Enlarge /. The RNA to be copied is dark blue; the copy is in turquoise; the enzyme is light green; and the drug is pink.
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Just this week, we had the first promising report on a drug that appears to improve recovery time for patients with COVID-19. Shortly after this announcement, a scientific journal published a paper describing how the drug interferes with the virus. While there are no real surprises in what has been revealed, it does contain important details on how SARS-CoV-2 can be blocked.
Copier
Combating a virus with a drug is a challenge. Viruses make a living from using their host's proteins to do most of the work involved in creating new viruses. This means that a drug must target some of the few proteins encoded by the virus without disrupting one of the far more common host cell proteins. In the case of the corona virus, biologists have identified a number of different characteristics of the virus that can be targeted without the apparent risk of serious side effects.
Remdesivir, which has shown promising results in a large clinical trial, is a drug that targets one of these virus-specific vulnerabilities. The coronavirus genome is encoded using chemical RNA, as opposed to the DNA used for our genome. In fact, there is nothing in our cells that requires them to make an RNA copy of an RNA molecule. As a result, the coronavirus genome encodes proteins that perform this RNA-to-RNA copy, referred to as RNA-dependent RNA polymerase. Remdesivir should look like one of the building blocks of RNA, hoping that it will bind to and inhibit the polymerase of an RNA virus.
However, this drug was developed with the intent to inhibit the polymerase of another virus (Ebola), so there was no guarantee that it would work against the coronavirus. And our cells have to make RNA copies of DNA, a process that is similar enough that remdesivir could interfere with this as well.
Nevertheless, tests in cells were promising enough to drive tests on humans. As these tests began, a group of Chinese scientists decided to investigate how remdesivir actually works. To this end, they decided to find out how the drug interacted with the coronavirus RNA polymerase at the atomic level. And that requires a technique to determine where all of the atoms in the protein and drug are.
A few decades ago, finding out details at the atomic level of proteins would have painstakingly tried for many months to get the drug and protein to form clean, ordered crystals. Since then we have developed a combination of hardware and algorithms with which we can now essentially take electron microscopic images of individual proteins and combine them with sufficient precision to find out where all the atoms are located. The technology, known as cryo-electron microscopy, was so revolutionary that it won developers a Nobel Prize.
These scientists also benefited from previous work on other corona viruses that identified three different proteins that were crucial for copying the virus genome. One of them is the enzyme, which combines individual units, so-called "bases", into a new RNA molecule. The other proteins in question simply help him hold onto and move along the RNA he is making a copy of. So the researchers produced these three proteins, inserted an RNA template and a partial copy, and then added Remdesivir.
In the atoms
Well, technically they haven't added remdesivir. Bases are added to RNA in a form with three bound phosphates. This form is very negatively charged and does not easily make it across membranes. Instead, the drug is provided in a form with minimal charge that can travel across cell membranes. In the cell, the cell's own enzymes convert it into the charged form, which is then used by the RNA-copying enzyme. Since they did not work in cells, the researchers had to do this conversion themselves. It's a bit aside, but it shows some of the challenges people who develop drugs face.
In any case, imaging technology requires the acquisition of thousands of electron micrographs of individual protein-RNA drug complexes, all of which are randomly aligned. While none of these alone is enough to find out where the atoms are, computer algorithms can combine all of these images and find out which underlying structure is compatible with all of these different images.
After over 80,000 images were combined, the resolution of the structure they determined had an accuracy of approximately 2.5 angstroms (10 to 10 meters). For comparison, a carbon atom in the same molecules has a width of about 1.5 angstroms. Given that many configurations that match this resolution make no sense – placing two carbon atoms on top of one another or the like – the picture is quite detailed.
One thing they discovered is that the proteins involved incorporated zinc atoms into their structure. This will come as no surprise to biochemists, as zinc-containing proteins are common. But there has been a steady flow of marginal treatments for the disease – including some with chloroquine derivatives – where zinc has been a key component. We'll have to see if this changes now that it is clear that zinc is needed to make copies of the virus (provided that the fact registers with people who tend to promote marginal therapies).
The structure confirms that Remdesivir not only adheres to the enzyme and blocks it. Instead, the drug is chemically incorporated into the growing RNA chain. Once there, however, it didn't have the right chemistry to add another base afterwards. As a result, the RNA cannot continue to grow. Copying stops and the resulting genome is defective. This is the same type of drug mechanism that is behind some of the earliest anti-HIV drugs like AZT.
Why doesn't this completely block the virus? Probably because there is simply much more of the normal equivalent of RNA building blocks in the cell and it is difficult to bring the drug to concentrations at which it will consistently damage all viral copies made. An additional limitation is that once the drug blocks the copying of a molecule, it is essentially inactivated because it remains chemically linked to that molecule.
The researchers found that there are other similar drugs that bind to the coronavirus RNA polymerase even more effectively. So there is a possibility that we can determine the structure of some of them and find out whether there are rules for which type of chemicals can adhere particularly well to the coronavirus enzyme.
Science, 2020. DOI: 10.1126 / science.abc1560 (About DOIs).
