Towards the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared on the scene, and practically shut down the world for the next several months. Since then, a slow and incomplete recovery has been observed, due largely to the immunity gained at population level from widespread infections and from large-scale vaccination.
Vaccine hesitancy and the shortfall in vaccine supply to many developing countries threatens the success of public health measures intended to bring an end to the coronavirus disease 2019 (COVID-19) pandemic.
The repeated overstrain placed by high numbers of hospitalizations and the horrifying numbers of deaths on healthcare facilities and workers indicates the need to develop other effective measures to deal with the mounting waves of cases driven by the emergence of successive variants of the virus, with higher transmissibility and immune escape characteristics.
A new paper, published in Cells, discusses the role of calcium signaling pathways as targets of inhibition in potential new antiviral therapeutic pathways.
Calcium is involved in multiple essential physiological pathways, and is known as the second messenger, mediating the transmission of the signal from one excitable tissue to another via a chemical chain, as well as being the trigger for several cascading pathways. This is seen, for instance, in muscle contraction, cell signaling, and the immune response.
In keeping with the central place of calcium in bodily processes, the concentrations of calcium within the cell and within cell organelle compartments are maintained within very tight limits, using various energy-intensive methods including molecular pumps, ion channels and ATPases. Calcium ion movement is disrupted severely during viral infections, as the infectious particle hijacks cell signaling pathways to replicate itself within the infected cell.
Within coronaviruses too, the viral envelope proteins have been found to act as calcium ion channels within key protein processing organelles like the endoplasmic reticulum Golgi apparatus intermediate compartment (ERGIC). This results in the activation of inflammasomes of the NLRP3 type.
Inflammasomes are protein oligomers, the formation of which is triggered by the presence of pathogen-associated molecular patterns (PAMP). These complexes form receptors that activate caspase-dependent cytokine pathways that cause pyroptosis.
With SARS-CoV-2, too, calcium-binding is considered to promote virus-host cell interactions. For instance, calcium signaling pathways have also been considered therapeutic pathways.
Earlier, calcium channel blockers were successfully tested against flu viruses, the Japanese encephalitis virus, and the Ebola virus, among others. The current paper discusses the potential for the use of calcium inhibitors in SARS-CoV-2 infections.
What did the study show?
The entry of calcium into the cells occurs via several types of calcium channels.
Voltage-gated calcium ion channels are found mostly in the cell membrane of excitable cells. Membrane depolarization causes the rapid influx of calcium, shifting the membrane potential towards the positive side. These are the channels allowing for the fastest movement of calcium ions in the cytosol and are thus found in excitable tissues, including pacemaker, neuronal or certain types of cardiac cells, as well as skeletal muscle cells.
They mediate hormone secretion, heart contraction, visual signals and neurotransmitter release, and are targeted in the treatment of several diseases. Several inhibitors of these channels, such as amlodipine, nifedipine, felodipine, verapamil and diltiazem are being tested for the current infection as well.
Among these, nifedipine and felodipine have highly selective activity, while the former, along with amlodipine, also reduces the risk of death and intubation among the elderly with severe COVID-19. This might be due to smooth muscle relaxation of the pulmonary vasculature, improving hypoxia. Treatment with calcium channel blockers was associated with an anti-inflammatory effect and thus a reduced likelihood of cytokine storm.
A new plant-derived natural small molecule, called neferine, was also identified as a potential entry inhibitor of the virus, and showed 75% inhibition of infection in cell cultures in a pseudovirus assay, by inhibiting calcium channels in the cell membrane. A calcium chelator, BAPTA-AM, was also found to significantly inhibit viral entry.
Store-operated channels open when intracellular endoplasmic reticulum (ER) calcium stores are depleted, producing a Ca2+-selective CRAC current (ICRAC). The effect is phospholipase C (PLC) activation, with subsequent release of cytosolic inositol trisphosphate (IP3), and the exit of calcium along the IP3 receptors. This leads to CRAC channel activation.
CRAC channels regulate calcium channels in multiple cells including T cells, where they are activated by T cell receptor (TCR) stimulation. Anomalies in this pathway lead to the occurrence of severe combined immunodeficiency (SCID) following single point mutations at strategic sites. The T cells formed in such patients show reduced proliferative and functional activity following antigen exposure, resulting in chronic and recurrent viral infections.
CRAC channels are thus involved in the cytotoxic T cell activity against viruses. These cells also provide durable protection against reinfection via memory T cell generation and maintenance, in collaboration with T helper cells. Memory T cells are part of a robust adaptive cellular immune response, expanding rapidly on exposure to the same virus or a secondary virus and then forming effector T cells that eliminate the infected cells.
CRAC channels are also important to antigen-TCR interaction that results in T cell activation, which supports the presence of a positive feedback loop between the TCR-increased calcium signaling following repeated antigen exposure-T cell activation. Besides this obvious role in antiviral immunity, CRAC channels may also be involved in the hyperinflammatory injury to lung endothelium, and the cytokine storm, seen in some patients following SARS-CoV-2 infection.
With the knockout of a key protein in this process, the STIM1 protein, the cell displayed high interferon-1 responses that led to robust resistance to the virus. conversely, the knockout of the Orai1 gene led to ready infection with the virus due to the lowered expression of calcium-dependent transcription factors that encode antiviral molecules.
CRAC channel inhibitors could be useful in treating cases of severe COVID-19. A novel nanoemulsion formulation of CM4620, named Auxora, is now in phase 2 trials for acute pancreatitis treatment. However, in such conditions, it inhibits inflammation in the lung caused by calcium influx, simultaneously lowering cytokine-mediated inflammation.
Encouraging reductions in recovery time were observed in severe COVID-19 pneumonia when treated with Auxora vs standard of care, with similar reductions in the proportion that required intubation. This agent may protect the lung endothelium and also suppress the spike in inflammatory cytokine production.
Other auxiliary proteins could regulate the CRAC channel traffic, such as the chaperone heat shock protein 27 (HSP27) that promotes STIM1 expression. HSP27 vaccines are being considered as a potential agent to prevent severe COVID-1-associated inflammation, promote endothelial repair and regrowth, and increase the production of white cells.
Cav-1 (Caveolin-1) is another multifunctional calcium channel auxiliary protein, with multiple binding sites on SARS-CoV-2. However, its absence on placental syncytiotrophoblast may explain the lack of vertical transmission of the virus in pregnant women.
Transient receptor potential (TRP) channels form a large family, of which especially TRPV5 and TRPV6 selectively transport calcium ions. The expression of TRP channels in virally infected tissues may be linked to viral entry, trafficking within the endosomes to promote viral entry, and the systemic response to the infection. These, therefore, provide a favorable environment for viruses, and they contribute largely to the calcium signaling system in infected hosts.
Black cumin has been reported to reduce COVID-19 symptom severity and clear the virus, when given over two weeks, possibly due to the presence of thymoquinone, and possibly involving TRP channel suppression. Other potential candidates include cannabidiol (CBD) from cannabis, which uses several TRP channels as its receptors, as well as berbamine, resveratrol, quercetin, and curcumin, among others. Spices like ginger, hot peppers, turmeric and pepper, as well as onions, contain anti-inflammatory compounds that interact with these channels as well.
The use of calcium channel blockers may be helpful in treating SARS-CoV-2 infection by preventing pathological changes in the infected cell, mediated by calcium ions. Calcium trafficking within the infected cell is a subject worthy of further study, and its inhibition may thus be a valuable method to target the virus at different levels, exploiting the various types of calcium channels.
Inhibition of such channels must also take into account the many and diverse off-target effects, such as those following upon CRAC channel inhibition, since these are found throughout the host organism. Complete CRAC inhibition is therefore not required, in all probability, to achieve antiviral efficacy.
On the other hand, Auxora has shown a potential benefit in the treatment of acute pancreatitis, and its role in COVID-19 remains to be demonstrated. Further detailed exploration of target and side effects will need to be carried out to elucidate the value of calcium channel inhibitors, as well as therapeutic hypocalcemia or calcium supplementation.
Overall, given the multitude of indications that CCBs [calcium channel blockers] may contribute to a better outcome of COVID-19 disease, it might be worthwhile to focus research in this direction while keeping an eye on the future endemic state of SARS-CoV-2.”