Host kinase identified as an inhibition target for SARS-CoV-2

Coronaviruses are positive-sense viruses with a ribonucleic acid (RNA) genome, with a host of members, which infect humans and animals. Among these, the alpha- and beta-coronaviruses are known to infect mammals. The current coronavirus disease 2019 (COVID-19) pandemic is due to a beta-coronavirus thought to have jumped across the species barrier from an as-yet-unknown mammalian animal host.

Study: Host kinase CSNK2 is a target for inhibition of pathogenic β-coronaviruses including SARS-CoV-2. Image Credit: Mark Umbrella/Shutterstock

Called the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this pathogen has taken over 5 million lives so far in just two years. Although vaccines were developed and rolled out in many countries, the emergence of more transmissible and/or immune escape variants of the virus threatens to prolong the pandemic. This indicates the urgent need for effective antivirals to help treat this and other pathogenic beta-coronaviruses that may emerge later.

Background

The coronavirus envelope bears multiple spike proteins composed of a trimer of spike monomers. The spike engages angiotensin-converting enzyme 2 (ACE2) receptor molecules on the host cell surface. Since the spike is typically restricted in its binding to ACE2 molecules of one or a few species of animals, it determines the spectrum of infectivity of each beta-coronavirus.

With the earlier SARS-CoV and MERS-CoV (Middle East coronavirus) outbreaks, the cognate receptors were ACE2 and dipeptidyl peptidase 4. Conversely, the mouse hepatitis virus (MHV) spike recognizes the mouse carcinoembryonic antigen-related cell adhesion molecule 1 (CAM1) receptor. The MHV is often used to study the features of the beta-coronaviruses since, unlike the SARS-like viruses, it can be handled in a biosafety level 2 environment.

Spike-receptor binding is followed by endocytosis of the receptor-virus complex, with the virus subsequently undergoing release and uncoating of the coronavirus genome. The encoded viral replicase and RNA-dependent RNA polymerase enzymes are transcribed and then translated into structural and accessory viral proteins.

The outcome is the assembly of new viral particles within the infected host cell, translocation of the new virions in vesicles to the host cell membrane, and their release to the outside via nonlytic exocytosis. During this process, the virus hijacks a number of host proteins to accomplish the various steps of its life cycle and suppress and evade immune response pathways.

The development of antiviral agents is a pathway often hampered by the negative selection pressure of currently active agents. However, resistance is unlikely to emerge rapidly against agents targeting highly conserved host cell proteins essential for viral replication or host immune evasion. Protein kinases are one such family of enzyme.

These enzymes occur in almost every cell signaling pathway and are often the targets of viral activity during infection. One of these is casein kinase 2 (CSNK2), a serine/threonine kinase occurring in tetrameric form with two catalytic sites. Multiple viruses undergo protein phosphorylation catalyzed by CSNK2, though all may not be essential events for replication.

Recent studies suggest that CSNK2A1 and CSNK2A2 interact with the nucleocapsid protein of beta-coronaviruses, including SARS-CoV-, SARS-CoV and SARS-CoV MERS-CoV, and potentially other members of this family. Multiple substrates have been identified for this enzyme in SARS-CoV-2-infected cells, indicating that its expression increases following infection with this virus.

Small molecule inhibitors of CSNK2 may thus prevent viral replication since SARS-CoV-2 hijacks this host cell enzyme to achieve infectivity and replication. The researchers used chemogenomics to identify viable drug candidates, a method whereby drug targets are validated. The underlying principle is to link the disruption of cell phenotype to a particular molecular target, which is affected by a selective small-molecule inhibitor.

In the current study available as a preprint on bioRxiv*, such an approach was used, with multiple inhibitor series including many small molecule inhibitors with their inactive analogs to identify off-target kinase inhibition or other unintended effects. The molecules tested include Silmitasertib, which has only modestly selective inhibitory activity against this enzyme, showing cytotoxic effects in cell culture at micromolar doses.

What did the study show?

When tested in an optimized cell line, the researchers observed, for the first time, that silmitasertib could exert a selective antiviral effect against beta-coronaviruses without cytotoxicity.

They also tested a series of CSNK2 inhibitors based on a known series of pyrazolo[1,5-a] pyrimidine compounds with potent, selective inhibitory activity against this enzyme but with no structural or physicochemical similarity to silmitasertib. The pyrazolo[1,5-a] pyrimidines 1–6 demonstrated potent inhibition of viral replication without cytotoxicity.

Among all the compounds of this series, SGC-CK2-1, which contains para-methyl and meta-propionamide aniline substituents, is the most selective of all known ATP-competitive small molecule CSNK2A inhibitors. At the same time, the structural analog SGC-CK2-1N is used as a negative control. While SGC-CK2-1 inhibited viral replication, the negative control was inactive.

Again, a series of analogous compounds were tested, with the aniline para-methyl group of SGC-CK2-1 replaced by a basic side chain. These were found to show decreased potency with increasing substituent size. The potency of CSNK2 inhibition correlated well with MHV inhibition.

The third series of potent inhibitors of CSNK2 was based on substituting a 6-(acetamino) indole as the 5-substituent on the pyrazolo[1,5-a] pyrimidines core, with similar results.

The cyclopropylamine group is crucial to solvent accessibility and its inhibitory activity. Therefore, even small changes to this group affect the binding of the cell CSNK2 target.

The findings of all three experiments show that the potency of CSNK2 inhibition is highly correlated with the suppression of beta-coronavirus replication over a range of 4 logs of activity.

This was validated by the knockdown of the genes encoding the various subunits of the enzyme. Knockdown of CSNK2A1 led to a 40% inhibition of MHV replication compared to a non-inhibitory control molecule. CSNK2A2 knockdown failed to affect MHV replication.

The knockdown of CSNK2B inhibited MHV replication by 85% compared to the control. Thus, it is evident that a functional tetramer of the CSNK2 enzyme may have two copies of either CSNK2A1 or CSNK2A2 or one of each, but must have two CSNK2B subunits for its function. When both CSNKA1 and CSNKA2 were depleted together, MHV replication decreased by 90%, confirming the crucial role played by CSNK2 in this process.

Under biosafety level 3 conditions, the potent pyrazolo[1,5-a]pyrimidine CSNK2A inhibitor 2 was tested on the A549-ACE2 cell line infected with SARS-CoV-2. This resulted in complete suppression of viral replication at concentrations above 700 nM but accompanying cytotoxicity.

When switched to primary human airway epithelial cells (HAE) grown in culture on an air-liquid interface to simulate the lung architecture at conducting airway level, they found that this compound reduced the level of the virus by 1.5-2-log without cytotoxic effects.

The researchers also examined the impact of inhibition of this enzyme on the viral spike protein. They observed that spike protein uptake by cells treated with this inhibitor showed a 70% to 80% reduction in spike protein uptake, which led to the conclusion that its suppressive effects on viral replication were partly due to its ability to block viral entry into the host cell.

What are the implications?

The use of kinase inhibitors that compete with ATP via a chemogenomics approach helps rule out off-target effects and trace a particular outcome to the drug's effect on the target enzyme. In this experiment, the chemical probe SGC-CK2-1 inhibited virus replication at nanomolar concentration, showing a highly selective effect on the kinase.

Secondly, its structural analog SGC-CK2-IN did not affect viral replication even when the dosage was increased by 100-fold. Thirdly, silmitasertib, which has the same enzyme target but a different physical structure and chemical activity, also inhibited viral replication. And finally, three series of ATP-competitive inhibitors of the enzyme, with various targeted substitutions on the core, showed a remarkable correlation between the alteration in structure and the activity of the compound against the virus, in keeping with its ability to inhibit the enzyme.

The combined chemogenomic evidence strongly implicates CSNK2A inhibition as the molecular mechanism of action of antiviral activity.”

This was confirmed by knockdown experiments as well.

The potential involvement of the enzyme in virus entry suggests that this may be a common beta-coronavirus entry target. Unlike other kinases like AAK1 and GAK, this shows antiviral activity at nanomolar concentrations, with this effect showing a clear and significant correlation with the inhibition of enzyme activity.

Additional factors that contribute to the observed potency of inhibition of viral entry may be in operation, and these remain to be explored.

While further studies will be required to dissect the molecular details of the signaling pathway that requires CSNK2 for virus uptake and its relative contribution to b-CoV replication, the potent anti-[beta-coronavirus] activity of CSNK2A inhibition suggests that it may be a viable broad spectrum antiviral therapy for current and future zoonotic diseases at doses that could be attainable in clinical setting.”

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.