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This post was reviewed by Dr. Kelly Summers and Dr. Andrey Kovalevsky, some of our subject matter experts.
3CL Mpro is a SARS-CoV-2 enzyme (the virus causing COVID-19) that processes other SARS-CoV-2 polyproteins (by slicing them up into smaller pieces) critical for the viral life cycle and infection. Researchers at the Oak Ridge National Laboratory have discovered the structure of 3CL Mpro at room temperature, with a technique called X-ray crystallography. This is the first step for designing an antiviral small molecule drug to treat COVID-19.
Introduction: Blocking Harmful Proteins with Small Molecules
Antiviral drugs and other pharmaceuticals are mainly small molecules made up of less than a few dozen atoms. They are structurally designed to interfere with viral proteins to block viral functions like entering, replicating within, and exiting human cells (see Lasya’s Sidebar: How Coronaviruses infect cells for more information). By releasing small molecules that only bind to viral — and not human — proteins, infection can be slowed or prevented without harming the human host.
Before targeting a viral protein, scientists must determine its structure and, most notably, its active site structure. The active site is the part of the protein where interactions with another protein or molecule occur. Enzymes are proteins that speed up biochemical reactions in the body; the active site is the part of the enzyme where the chemical reaction takes place. If the active site is blocked, function is blocked. Therefore, the first step in creating antiviral small molecules is often to determine active site structure. That is exactly what researchers at the Neutron Scattering Division, Oak Ridge National Laboratory in Tennessee have done for the Novel Coronavirus 3CL Mpro enzyme (1).
Research Rehashed: X-Ray Crystallography and 3CL Mpro Active Site Structure
3CL Mpro is a SARS-CoV-2 protease enzyme; in other words, it is a protein that cuts up other proteins during SARS-CoV-2 infection. Proteases, such as 3CL Mpro, do not cut randomly. They cut proteins at specific locations of amino acids (see Lasya’s Sidebar: Central Dogma for more information about amino acids and proteins); often, this trimming turns premature proteins into mature forms. 3CL Mpro is produced from SARS-CoV-2 RNA within a long polyprotein chain (a premature chain of multiple proteins). 3CL Mpro cuts itself free from the polypeptide chain, and then proceeds to cleave the chain further, freeing other non-structural viral proteins (proteins involved in functions other than maintaining the shape and structural integrity of the virus) involved in viral replication. Therefore, blocking 3CL Mpro function within human cells could potentially prevent viral replication, making it a good target for antiviral small molecule design.
Protein X-ray crystallography is a process that uses X-ray diffraction (interference of X-rays passing through obstacles which sizes are of the same magnitude as the X-ray photon wavelength) to discover the 3D structure of proteins. To analyze the 3CL Mpro protein with X-ray crystallography, the researchers made copies of the 3CL Mpro gene (see Lasya’s Sidebar: On Mutation for more information about DNA, and genes). To make the gene copies, the researchers inserted the 3CL Mpro gene into a plasmid vector (a circular piece of DNA, found within bacteria and modified to carry non-bacterial DNA for lab experiments), and performed Polymerase Chain Reaction (PCR). PCR is a laboratory technique used to copy and produce large amounts of specific DNA. The PCR copies of 3CL Mpro gene-containing plasmids were then inserted into E. coli bacteria. E. colinaturally makes proteins from genes within plasmids (see Lasya’s Sidebar: Central Dogma for more information on protein production), so it is grown in culture to produce an ample supply of 3CL Mpro protein for analysis using X-ray crystallography – a method called recombinant protein production. The researchers performed X-ray crystallography at room temperature (20oC), rather than low temperatures, to better represent the 3CL Mpro structure at physiological temperature (37oC).
The X-ray crystallography results were converted into the computer-generated image of 3CL Mpro shown below. 3CL Mpro is a protein dimer, meaning it is made up of two identical subunits (one in orange and one in teal). In the image on the left (a), the orange subunit shows the internal structure of the folded protein chain, whereas the teal subunit shows the texture of the surface. In the image on the right (b), the active site of the protein and the exposed amino acids can be seen. The amino acid types (see Lasya’s Sidebar: CentralDogma) and positions are labeled. Each amino acid looks different and has different functions; how the amino acids are arranged in the active site determines the specific binding and enzymatic abilities of proteins and enzymes.
The structure of 3CL Mpro analyzed at room temperature was compared to the structure found at a low temperature (-173oC) in another study (2). There were notable differences in the structure of the active sites across temperatures. These new results at room temperature may provide more understanding into the structure of 3CL Mpro at physiological temperature.
The Oak Ridge National Laboratory researchers also studied what happens when their newly-discovered 3CL Mpro structure is compared to that of the enzyme with bound peptidomimetic inhibitor N3, so they used a computer software to superimpose the two structures on top of each other. Peptidomimetic inhibitor N3 is a piece of a protein that was created by other researchers (for research purposes) to bind to 3CL Mpro’s active site (note, although it can bind the 3CL Mpro active site, it may not make a good drug because it has to first go through a series of tests on living cells, non-human organisms and then when given to people. When analyzing 3CL Mpro bound by N3, the researchers determined that the structure of 3CL Mpro changes substantially when this ligand bounded. This is an important discovery because understanding how the structure of 3CL Mprowhen bound to an inhibitor changes is useful during the design of antiviral pharmaceutical small molecules. However, depending on the inhibitor bound to the active site, the protein may change its shape in different ways. Therefore, comparing unbound and bound structures (see Figure 1) is an important step in antiviral drug design.
Limitations and Future Directions
It is important to understand that these results provide just one piece of the puzzle; much more research must be done to discover a useful antiviral small molecule to block SARS-CoV-2 infection. Additionally, the X-ray crystallography results were taken at room temperature, which is described as near-physiological by these researchers. However, the standard room (20oC) is a little colder than physiological temperature (37oC), meaning the structure of 3CL Mpro within the body may be slightly different than determined in this study. As a result, the chemical structure of the antiviral molecule designed based on this structural analysis may have to be altered several times before the right one is designed that is effective in the body.
While further pre-clinical and clinical studies are required before a pharmaceutical can be released for public use, research such as this is critical to furthering our understanding of SARS-CoV-2, bringing us closer to the discovery of an antiviral small molecule that can safely treat COVID-19.
- Kneller, D.W., Phillips, G., O’Neill, H.M. et al. Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography. Nat Commun 11, 3202 (2020). https://doi.org/10.1038/s41467-020-16954-7
- Zhang, L. et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors. Science 368, 409–412 (2020). https://doi.org/10.1126/science.abb3405