The paper we’re demystifying can be found here, if you would like to follow along.
This post was reviewed by Dr. Derek McLachlin, one of our subject matter experts
The race for a drug or a vaccine against SARS-CoV-2, the virus that causes the disease COVID-19, is on. Vaccine trials are underway and teams around the world are searching for the solution to a problem that’s become a worldwide pandemic.
Yesterday, a group of scientists reported a big leap forward.
Linlin Zhang et al. (et al. is a fancy way of saying, ‘and the rest of the team’) reported the development of a chemical compound that slows down how quickly the virus can grow and infect lung cells. This type of drug is called an inhibitor. This research was reported in Science on March 20, 2020.
Whenever a new disease-causing something comes along, scientists all over the world immediately start looking for drug targets on that something. A drug target is exactly what it sounds like- a big bullseye for newly developed drugs to stick to and do something to prevent the disease. In the case of coronaviruses (like the ones that cause SARS, MERS and COVID-19), one of the most popular drug targets is a protease called Mpro. A protease is a protein that behaves like scissors. It acts to break down bigger proteins into smaller pieces called peptides or even into individual building blocks, called amino acids. Proteases are in all sorts of life forms, including humans. In the case of viruses, their genetic material codes for proteases. When the viral genome is copied by the host cell, the host cell makes the virus proteases too.
Previous research has shown that if Mpro is messed up then the process of making more viruses, infecting more cells, causing damage and jumping to other hosts is blocked. The best part is that the coronavirus proteases don’t look anything like human proteases! This means that a drug that takes out coronavirus proteases won’t hurt the human ones. These drugs have a very low likelihood of being toxic to human life processes. Making one of these drugs would be a big step forward.
First off, this group of researchers figured out the crystal structure of the Mpro of SARS-CoV-2, the virus that causes COVID-19 (this is a much bigger deal than I can express, solving crystal structures is hard people). They found that it was very similar to the Mpro of SARS-CoV (AKA the SARS virus). Crystal structures give scientists information on what a protein looks like and locations on the protein that are critical to its function. Having this crystal structure allowed them to understand more about how the protease works and what pieces of its ‘machinery,’ so to speak, they could aim to mess up with a potential drug.
They describe that the protease must bind with another copy of itself, or dimerize, in order to actually cut any proteins up. You can think of it as a pair of scissors- there has to be two blades for it to actually cut anything efficiently. They also found that despite small differences in amino acid sequence, the Mpro of SARS-CoV-2 (the novel coronavirus) is only slightly more efficient than that of SARS-CoV (SARS).
Now that the group knew what their target looked like, they got to work on making a drug to mess it up.
This group of researchers is no stranger to drug design. In the past they’ve designed and made α-ketoamides that mimic proteins and inhibit the main proteases of different sorts of coronaviruses and of enteroviruses. Basically, this group managed to make a drug that acts across a broad range of viruses. Their best compound had an EC50 in the picomolar range against MERS-CoV and low micromolar EC50s against SARS-CoV. They took this compound, which they called 11r (see the figure below), and decided to make it better. How? With a lot of chemical sleuthing.
The first thing they wanted to do was improve the half-life of the compound while in plasma- the largest component of blood. A half-life is the amount of time it would take for half of the drug to be broken down. It’s important for a drug to have a good plasma half-life because the drug has to stay stable in the bloodstream for long enough for it to reach the part of the body that it is targeting. To improve the half-life of 11r the researchers did two things:
- They ‘hid’ one of the easily broken bonds underneath a larger chemical ring. This big ring makes it harder for the proteases within human cells to find the bond and break it down.
- They replaced a hydrophobic (water-hating) part of the drug with a piece that’s a little less hydrophobic. The new, less hydrophobic piece is called a boc group and also acts as a protecting group. This would keep the drug from binding to free proteins in the plasma and getting tangled up before it can reach its target.
Once they finished making these changes, they used the crystal structure to see how the new and improved compound (13a, see the figure below) would bind to the Mpro of SARS-CoV-2. They saw some suggestion of clashes- a chance that one of the big rings of the chemical compound would bang into a jutting side chain of one of the amino acids of the protease. However, based on their previous research the scientists knew that the amino acid side chain was usually very flexible. From this, they decided 13a was fine to go ahead and conduct further experiments with.
When testing 13a in mice, the researchers found that the two changes described above successfully increased how long the drug is stable in plasma and also decreased how much the compound binds to plasma proteins- successful so far! However, they found that the new structure wasn’t as effective at inhibiting the Mpro of SARS-CoV-2 as the initial molecule.
Seeing this, they went back to the drawing board. The researchers made a big decision- they wanted to focus only on compounds that act against SARS-CoV and SARS-CoV-2. They were willing to sacrifice the idea of making a broad-spectrum silver bullet against a lot of different types of viruses. With this more narrow target in mind, the researchers replaced a big six carbon ring with a smaller one- only three carbons bound into a triangle shape.
This new compound (13b in the figure) successfully inhibited the Mpro of SARS-CoV-2 with an IC50 of 0.67 (±0.18) μM. This means that the new compound is pretty efficient- 0.000000000067 moles of the drug per millilitre is enough to inhibit the protease’s cutting activity by half. A mole is a unit of measure commonly used in chemistry.
They found that when the drug was given subcutaneously (under the skin) to mice, the compound seemed to be lingering in the lungs. This is great, because it means that 13b is successfully getting to where it needs to go- to the lungs, to fight the virus that replicates in the lungs. When 13b was made into a mist, or nebulized, and given to the mice through inhalation, the mice didn’t seem to have any bad side effects. Also, after 24 hours a decent amount of 13b was still detected in the lungs.
The graphs above show that 13b is very good at lowering SARS-CoV-2 replication in cultured lung cells. The EC50 of 13b in cells was calculated as 4-5 uM, which means that 4-5 uM of the compound is enough to lower the viral RNA replication efficiency by 50%.
The researchers finish their report by reflecting on the pharmacokinetic properties of their new drug, 13b. “Pharmacokinetic” is a word that describes the way the body responds to a drug and the way the drug responds to the body. The favourable pharmacokinetic properties of compound 13b might give other groups a useful starting point to develop more chemically-similar drugs that could act against coronaviruses.