Calcium Channel Blockers Could Be Potential COVID-19 Drugs

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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.

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 theFP-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 viral spike protein (S) is a CoV glycoprotein located on the viral envelope. It is required to mediate the viral entry into the host cells and is a major pathogenicity determinant2, 3.

The S proteins of SARS-1, SARS-2 and MERS anchor on the viral envelop membrane in the form of trimers. Each monomer consists of S1 and S2 subunits.

The two major steps in viral entry are

1) receptor binding, in which the S1 subunit recognizes a receptor on the host cell membrane, e.g., ACE2, and attaches the virus to the host cell, followed by

2) membrane fusion, in which the S2 subunit mediates the viral envelop membrane and the host membrane fusing together and releasing the virion into the host cell. Membrane fusion is a required stage in viral entry4; thus, the blocking of this membrane fusion could be an objective leading to vaccines and therapies to combat COVID-195.

The major region of S that interacts with lipid bilayers of the host is called the “fusion peptide” (FP). Almost every enveloped virus has its glycoprotein and the corresponding FP, as it is critical for membrane fusion as it inserts into the host lipid bilayer upon activation of the fusion process, perturbing the membrane structure, and initiating membrane fusion6, 7. Previously, we used phospholipid (PC) spin labels to detect the local perturbation of the membrane by viral FP and TMD8–14.

The S proteins and FP of SARS-1, SARS-2 and MERS have several distinct features comparing to the other class I viral fusion protein, which includes the influenza hemagglutinin (HA) and the HIV envelope protein (Env). First, influenza and HIV envelope proteins are known to be activated via cleavage by host cell proteases to cleave the proto-glycoproteins into two subunits; this occurs at a single, restricted site directly upstream of their FP.

In contrast, coronaviruses typically have two distinct cleavage sites (S1/S2 and S2’) that can be activated by a much wider range of proteases. As a result, we have shown that the FP is only exposed after the cleavage event at the S2’ site, and using ESR (Fig 1A) we have identified that the bona fide FP is directly downstream of the S2’ site for all of SARS-1, SARS-2 and MERS 12, 15, 16. This gives coronaviruses unique flexibility in the ability to invade new cell types, tissues and host species.

Fig. 1. A) Diagram of subunits and domains of the S proteins of the SARS-1 and SARS-2 (upper panel) and the sequence alignment of the SARS fusion peptide domain (lower). The S protein is cleaved at the S1/S2 site into S1 and S2 subunits. The cleavage at the S2’ site exposes the N- terminal FP domain in the S2 subunit. We designate the FP1 and FP2 segment in the FP (previously designated as FP1_2) as shown in the lower panel. The residues in bold are the negatively charged residues. The residues in red highlight the difference between SARS-2 and SARS-1 FPs. B) The mutations for the SARS-1 FP used in this study. The residues in bold in sequence of the wild type (WT) are the negatively charged residues. The residue(s) in red indicates where the substitution occurs. C) The structure of spin labeled lipids used in this study: DPPTC, 5PC, 10PC and 14PC.

Second and more directly relevant to this study, we discovered the viral entry of the CoVs are Ca2+ dependent12, 15, 16, which is quite rare among the enveloped viruses13. We also discovered that the function of the CoV FPs is Ca2+ dependent, and while the SARS-1 and SARS-2 FPs bind Ca2+ at an 1:2 (FP:Ca2+) ratio, the MERS FP binds Ca2+ at an 1:1 ratio. Presumably, the FP is where the target for the Ca2+ in this Ca2+-dependent viral entry mechanism.

This is important because the FP domain is very conserved comparing to other parts of the S protein, thus making it an ideal target for vaccine and antiviral drugs. It also raises the possibility of repurposing FDA-approved drugs in blocking Ca2+ channels for COVID treatment. However, how the CoV FPs interact with Ca2+ and how this interaction affects the function of the FP and the viral entry both remain unknown.

There is no crystal structure of intact S, severely limiting our mechanistic understanding of coronavirus fusion. While the basic structure of the S of SARS and SARS-2 can be revealed using cryo-EM techniques17–20 and part of the SARS-2 S2 subunit has been solved by X-ray crystallography21, 22, many structural and functional aspects remain undetermined.

Furthermore, the FP adopts its active conformation only in the membranes12, which is generally not determined in the crystallographic structure as they are hydrophobic and intrinsically disordered. The structure of the SARS-2 FP has been solved only recently, using NMR in bicelles23. ESR is a useful technique to study the effect of FPs on the membrane structure with implications for the mechanism leading to membrane fusion. It can also be used to determine the peptide structure in the membrane in the form of Pulse-Dipolar ESR24, 25 and Power Saturation ESR26.

In this study we focus on the SARS-1 FP-Ca2+ interaction and its effect on membranes, using mainly ESR. We try to answer four questions in this research. First, as there are six negatively charged residues on the FP, which are involved in the Ca2+ binding? Second, what is the topology model of Ca2+ binding?

Third, how is the Ca2+ binding enhanced the FP-induced membrane ordering? Fourth, does the separate FP function the same way as the FP on the whole S protein on the viral membrane does? For this purpose, we systematically introduced mutations on those residues, and examined their effect on membrane structure as well as their Ca2+ binding.

We also used a more advanced pseudotype viral particle-SUV system to investigate the function of FP in the context of the whole protein, which better simulates the “biological scenario” 13 and compare the results with the those of separate peptides. In addition, we also show the study on a few corresponding mutants of the SARS-2 FP and prove this research on SARS-1 is helpful for the SARS-2 FP case.

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