Article Review: Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants
It seems as though one could not visit any news site or social media network in January 2022, without seeing an article or post stating a "new study shows cannabis fights COVID." While this statement may accurately reflect the study, it is overly simplistic and optimistic. Let's dig into the actual study and see what the researchers from Oregon State University and Oregon Health and Science University discovered.
A quick recap on how SARS-CoV-2 is structured and interacts with human cells. SARS-CoV-2 is an RNA virus that is not technically alive, it requires a host organism to incorporate and replicate its genetic material to produce more virus. As seen in the above image, SARS-CoV-2 is characterized as a spherical virus with spiky structures dotting the surface. These are the infamous spike proteins that allow the virus to attach and infect host cells.
SARS-CoV-2 infection has a number of steps that could be targeted for antiviral intervention, including: binding of the viral spike protein, cell entry, genome replication, viral maturation, and viral release. This means that scientists have a number of potential areas to focus treatment options on that could prevent infection, prevent replication of foreign genetic material, or prevent the manufacture and release of new virus.
Infection through the binding of the viral spike protein is often seen as the most promising intervention for a number of reasons, not least of which is its presence at the beginning of the viral infection life cycle. On certain human cell types, including endothelial cells of the lungs, heart, kidneys and intestines, a cell surface receptor known as ACE2 (angiotensin converting enzyme-2) is the target that the SAR-CoV-2 viral spike proteins interact with.
The mechanism by which SARS-CoV-2 infects human cells is fairly straightforward, the S1 subunit in the above image binds to the human ACE2 receptor and initiates infection. When the S1 subunit is bound to the ACE2 receptor two thing appear to happen, first a protease on the human cell membrane cleaves the S1 subunit and then the S2 subunit facilitates fusion of the membrane of the virus to the human cell membrane.
To use an analogy, the S1 subunit is the attach point for a spaceship docking at the International Space Station and the S2 subunit serves as the airlock.
From the understanding of this interaction, it is safe to say that the S1 subunit is critical in detecting and binding with target cells and if an analog could be found that would outcompete the ACE2 receptors on the human cells, infection could potentially be minimized. This method of using small molecules to bind to virus and prevent infection has been effectively used in treatments for both hepatitis and HIV.
So how does one scan molecules for suitability in serving as an analog in this situation?
There are a number of methods that could be used to scan and detect molecules that would serve to bind with the S1 subunit. In this experiment, van Breeman et al., bound the S1 subunit to a magnetic bead and then incubated them with a variety of potential analogous molecules. After the incubation, they washed each well in the plate (see above) with a buffer to remove any non-bound molecules and then the remaining molecules bound to the S1 subunit were released with a solvent. The recovered solvent-ligand (ligand is in this case refers to any molecule that is bound to the target S1 subunit) is then scanned using UHPLC-MS.
UHPLC-MS stands for ultra-high-pressure liquid chromatography-mass spectrometry. Liquid chromatography is a method by which a solution of different molecule types can be separated based on their affinity to porous surface they are traveling through. Mass spectrometry, at its most basic level detects the molecular weight of molecules in a solution.
The steps to find analogous ligands for the S1 subunit that can outcompete the human ACE2 receptor is as follows:
- Bind S1 subunit to magnetic bead
- Add solution of potential ligands
- Wash off unbound ligands
- Remove bound ligands into solution
- Run solution through UHPLC system to separate ligand species
- Run these species on Mass Spectrometer to identify based on molecular weight
van Breeman et al., conducted this experimental procedure on a wide range of substances isolated from the Cannabis sativa plant. There rational was based on the fact that C. sativa is known to have at least 170 biologically unique compounds and would allow for testing of a number of compounds that have been shown in previous research to positively impact human health.
Upon completion of the magnetic affinity binding and UHPL-MS protocols, a number of candidates were found from the extract of C. sativa, but through repeated experimentation, three showed the most promise: CBDA, CBGA, THCA-A. These three molecules bound most readily to the S1 subunit and stayed bound for the longest time. To determine affinity, an equal amount of various molecules that were found to bind to the S1 subunit were added and these three ligands outcompeted them. This can be seen in the above image, where the three ligands (CBDA, CBGA, and THCA-A) all bind more readily and stay bound longer than the ACE2 human receptor.
What does this mean? In theory, these compounds should outcompete ACE2 human receptor binding and prevent most of the infecting SARS-CoV-2 virus from infecting cells. Due to the amount of virus, it would be impossible to prevent any host cell infection due to the random nature of cell-virus interaction, but a substantial reduction in infected cells should also lead to less new virus and thus fewer new infections.
The journal article then goes into a bit of information about why the isolated C. sativa compounds bind so well to the S1 subunit, using molecular protein modeling and ligand docking. I think this is beyond the necessary scope for this review article, but if anyone is interested, reach out and I can discuss it further.
If the three identified compounds can actually outcompete binding at the ACE2 human receptor, then adding them to a mixture of human cells that have the receptor and live SARS-CoV-2 virus should paint a pretty clear picture.
This image shows three different treatments applied to a human analogous cell line called Vero e6. These are kidney cells descended from an African green monkey lineage and can serve as the first step toward human treatment because of their similarities to our own cells. In these images, the blue are cell nuclei and the red is viral RNA. As you can see in the control image, there are a number of replication sites that have formed after infection with the virus. In both the CBDA and CBGA images, the level of infection is substantially decreased.
In this last image we can see just how effective each compound is at neutralizing infection by SARS-CoV-2 and the beta and delta variants at various concentrations. In both cases as concentration of the analogous ligand is increased, the level of neutralization is also increased. My best guess is this is a measure of infection compared to control infections.
While this information is promising, the effective dosing levels appear to be quite high, with the journal article itself claiming that "concentrations needed to block infection by 50% of viruses is high but might be clinically achievable." This indicates that the dosage needed to see an appreciable decrease in the amount of infected host cells is quite high and needs to be tested further. In tests on humans, CBDA concentrations in the body have been detected at 0.21uM. Based on the information in this article, the concentration necessary to see reduction in infection of at around 50% is 31.6ug/mL which for CBDA is a molarity of 88.1uM. This means the detected amount in humans to this point is 1/440th of what is needed.
Based on this information do compounds extracted from C. sativa reduce infection from SARS-CoV-2 and its variants? It appears so, but like much research about cannabis and medicine, the headlines don't tell the full story. At this point the amount of ingested CBDA or CBGA necessary to outcompete ACE2 human receptors is far beyond what humans have taken and thus even the most ardent of marijuana enthusiasts will see no benefit from CBD oils, edibles or smoking of the plant. This doesn't mean that marijuana has no potential health benefits, but in this case, the numbers are simply too great to overcome without further study and intervention.
 - Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants