“It is much more critical to identify where that drug is working, because ultimately you don’t want to blindly go to a human trial or even an animal experiment, “says Jason Kindrachuk, a microbiologist at the University of Alberta who is not involved in the research. The approach It is not a guarantee at all, but it is a good way to generate leads that researchers can study more deeply. “All this information is incredibly useful,” he says.
The new UCSF-led research takes a slightly different approach. This time, they measured the changes in an activity called phosphorylation, which plays a role in activating and deactivating cellular processes: everything from how the cell grows and divides and dies to, yes, how filopodia develops. Activity is handled by a type of protein called kinases. The researchers infected cells of African green monkeys, known for their high susceptibility to SARS-CoV-2, and, using mass spectronomy, observed how phosphorylation changed over time. They then mapped activity to analog kinases in human cells, finding substantial changes in 49 of them.
Some of the affected pathways seemed intuitive, inhibiting cellular processes such as division and apoptosis or cell death. “It’s a great photo,” says Julia Schaletzky, a UC Berkeley drug discovery researcher who was not involved in the study. “The virus just wants to make sure that the cell starts working for you, the virus, as soon as possible. Drop everything except make more particles. “
The next task was to find drugs that block kinase misbehavior. “What’s great is that there are so many different medications that work against them,” says Krogan. Many of the team’s candidates were cancer drugs, designed to stifle cellular processes that drove them crazy. To obtain them, Krogan turned to a UCSF colleague, chemist Brian Shoichet. “You go into their freezer and you can find an inhibitor for any kinase. And if you don’t have it, you know how to get it, ”he says.
Collaborators in Paris and New York tested 68 compounds known to inhibit kinases, looking for those that could kill the virus at a dose that didn’t kill monkey cells. In that sense, the majority did not pass the meeting. But six or seven of the compounds, Krogan says, were particularly promising, including some that were “more potent than remdesivir,” which they used as a control.
Among them was a compound called silmitasertib, which inhibits a kinase called CK2, and is being tested as a cancer drug. The researchers had already identified it in their previous Nature study based on protein interactions. And in the course of that investigation, they also noticed interesting structures emanating from the infected cells. They turned to more collaborators, this time at the University of Freiburg in Germany and at Rocky Mountain Labs in Montana, to obtain images of the infected cells in greater detail using electron microscopes. It was then that they found those unusually thin filopodia, along with clumps of viruses and CK2.
“It’s exciting but preliminary,” says Jenny Gallop, a biochemist at the University of Cambridge who studies filopodia. With her own laboratory temporarily closed due to the pandemic, she had been searching the internet for signs that filopodia might play a role in SARS-CoV-2. She had reason to suspect that they might. Viruses, including Marburg and Ebola, have been found to hijack filopodia, forcing them to grow larger, branch out, and transport virus particles to their neighbors. The mechanism is especially well studied in another virus called vaccinia, she notes, where research has suggested that it plays a role in “super-spreading” the virus from one cell to another.
The difficulty, Gallop says, is that tiny filopodia imaging is an arduous process. The level of detail the researchers achieved in a short time is no small feat, but more experimentation and imagination is needed to confirm whether the researchers have identified the correct mechanism. CK2 is an important kinase that plays a role in many cellular processes, he notes; Furthermore, other kinases have roles in the development of filopodia. A logical next step in reaffirming the link would be to see if silmitasertib, as a CK2 inhibitor, actually limits the growth of filopodia in infected cells.
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