Hydroxychloroquine: blocking a portal to viral entry.

Early in the SARS-CoV2 pandemic hydroxychloroquine gained tractions as a potential treatment, but despite its effectiveness in cell culture, the effectiveness in clinical trials proved more complicated. We sought a mechanism of action with a potential to resolve the apparent discrepancy.
Hydroxychloroquine: blocking a portal to viral entry.

Hydroxychloroquine has been used as an effective anti-malaria drug for many decades and more recently as an anti-inflammatory treatment in Niemann-Pick syndrome and lupus.  Most of what is known about hydroxychloroquine relates to its anti-malarial properties in the mosquito, not mammalian cells.      

How does hydroxychloroquine work in a mammalian cell?

Unknown to most, hydroxychloroquine is also a local anesthetic. Anesthetics are hydrophobic molecules that partition into the membrane. Could the lipid partitioning properties of hydroxychloroquine contribute to its mechanism of action in humans? Recently my lab showed a membrane-mediated mechanism for local and general anesthetics—the anesthetics displace proteins from cholesterol dependent domains (pockets of saturated lipid) in the membrane (Pavel PNAS 2020).

Like anesthetics, hydroxychloroquine displaces the SARS-CoV2 receptor from cholesterol dependent domains. The displacement is important since the cholesterol dependent domains act as portals for the virus to enter the cell (see white circles in the figure). Thus, bumping the receptor out of the entry way, reduces cellular uptake of the virus.

 Understanding the amount of cholesterol in the tissue is key. We found very high levels of cholesterol in the lungs of patients with chronic obstructive pulmonary disease (COPD), a lung disease that increases the severity of COVID19. In comparison to COPD patients, cultured lung cells had low cholesterol. The low cholesterol is typical of a healthy young child.

 What are hydroxychloroquine’s side effects? Reviewers asked us to test the toxicity of hydroxychloroquine at high concentration. Previous studies showed no toxicity in VeroE6 cells up to 50 µM, the same cells and concentration we used, so we were a little annoyed at the request. While repeating the VeroE6 experiments, we included an HEK293T cell line since we used both in our experiments. Like the previous studies we saw no toxicity with VeroE6 cells (yes science is reproducible at times), but we did see toxicity in HEK293 cells.

 So what was different in the two cell types? We found uptake of cholesterol into most cell types attenuated the effect of hydroxychloroquine. If we look to Niemann-Pick, that disease is a cholesterol transport disease with very high tissue cholesterol. VeroE6 also have relatively high free cholesterol and readily available endocytosis. Taken together, high cholesterol appears to protective against hydroxychloroquine toxicity. Our data suggests individuals with low tissue cholesterol (e.g., young healthy adults and children) are likely to see the most toxicity.

 How does this knowledge benefit the clinic and future research?  From the outset we aimed to do something that would benefit the clinic. As biophysicists, we use molecular mechanism of action to guide in vivo experimentation. If the clinic behaves like the cell culture, hydroxychloroquine will need to be dosed based on the levels of tissue cholesterol. Most doctors only look at cholesterol in the blood. How the blood cholesterol correlates to the tissue cholesterol is still an open question. The blood is a transport mechanism that may not reflect the years of potential buildup in the tissue. This is especially true of aged individuals with chronic disease. 

 In the end, the cross pollinating between anesthesia and viral entry was very fruitful. The effect of anesthetics on the endocytosis of viral particles paves the way for an understanding of anesthetics on endocytosis of ion channel, key effectors of anesthesia.

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