity between human and mouse PIKfyve. The optimal titer and time point to analyze viral replication were determined via inoculation of mouse derived-from-brain-tumor (DBT) cells by MHV-NLuc . Accordingly, DBT cells were inoculated by MHV-nLuc with an MOI = 0.1 and luciferase measured at 10 h post-infection.…”

“…PIKfyve inhibitors were diluted in DMEM, preparing 4-fold serial dilutions over a concentration range of 15–0.22 μM. The assay protocol was executed as previously described . An LDH assay was run in parallel to assess the viability (Figure S3).…”

“…At 46 h post-infection, Nano-Glo reagent (Promega) was added to each well according to the manufacturer’s protocol (Promega) and relative light units (RLUs) were measured using a Promega GloMax plate reader. An LDH assay to assess viability ( n = 2, with two technical replicates per assay) was run in parallel using 20 000 uninfected A549-ACE2 cells per well in 96-well clear plates . After 46 of treatment with compounds in dose–response, an aliquot of the supernatant from each well was taken, centrifuged to remove cell and debris, and transferred to a clean plate.…”