COVID-19 News: Cholesterol And Ceramides Facilitate SARS-CoV-2 Spike Protein-Mediated Membrane Fusion Which Can Be Inhibited By Chlorpromazine!

COVID-19 News: A new study by researchers from Inserm (French National Institute of Health and Medical Research) has validated that cholesterol and ceramide lipids facilitate SARS-CoV-2 spike protein-mediated membrane fusion.


 
The researchers also found that Chlorpromazine (CPZ), a commonly used antipsychotic drug, could be used to inhibit this fusion and also prevent SARS-CoV-2 cellular entry.
 
It is already well established that SARS-CoV-2 entry into human host cells is mediated by the Spike (S) protein of the viral envelope.
 
The S protein is composed of two subunits: S1 that induces binding to the host cell via its interaction with the ACE2 receptor of the cell surface and S2 that triggers fusion between viral and cellular membranes. Fusion by S2 depends on its heptad repeat domains that bring membranes close together, and its fusion peptide (FP) that interacts with and perturb the membrane structure to trigger fusion.
 
Numerous studies have previously suggested that cholesterol and ceramide lipids from the cell surface may facilitate SARS-CoV-2 entry into host cells, but their exact mode of action remains unknown.
 
Such studies were covered in our past COVID-19 News coverages:
 
https://www.thailandmedical.news/news/sars-cov-2-news-phosphatidylinositol-phosphate,-cholesterol-and-genes-tmem106b,-vac14,-scap-used-by-sars-cov-2-to-infect-human-cells
 
https://www.thailandmedical.news/news/covid-19-research-study-shows-that-high-density-lipoproteins-hdl-or-good-cholesterol-facilitates-sars-cov-2-cell-entry
 
https://www.thailandmedical.news/news/covid-19-research-scripps-research-indicates-that-host-cells-laden-with-cholesterol-enhances-facilitation-of-sars-cov-2-viral-entry
 
https://www.thailandmedical.news/news/breaking-spanish-study-shows-that-sars-cov-2-spike-glycoproteins-can-interact-with-oily-human-skin,-sebum-and-skin-ceramides
 
https://www.thailandmedical.news/news/university-of-pennsylvania-study-shows-that-lipids-pay-a-critical-role-in-sars-cov-2-replication-and-human-immune-response
 
The study team utilized a combination of in vitro liposome-liposome and in situ cell-cell fusion assays to study the lipid deter minants of S-mediated membrane fusion.
 
The study finding showed that cholesterol and ceramide both facilitated fusion, suggesting that targeting lipids could be effective against SARS-CoV-2.
 
The study team explored the effect of chlorpromazine (CPZ), an antipsychotic drug known to perturb membrane structure, as proof of concept.
 
The study findings showed that chlorpromazine or CPZ inhibited S-mediated membrane fusion and thus potentially SARS-CoV-2 entry.
 
The study findings were published on a preprint server and are currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2022.12.16.520599v1
 
The study finding show that the fusion peptide FP1 of the S protein from SARS-CoV-2 mediates the fusion between liposomes to which it is membrane-anchored (v-liposomes mimicking the viral envelope) and protein-free liposomes (c-liposomes mimicking the cellular plasma membrane).
 
However, the fusion peptide FP2 could not induce fusion between v- and c-liposomes.
 
A structural study on FP1 and FP2 of the S protein from SARS-CoV found that FP1 and FP2 can cooperate and penetrate deeper into the lipid bilayer structure when they are together.
 
In the current study’ liposome fusion assay, FP1-FP2 displayed the same fusion activity as FP1. The synergy between FP1 and FP2 was thus not observed in the experiments with FP1 and FP2 from SARS-CoV-2 or at least it did not translate into stronger fusion activity.
 
The study team also observed that adding phosphatidylethanolamine or PE and cholesterol or CHOL into the c-liposome membrane to better recapitulate the lipid composition of the outer leaflet of mammalian plasma membranes strongly enhanced FP1-induced fusion between v- and c-liposomes.
 
 In the cell-cell fusion assay, depletion of plasma membrane CHOL in the presence of MβCD induced a dramatic (~70%) decrease of fusion between cells expressing S proteins and cells expressing ACE2 receptors.
 
In vitro liposome-liposome fusion and in situ cell-cell fusion assays therefore both show that CHOL facilitates S-mediated membrane fusion.
 
This validates two recent studies which found that efficient SARS-CoV-2 fusion with the cell membrane requires CHOL.
https://pubmed.ncbi.nlm.nih.gov/33890572/
 
https://pubmed.ncbi.nlm.nih.gov/32944968/
 
Importantly, cellular infection by SARS-CoV-2 was also previously found to be facilitated by the presence of ceramide lipids or CER in the cell plasma membrane.
https://pubmed.ncbi.nlm.nih.gov/34608263/
 
Typically, CER molecules are known to assemble into large hydrophobic gel-like domains in the outer leaflet of the cell plasma membrane that can serve to sequester specific surface proteins. It was proposed that such CER-enriched domains could trap ACE2 and TMPRSS2 to facilitate S protein binding and priming, respectively.
 
The study team hypothesized that CER-enriched domains, because of their high hydrophobicity, could also favor FP1 interaction with the cell membrane and thus membrane fusion.
 
It was found that addition of CER into the c-liposome membrane in fact increased FP1-mediated fusion between v- and cliposomes in vitro, and depletion of CER upon treatment of cells with Fumonisin B1 decreased S-mediated cell-cell fusion in situ.
 
Thus, CER could thus promote SARS-CoV-2 infection in two distinct ways:
 
- i) by facilitating S protein binding/priming, and
 
-ii) by directly activating the fusion step.
 
The observation that CHOL and CER facilitate S-mediated membrane fusion prompted the study team to investigate the effect of approved drugs that can modify membrane lipid composition and/or structure and could thus be repurposed to inhibit SARS-CoV-2 infection.
 
The study team focused on the antipsychotic drug (AP) Chlorpromazine or CPZ because of its previously proposed antiviral activity and its known effects on membrane biophysical properties.
 
Numerous past studies revealed that such AP drugs could be effective in reducing SARS-CoV-2 infectivity.
 
A detailed screening assay based on morphological profiling of cells infected by SARS-CoV-2 identified two APs, domperidone and metoclopramide, exhibiting antiviral effects.
https://pubmed.ncbi.nlm.nih.gov/34413211/
 
Yet, another study searching for drugs targeting SARS-CoV-2 proteins interactome found that the AP haloperidol (HAL) displayed some antiviral activity.
https://pubmed.ncbi.nlm.nih.gov/32353859/
 
Chlorpromazine or CPZ was found to inhibit the replication of MERS-CoV, SARS-CoV and SARS-CoV-2 in cultured cells.
https://pubmed.ncbi.nlm.nih.gov/33387629/
 
https://pubmed.ncbi.nlm.nih.gov/24841269/
 
https://pubmed.ncbi.nlm.nih.gov/24841273/
 
Some researchers also suggested that CPZ might block viral replication at an early entry stage, which could be the fusion step.
 
The current study’s in vitro and in situ fusion data both unambiguously show that addition of CPZ reduces S-mediated membrane fusion.
 
The study findings also show that adding CPZ produces the same effect on fusion as depleting the membrane of CHOL.
 
Chlorpromazine or CPZ was previously found to modify the lateral organization of CHOL-containing membranes and to perturb raft domains, which could be attributed to its capacity to compete with CHOL since CPZ and CHOL have a similar ring-like planar structure.
 
Such competition between CPZ and CHOL could explain the inhibitory effect of CPZ on S-mediated membrane fusion observed in the study. This would also explain why CPZ had no effect on S-mediated cell-cell fusion when cells were treated with MβCD.
 
However, In vitro, the inhibitory effect of CPZ persisted regardless of the lipid composition of the liposomes. This could be because (i) CPZ was also shown to increase lipid order in membranes or membrane regions lacking CHOL2 and (ii) in vitro systems, which are intrinsically simpler in terms of their lipid and protein composition, are more sensitive to perturbations of the membrane structure.
 
The inhibitory effect of CPZ on FP1-induced fusion could therefore result from its ability to increase lipid order and thus counterbalance the perturbing effect of FP1 on membrane structure.
 
To date, therapeutic strategies targeting the S2 fusion machinery mainly focused on the development of anti-fusogenic synthetic peptides mimicking the HR2 domain. Such peptides were shown to inhibit formation of the native HR1/HR2 six-helix coiled-coil hairpin complex, thus preventing viral fusion and infection by several human coronaviruses in situ, including MERS-CoV, SARS-CoV and SARS-CoV-2.
https://pubmed.ncbi.nlm.nih.gov/30989115/
 
https://pubmed.ncbi.nlm.nih.gov/32231345/
 
https://pubmed.ncbi.nlm.nih.gov/36122200/
 
Interestingly, intranasal injection of a synthetic lipopeptide derived from HR2 was found to protect ferrets against infection by SARS-CoV-2.
https://pubmed.ncbi.nlm.nih.gov/33597220/
 
However, these promising effects of anti-fusogenic synthetic HR2 peptides remain to be evaluated in the context of human clinical trials.
 
In order to circumvent the complex processes associated with the development of new drugs, an alternative strategy consists in repositioning existing approved drugs by testing their capacity to block SARS-CoV-2 fusion with target cells. The current study is in line with this approach and suggests that amphiphilic molecules with a planar shape such as CPZ could be effective inhibitors of SARS-CoV-2 infection due to their ability to modify the structure of membranes.
 
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