As I explained in the last two updates (here and here) I've been studying hydroxychloroquine and chloroquine largely because I'm investigating whether their mechanisms shed any light on whether quercetin (rich in onions, nuts, and a variety of vegetables) and EGCG (abundant in green tea), are antiviral toward SARS-CoV-2 (the coronavirus that causes COVID-19) by acting as zinc ionophores. If they are, I might add them to the protocol in The Food and Supplement Guide for the Coronavirus.
In those two updates, we discussed how there remains no evidence that hydroxychloroquine or chloroquine are effective against COVID-19 in humans, and that their in vitro (in a lab dish) antiviral activity toward SARS-CoV-2 is likely a result of increasing the pH of endosomes and possibly a result of increasing the pH of the golgi apparatus, not by acting as zinc ionophores.
The endosome is a little bubble the virus uses to get into cells, and raising its pH (making it more alkaline and less acidic) prevents the virus from using it to get into the cell. The golgi apparatus is where the cell adds sugars to proteins in a process known as glycosylation, and raising the pH of the golgi interferes with that process. These drugs interfere with the glycosylation of ACE2, the protein the virus uses to attach to a cell. Although this hasn't been shown yet, decreased glycosylation of ACE2 might make it harder for the virus to attach to the cell.
A zinc ionophore is something that can bring zinc across a membrane into a cell or a specific compartment within the cell. Chloroquine has only been shown to do this at concentrations far higher than those needed to kill SARS-CoV-2 in vitro, and when it does so it traps the zinc in digestive compartments known as lysosomes, where the zinc would not be expected to kill the virus.
Quercetin and EGCG as Zinc Ionophores
Quercetin and EGCG have been also been shown to act as zinc ionophores.
However, I am even more doubtful that this is relevant to COVID-19 than I am for chloroquine. Here's why:
- The concentrations used are far higher than those even reachable with supplements; at least the concentrations of chloroquine that kill SARS-CoV-2 and begin to act as a zinc ionophore are reachable by taking the drug.
- In order for the effect to occur, zinc concentrations also have to be far higher than they ever are in humans.
- It is not clear whether the zinc would be in a relevant location within the cell.
Unrealistic Concentrations of Quercetin and EGCG
The study used 100 micromoles per liter (a measure of how many molecules there are per liter, abbreviated uM) of quercetin, EGCG, or an antitumor drug known to act as a zinc ionophore, clioquinol.
Supplementation with 1095 milligrams of quercetin only raises plasma concentrations to a little over 1 uM, which is 100 times lower than the concentrations used in this study. Supplementation with 500 milligrams of EGCG only raises plasma concentrations to 1.75 uM, which is 57 times lower than the concentrations used in this study.
Unrealistic Concentrations of Zinc
The study found no effect of quercetin or EGCG at 5 uM zinc. These compounds only enhanced zinc transport at 50 uM zinc.
Normal plasma concentrations of zinc range from 12 to 18 uM. Even in the hours just following a high-dose zinc supplement of 45 milligrams, plasma zinc does not go any higher than this range. Plasma zinc will never reach 50 uM with zinc supplements.
Furthermore, the zinc in this study was supplied as zinc chloride, which will be present in the freely available ionic form. By contrast, in plasma, 75-90% of zinc is bound to a protein known as albumin, and most of the rest is bound to α2-macroglobulin, a protein that prevents blood clotting, or retinol-binding protein, a protein that carries vitamin A. Freely available ionic zinc is only about 80 nanomoles per liter. A nanomole is one thousandth of a micromole, so the freely available zinc concentration used in the study was 60 times the concentration in plasma when EGCG and quercetin had no effect and 603 times the concentration in plasma when EGCG and quercetin did have an effect.
Now, it's not 100% clear to me that we should disregard the protein-bound zinc within plasma. It's conceivable that EGCG and quercetin could siphon zinc off from albumin to bring into the cell. However, even assuming all of the protein-bound zinc in plasma is just as available as the zinc chloride used in the study, the study still needed to use 3.3 times the total zinc found in plasma.
So far to show a zinc ionophore effect of these molecules we need two things:
- 57 times as much EGCG or 100 times as much quercetin as achievable with oral supplements.
- At least >3 times the total zinc found in plasma, and possibly >600 times the ionic zinc found in plasma.
Is the Location of the Zinc Relevant?
Now we can borrow one key insight from our study of chloroquine. One of the reasons chloroquine probably doesn't kill SARS-CoV-2 in vitro by acting as a zinc ionophore is because chloroquine traps zinc in digestive compartments known as lysosomes, where the zinc would have no effect on the virus. In order to stop the replication of the virus, we want the zinc to be in the cytosol, the main compartment of the cell.
Do EGCG and quercetin increase cytosolic zinc (good) or lysosomal zinc (useless)?
We really have no idea.
The method used to detect zinc in this study was called FluoZin-3. When it encounters “labile zinc,” a small minority of intracellular zinc that is either free ionic zinc or loosely bound zinc, FluoZin-3 fluoresces. Unfortunately, FluoZin-3 is not able to distinguish between cytosolic zinc and lysosomal zinc.
Whether the zinc is cytosolic (good) or lysosomal (useless) is probably irrelevant because the effect requires 50-100 times the reachable concentrations of quercetin or EGCG and either 3 times the normal concentration of total zinc or possibly more than 600 times the normal concentration of free zinc.
Quercetin as a Potential Inhibitor of the Viral 3CL Protease
A more promising paper used computer modeling software to predict that quercetin and EGCG inhibit one of the enzymes that SARS-CoV-2 uses to replicate, known as 3CL protease. The model predicts that EGCG would inhibit 50% of the enzyme's activity at a concentration of 13 uM, which is still seven times what can be reached in plasma with supplements. By contrast, it predicts that quercetin inhibits the enzyme at 0.7 uM, well within the concentrations that can be reached in plasma using 1000 milligram doses.
This might make quercetin, but not EGCG, useful for COVID-19 prevention or early treatment, but it should be regarded as very preliminary.
As noted yesterday, the SARS-CoV-2 3CL protease is found embedded in the membrane of the endoplasmic reticulum, with its enzymatic activity facing the cytosol. When cells are incubated with quercetin, much of it winds up embedded in the membrane of the endoplasmic reticulum as well. If we take the computer model's prediction at face value, it still isn't clear whether the quercetin embedded in the membrane would have the opportunity to inhibit the viral enzyme, given that the active part of the enzyme is facing the cytosol rather than the interior of the membrane. Still, it does make it's way to the correct location.
Whether quercetin inhibits the enzyme at all, however, still isn't known. Computer modeling is only a way of brainstorming possible interactions between molecules that can later be tested directly. The pharmaceutical industry uses the technique to come up with lists of drug candidates that can then be selected to undergo further testing to determine whether they actually have the activities the computer model predicted. To learn more about the limitations of this modeling and why its predictions can be wrong, see here, and skip to the fourth instance of “limitations” when keyword searching the page.
At the moment, I am exceedingly doubtful that quercetin or EGCG can kill SARS-CoV-2 by acting as zinc ionophores. I find the predicted inhibition of the 3CL protease by realistic concentrations of quercetin to be promising, but I would like to see at a minimum a paper showing that quercetin has this effect in cells infected with the virus before I would consider adding it to my prevention regimen.
I will continue to monitor the research, especially on the potential of quercetin to inhibit viral replication enzymes, but at this time, I will not be updating The Food and Supplement Guide for the Coronavirus to include EGCG or quercetin.
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These research updates are made possible by purchases of The Food and Supplement Guide to the Coronavirus. The guide contains my most up-to-date conclusions about what we should be doing for nutritional and herbal support on top of hygiene and social distancing for added protection. Due to the absence of randomized controlled trials testing nutritional or herbal prevention, these are my best guesses for what is likely to work without significant risk of harm, based on the existing science. Purchasing the guide is also a way to donate $9.99 toward this service of providing free research updates, as the financial support provided by the guide is what enables me to spend time staying on top of the research.
I am not a medical doctor and this is not medical advice. I have a PhD in Nutritional Sciences and my expertise is in conducting and interpreting research related to my field. Please consult your physician before doing anything for prevention or treatment of COVID-19, and please seek the help of a physician immediately if you believe you may have COVID-19.
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