BREAKING! Cornell Study Find That Entry Of Coronaviruses Is A Ca2+-Dependent Process. Calcium Channel Blockers Could Be Potential COVID-19 Drugs

COVID-19 Drugs: A new study by researchers from Cornell University -New York has found that that entry of coronaviruses such as SARS-1, MERS and even SARS-CoV-2 is a Ca2+-dependent process. The study findings also propose that existing FDA approved calcium channel blockers could be repurposed as potential COVID-19 drugs.

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Entry of coronaviruses into host cells is mediated by the viral spike (S) protein.
 
The study team previously identified that the domain immediately downstream of the S2’ cleavage site is the bona fide fusion peptide or FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology.
 
The team also found that the SARS-1 FP induces membrane ordering in a Ca2+ dependent fashion.
 
In the current study, the team wanted to know which residues are involved in this Ca2+ binding, to build a topological model and to understand the role of the Ca2+.
 
They performed a systematic mutation study on the negatively charged residues on the SARS-1 FP.
 
Although all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue.
 
The study team provided a topological model of how the FP binds Ca2+ ions: both FP1 and FP2 bind one Ca2+ ion, and there are two binding sites in FP1 and three in FP2.
 
The team also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC (Isothermal titration calorimetry) experiments show that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca2+, the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca2+ is to help to folding of FP in membranes to enhance its activity.
 
Utilizing a novel pseudotyped virus particle (PP)-liposome methodology, the study team monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time.
 
The study findings revealed that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization.
 
Importantly the study findings could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel to combat the ongoing COVID-19 pandemic.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.11.03.467161v1
 
The study findings suggest targeting fusion peptide-calcium interaction may help treat COVID-19.
 
Most coronaviruses pose a major threat to the global healthcare systems and include zoonotic pathogens such as severe acute respiratory syndrome (SARS-1), severe acute respiratory syndrome coronavirus 2 (SARS-2) and Middle East respiratory syndrome coronavirus (MERS). Coronaviruses enter the host cells using the viral spike (S) protein with the fusion peptide (FP) of the S protein interacting with the lipid bilayers of the host.

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The study team discovered the entry of coronaviruses is a Ca2+-dependent process. This study mainly focused on the SARS-1 FP-Ca2+ interaction and its effect on membranes using electron spin resonance (ESR).
 
The study team performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. Since there are six negatively charged residues on the FP, they investigated the residue involved in the Ca2+ binding. The topology model of Ca2+ binding was also identified in this study. In addition, the influence of Ca2+ binding on the FP-induced membrane ordering was determined.
 
The study team also investigated if the separate FP function the same way as the FP on the whole S protein on the viral membrane.
 
The S proteins are available on the membrane in the form of trimers and each monomer consists of S1 and S2 subunits. The virus enters the host cell through receptor binding, followed by membrane fusion.
 
The study team systematically introduced mutations on the residues and observed their effect on membrane structure as well as Ca2+ binding.
 
Furthermore, a more advanced pseudotype viral particle (PP)-SUV system was used to examine the function of FP in the context of the whole protein, which better simulates the “biological scenario” and the results with those of separate peptides was compared.
 
The two distinct cleavage sites (S1/S2 and S2’) of coronaviruses are activated by a much wider range of proteases. This resulted in the exposure of FP after the cleavage event at the S2’ site and it was identified through ESR that the bona fide FP is directly downstream of the S2’ site for all of SARS-1, SARS-2, and MERS. This provides coronaviruses unique flexibility to invade new cell types, tissues, and host species.
 
The study team also found that the SARS-1 and SARS-2 FPs bind Ca2+ at a 1:2 (FP: Ca2+) ratio, the MERS FP binds Ca2+ at a 1:1 ratio which implied that the function of the CoV FPs is Ca2+ dependent.
 
This finding increases the potential of repurposing FDA-approved drugs that block Ca2+ channels for COVID-19 treatment.
 
The study findings showed that although all six negatively charged residues contributed to the membrane ordering activity of the FP to some extent, D812 was the most important residue.
 
Both FP1 and FP2 bound one Ca2+ ion, and there were two binding sites in FP1 and three in FP2.
 
The study team found that a similarly critical role was played by the corresponding residue D830 in the SARS-2 FP. The isothermal titration calorimetry (ITC) experiments depicted that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreased for all mutants.
 
As the binding of Ca2+, the folding of FP, and the ordering activity correlated across the mutants, it can be suggested that the Ca2+ helped fold FP in membranes to increase its activity.
 
With the help of novel pseudotyped virus particle (PP)-liposome methodology, the study team found that similar to separate FPs, the SARS-1 and SARS-2 PPs also induce membrane ordering, which declined due to the mutations of the negatively charged residues.
 
The difference in kinetics between the PP and FP could be attributed to FP trimerization.
 
Therefore, the study findings provided therapeutic solutions to control the ongoing coronavirus disease 2019 (COVID-19) pandemic by either targeting the FP-calcium interaction or blocking the Ca2+ channel.
 
The study findings on the SARS-1 FP can also be applicable for SARS-2 FP since their sequences are similar. Although the SARS-2 and SARS-1 FPs vary only in three residues, the SARS-2 FP has a notably greater ordering effect.
 
The study findings suggest that all negatively charged residues are involved in Ca2+ binding, though their contributions are different.
 
The study team also recommends that further studies be conducted on the repurposing of existing FDA approved calcium channel blockers to treat COVID-19.
 
Examples of such existing FDA approved calcium channel blockers includes Nifedipine (Adalat CC, Afeditab CR, Nifediac CC, Nifedical XL, Procardia XL), Clevidipine (Cleviprex), Amlodipine (Norvasc, Katerzia), Felodipine (Plendil), Diltiazem (Cardizem CD, Cardizem LA, Cartia XT, Dilacor XR, Dilt-CD, Dilt-XR, Matzim LA, Taztia XT, Tiazac) and Verapamil (Calan, Calan SR, Covera-HS, Isoptin SR, Verelan, Verelan PM).
 
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