Tennessee scientists have partnered on cutting-edge discoveries in a race against COVID-19

Scientists at Oak Ridge National Laboratory have taken an important step in the race to understand SARS-CoV, the virus that causes COVID-19. Using sophisticated techniques, the scientists mapped out the structure of a critical protein of the coronavirus.

They're hoping to answer this age-old scientific question that's more pressing than ever in this pandemic: How do you kill something that's not really alive?

The results of the groundbreaking work at Oak Ridge National Laboratory were published in Nature, a leading science journal.

Without this protein, the coronavirus cannot replicate. The scientists hope that by studying this structure, they will be able to find drugs that can stop it.

"The most important part is probably the fact that this protein is essential for the replication of this virus," said Dr. Daniel Kneller, a researcher at Oak Ridge National Lab and the first author of the study. "If you inhibit this protein you're preventing the virus from assembling. Period."

While this study is focused on a very specific aspect of COVID-19, it opens a window onto the immense network of scientists and scientific organizations working on the pandemic. Tennessee is home to a state-spanning, drug development pipeline that is a microcosm of national and global research.

The COVID-19 protease protein is both shaped like a heart and functions as one. Without it the virus cannot grow and spread.

Understanding the COVID-19 mechanics

Viruses are hard to treat because they aren’t like other things that cause diseases. Antibiotics, antifungals and antiparasitic drugs stop cells from replicating or kill them outright. But viruses aren’t made of cells. They are bundles of proteins and genetic material that hijack cells, forcing them to produce viral particles. Viruses cannot replicate on their own.

There’s debate among biologists as to whether viruses are actually alive because of this.

"How do you kill something that's not really alive?" said Dr. Martha S. Head, director of the Joint Institute for Biological Sciences at Oak Ridge National Lab. She oversees Oak Ridge's COVID-19 molecular design research projects. She explained that this study was part of a push to shut down viral replication at multiple stages, which is how HIV is treated.

"That combination (of drugs) shuts down so many parts of the (HIV) life cycle that you drive down viral loads to where they don't matter," Head explained.

To do this, the Oak Ridge team went right to the heart of the coronavirus — its proteins. When a virus infects a cell, it forces the cell to produce viral proteins. But host cells often can’t create finished viral proteins, just long strands of unfinished, conjoined, protein.

Why target this part of the virus?

Proteins are a bit like self-folding origami. Once they’re assembled they fold into their final shape. Some proteins need to be cut by enzymes, like a protease, to get them into their final shape. Viral proteins can’t do that when they're conjoined.

To make finished protein, each viral particle carries a protein enzyme called protease. The COVID-19 protease cuts unfinished viral protein, freeing it to fold itself into a final shape. If the protease can’t do this, new virus cannot be made.

It is the heart of viral replication. Finding a chemical compound that can attach to and stop the heart of COVID-19 could be critical for developing a treatment. This is actually the therapeutic approach for anti-HIV drugs like Atazanavir.

But to quickly discover a drug, scientists it helps to have an accurate map of the protease.

"If part of the protein is incorrectly modeled when you try to design drugs you may miss interactions that would otherwise form (between the drug and the protein)," explained Dr. Andrey Kovalesky, a researcher at Oak Ridge who worked on the study. He explained that without an accurate structure model, researchers might miss a potential drug or get bogged down in false positives.

To find the structure, the scientists grew large crystals made of viral protein in the lab. They took these crystals and exposed them to x-rays. When x-rays hit the protein crystal, they bend and scatter in different directions based on the shape of the protein. After they scatter they hit sensitive x-ray detectors. The scientists use the pattern of x-ray hits to ultimately figure out the shape of the protein.

This is called x-ray crystallography and was famously used to discover the structure of DNA.

The technique is like taking multiple photographs from different angles of the same object. By looking at all angles of the object you can figure out its 3-D structure.

Usually this kind of experiment is done at very cold temperatures. That’s because protein molecules tend to move more at warm temperatures. It’s like photographing a moving object.

Unfortunately, getting a clearer picture can also mean missing the shape of the protein or how they move. Viruses often change shape but they can’t when they’re frozen. It’s like looking at frozen meat and expecting it to behave like a living muscle.

"You really have to appreciate that it's one single conformation that you're looking at," said Dr. Paul McGonigle, director of the Drug Discovery and Development Program at Drexel University. "You hope that this is the conformation the protein exists in most of the time, but you never know for sure."

Solving a major issue

The scientists at Oak Ridge did something special. They did this study at room temperature. Their equipment is more sensitive than the type typically used for this kind of experiment. Because of that, they could see the fuller range of motion in the of COVID-19 protein — that accuracy is very important for developing a drug.

"I think it's useful for them to have these different conformations to target," said Dr. Ole Mortensen, associate professor of pharmacology at Drexel University. Mortensen explained that his own drug development work was made more challenging because he only had a single snapshot of his target protein.

"I'm worried that I could be missing some of the other ones. I think it makes sense what they're doing. They're opening up more possibilities." Mortensen said.

How did Oak Ridge get involved?

Oak Ridge might not immediately come to mind when you think medical research. But the national lab system has played a role in medical science since its inception. Sex chromosomes were discovered by pioneering geneticist Liane Russell at Oak Ridge, for example.

When Congress injected hundreds of millions of dollars into COVID-19 research through the CARES Act, The Department of Energy received $99.5 million for the national lab system. Compared to other agencies like the National Institutes of Health or Department of Defense, which got $945 million and $415 million respectively, that might not seem like a lot.

But the national lab system has unique resources that can be quickly marshaled against COVID-19. The neutron facility has the kind of sensitive x-ray detectors necessary to scan a protein at room temperature. The facility houses a lab capable of quickly growing large protein crystals.

"Oak Ridge is uniquely good at growing really big protein crystals," said Charles Sanders, a professor of biochemistry at Vanderbilt University. "Because they have that general expertise, it lets them do room temperature crystallography."

"They also have a network of people around the country, so if the big dogs at these agencies want stuff to happen then it can be, a wartime response, basically," Sanders said.

When the CARES act passed, it let the scientists clear their schedule and focus on COVID-19. Ordinarily, research like this takes months if not years of applying for grants and negotiating for time on equipment. This study mapped and published the protease structure in about a month.

"This is different than our usual projects," said Dr. Head. "Across the Department of Energy as a whole, the speed (of organizing research) is astronomically fast."

Breaking a supercomputing record


Importantly, Oak Ridge houses the Summit supercomputer, one of the fastest supercomputers in the world. Once the structure was figured out by one team, the Summit team quickly screened it against a massive library of potential compounds, looking for potential matches.

"We broke a world record on the supercomputer," said Jeremy Smith, director of the Center for Molecular Biophysics at Oak Ridge. "We screened 1.2 billion compounds in less than a single day."

This is not the first time Dr. Smith has run a massive simulated drug test like this. Knox News covered his experiments back in March. The difference here is scale. Smith’s team screened the COVID-19 protease against 1.2 billion possible drugs in a single day using Summit's whole processing system. Now the most promising candidates are being sorted out for eventual testing against “live” COVID-19 virus.

Help from other Tennesseans

As impressive as Oak Ridge’s facilities are, they don’t have the ability to do that kind of testing in house. For that, Smith turned to Dr. Colleen Jonsson, a professor University of Tennessee Health Sciences Center in Memphis.

Dr. Jonsson is an experienced virologist and virus hunter. She also happens to be the director of the Southeast Regional Biocontainment Laboratory, one of a small network of labs authorized by the federal government to do research on dangerous biological agents and emerging infectious diseases.  She had the facility and staff to do what Oak Ridge could not: validate potential drug targets in the real world.

"A virtual screen (in a computer) is a theoretical screen." Dr. Jonsson said. "Once we find them we have to validate we're actually hitting the right thing."

Jonsson said that earlier in the year Smith reached out to her to test possible drugs targeting a different protein, the spike protein the coronavirus uses to attach to cells. Since then, her lab has expanded to validating other potential drugs targeting other proteins. While Dr. Jonsson hasn’t yet begun to work on the COVID-19 protease, she expects to shortly.

This part of the process, where drugs are tested in live cells to see if they stop a virus from replicating, is arduous. Scientists working at the Biocontainment Lab don full biohazard suits to run their tests. Even after they validate that a potential drug works on a virus in a dish, they still need to run extensive dosage and safety testing, a process that can take months if not years.

Then they have to see if the drug actually works in a real infection. For this they need to test the drug in an infected animal that gets infected with COVID-19 like a human would. Getting an accurate animal model is a difficult process that requires its own experiments and validation.

Dr. Jonsson's team has been busy with this, and other COVID-19 work, since the start of the pandemic. They are among the very few people reporting to work at the University of Tennessee Health Sciences Center campus during the initial lockdowns. The Biocontainment team worked quickly to get everything ready for coronavirus research.

"They worked every day through the Safer at Home order with remarkable dedication," said Dr. Jonsson. "Everyone was working seven days a week to get everything ready."

In spite of its critical role in coronavirus research, the Biocontainment Lab is operating semi-independently. It has not received any funding yet through the CARES Act and is working solely on University of Tennessee funding.

More work to come

None of this science is settled. The author of another structural study on the COVID-19 protease, Dr. Rolf Hilgenfeld of the German Center for Infection Research, was not convinced that the Oak Ridge study would amount to anything.

"I don't think this small difference, (between the shape of the proteins at different temperatures) whatever is its cause, matters for drug design," wrote Hilgenfeld in an email to Knox News.

The Oak Ridge team is planning to scan COVID-19 proteins using a higher resolution technique, neutron scattering at the Spallation Neutron Source, to get even better structures. Dr. Jonsson's team hopes to be running animal tests for possible COVID-19 drugs by the end of the year.

Source: Knoxville News Sentinel, by Vincent Gabrielle

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Published July 17, 2020