LONDON – Scientists investigating the impact of SARS-CoV-19 on protein expression in human cells have shown that infected cells develop virus-laden membrane protrusions, or filopodia, which may explain the rapidity of viral spread through the body.
They suggest SARS-CoV-2 hijacks casein kinase II (CK2), which plays a key role in cytoskeleton formation, cell growth and proliferation, co-opting it into creating the tentacle-like protrusions.
While reorganization of the actin cytoskeleton is a common feature of a number of viral infections, SARS-CoV-2 induced a dramatic and distinct increase in filopodia. Electron microscopy images show those structures are significantly longer and more branched, enabling more aggressive transmission than is the case for other viruses.
The discovery of the unusual structures was made as part of an evaluation of all human proteins that exhibit up- or down-regulation of kinase expression and consequent changes in phosphorylation, following SARS-CoV-2 infection.
The resulting “Global Phosphorylation Landscape,” published online in Cell on June 27, 2020, shows how the virus shifts host cell activity to promote its own replication and to infect nearby cells. Based on that, the researchers identified seven approved drugs that could disrupt those mechanisms.
“The distinct visualization of extensive branching of the filopodia once again [illustrates] how understanding the biology of virus-host interactions can illuminate possible points of intervention,” said Nevan Krogan, director of the Quantitative Biosciences Institute at UC San Francisco, who led the research.
The electron microscope images of the filopodia may be the most eye-catching feature, but the phosphorylation landscape also has delivered multiple new drug targets.
The researchers found that 40 of the 332 human proteins that interact with 27 SARS-CoV-2 viral proteins exhibit significantly differentiated phosphorylation as a result. At the same time, 49 human kinases out of a total of 518, were either up- or down-regulated.
In addition to CK2, kinases in the p38/MAP kinase pathway, cyclin-dependent kinases and phosphatidylinositol-5 kinase were the most likely to be hijacked by the virus.
“The virus prevents human cells from dividing, maintaining them at a particular point in the cell cycle. This provides the virus with a relatively stable and adequate environment to keep replicating,” said Pedro Beltrao, group leader at the European Molecular Biology Laboratory’s Bioinformatics Institute in Cambridge, U.K., who worked on the in silico screening project to look for existing compounds that would inhibit SARS-CoV-2-infected cells.
“We employed state of the art bioinformatics approaches to readily identify regulated kinases from sparse phosphorylation profiles,” Beltrao said. “Many of [these] are likely to be established drug targets with therapeutic potential.”
Alongside the finding that CK2 physically interacts with the SARS-CoV-2 nucleocapsid protein, the researchers showed that inhibiting p38/MAPK signaling in virus-infected cells suppressed the overproduction of inflammatory cytokines and also directly impaired viral replication. That implies p38/MAPK inhibition may target multiple mechanisms related to COVID-19 pathogenesis, they said.
Similarly, cyclin dependent kinases, which regulate the cell cycle and cell death, and phosphatidylinositol-5 kinase, which regulates cytoskeleton function, can be targeted by existing cancer and anti-inflammatory drugs, halting viral replication.
The researchers triangulated changes in phosphorylation in SARS-CoV-2-infected cells to specific kinase targets, identifying 87 FDA-approved drugs and compounds in development.
Those were narrowed down to seven drugs – silmitasertib, gilteritnib, MAPK13-IN-1, SB203580, ralmetinib, apilimod and dinaciclib – which all demonstrated anti-SARS-CoV-2 activity in cell lines.
It was encouraging to find drugs targeting differentially phosphorylated proteins inhibited infections in cell culture, said Kevan Shokat, professor of cellular and molecular pharmacology at UC San Francisco. “We expect to build up this work by testing many other kinase inhibitors, while concurrently conducting gene knockout experiments,” he said.
Senhwa Biosciences Inc.’s silmitasertib is the only CK2 inhibitor in development. The drug currently is in a phase II trial in cholangiocarcinoma. In April, the Taiwan-based company announced a partnership with the U.S. NIH to evaluate silmitasertib in COVID-19, based on earlier findings by the UC California researchers indicating CK2 involvement.
Another repurposed drug falls
One possible antiviral treatment for SARS-CoV-2 infection has fallen by the wayside after the steering committee of the large-scale U.K. Recovery trial reported on June 29 that there is no beneficial effect of the HIV combination drug lopinavir-ritonavir in patients hospitalized with COVID-19 infections.
A total of 1,596 patients randomized to lopinavir-ritonavir were compared with 3,376 patients randomized to usual care alone. Of those patients, 4% required invasive mechanical ventilation when they entered the trial, 70% required oxygen alone, and 26% did not require any respiratory intervention.
There was no significant difference in the primary endpoint of 28-day mortality, at 22.1% in the lopinavir-ritonavir arm vs. 21.3% for standard of care (p=0.58). The results were consistent in different subgroups of patients. There was also no evidence of beneficial effects on the risk of progression to mechanical ventilation or length of hospital stay.
The investigators said the data “convincingly rule out any meaningful mortality benefit of lopinavir-ritonavir in the hospitalized COVID-19 patients we studied.”
They note it was not possible to study a large number of patients on invasive ventilation because of the difficulty of administering the orally administered drug to patients on ventilators. “As such, we cannot make conclusions about the effectiveness in mechanically ventilated patients.”
The full results will be made available as soon as possible.
That is the third set of results from Recovery, following data on hydroxychloroquine, which found the malaria drug had no benefit on hospitalized COVID-19 patients; and dexamethasone, which did show benefit for patients needing oxygen or ventilation.
Peter Horby, professor of Emerging Infectious Diseases and Global Health at Oxford University, and chief investigator for the study, said, “In 100 days, the Recovery trial has provided results enabling change in global practice three times. This extraordinary national effort has shown that two drugs used to treat hospitalized COVID patients throughout the world, hydroxychloroquine and lopinavir-ritonavir, do not improve survival, whilst one drug that was not recommended, dexamethasone, saves lives.”
Deputy chief investigator, Martin Landray, professor of Medicine and Epidemiology at Oxford University, noted that in many countries, current guidelines recommend lopinavir-ritonavir as a treatment for COVID-19. “The results from this trial, together with those from other large randomized trials, should inform revisions to those guidelines and changes to the way individual patients are treated,” Landray said.