Copper gluconate supplementation could reduce SARS-CoV-2 infection in vitro

The second wave of the coronavirus disease 2019 (COVID-19) pandemic has affected many countries, costing thousands of lives and dealing further heavy blows to already reeling economies.

Scientists have been working hard to bring out new drugs and repurpose older ones to stop the virus in its tracks and help the world return to something resembling normality.

The highly infectious nature of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and the significant percentage of people who develop severe or critical disease following infection, has placed a heavy or overwhelming burden on healthcare providers and public health authorities. The search for pharmaceutical control measures requires in vitro studies of drug efficacy, for which not all laboratories are equipped.

Most predicted lead compounds have come from computational studies. Thus, there is little actual in vitro information on the efficacy of drugs thought to have potential activity against the virus, beyond a handful comprising remdesivir, hydroxychloroquine, lopinavir-ritonavir, favipiravir, interferon, camostat mesylate, tocilizumab, and other immunomodulators.

Among these, the SOLIDARITY and RECOVERY trials, which are still ongoing, have reported in preliminary findings that these drugs have no significant effect on the number of deaths or duration of hospitalization following SARS-CoV-2 infection.

Copper has long been known to exert antimicrobial and antiviral actions.

SARS-CoV-2 can be eradicated from a copper surface within 4 hours while it can survive up to 72 hours on stainless steel and plastic surface.”

Thus, it could be helpful to inactivate the virus on hospital door handles, face masks, and other surfaces that face contamination.

This research has been published in the preprint sever bioRxiv*.

Research Process

Research Process. Image Credit: https://www.biorxiv.org/content/10.1101/2020.12.13.422548v1.full.pdf

Copper in the body

Copper is a cofactor essential for the activity of multiple enzymes that take part in redox reactions. The concentration of copper within the cell must be high enough to prevent metabolic breakdown.

In adults, this would range from 650 to 1850 μg/L. Copper is bound by many proteins, including ceruloplasmin, albumin, and alpha-2-macroglobulin. The first bind 40 percent to 70 percent of total copper in the plasma.

Copper gluconate

The current study explores the use of copper to block cell infection by SARS-CoV-2. Copper gluconate could be a viral inhibitor, preventing SARS-CoV-2 infection. The researchers decided to use a novel confocal microscopy-based high content screening (HCS) technique to evaluate the effect of pre-and post-treatment with copper gluconate on cell infection with this virus.

The scientists used a cell system exposed to synthetic SARS-CoV-2, with a green fluorescent protein (GFP) as the reporter. The reporter facilitates the rapid detection of the virus by fluorescence.

The use of this method helps achieve separate analysis of each cell, with high reliability due to the simultaneous counting of thousands of cells at each well. The complete automation of this process eliminates multiple sources of bias. In short, this model is suitable for drug screening, as shown by its use for remdesivir.

In this study, the use of the GFP-SARS-CoV-2 was combined with the confocal microscopy method to evaluate the antiviral activity of copper gluconate. It can be further refined to examine other potential antiviral drugs in mammalian cell lines.

Copper gluconate and cell viability

The researchers tested for toxicity of copper gluconate using Vero E6 cells treated with copper gluconate at concentrations of 0 to 1600 μM for 24 hours. They measured the amount of XIT converted to formazan, yielding an orange color, with the CyQUANT XTT assay.

They found that at up to 200 μM, Vero cell viability remained intact. After this, it fell sharply from 400 μM onwards to 800 μM, being measured at <40 percent and almost zero, respectively.

Copper gluconate and viral entry

Next, they treated Vero E6 cells with copper gluconate from 0 to 100 μM at 18 hours and then infected them with the virus. After a one-hour gap to allow adsorption of the virus the cells were again exposed to a fresh culture medium with the same copper gluconate concentration for 48 hours more. At this point, confocal microscopy was performed to assess the level of infection and viral replication.

The results showed that cells treated with copper gluconate at 25 μM or more had 70 percent lower infection rates (number of cells infected). This dosage was the lowest to achieve a marked reduction in infection. Moreover, the mean intensity of GFP fluorescence was inversely proportional to the concentration of copper gluconate, indicating the limiting effect of copper on viral replication.

Nonetheless, even at 100 μM, copper gluconate did not achieve complete viral inhibition. Concentrations beyond this were not assayed, even though it seems to be non-cytotoxic even at concentrations of 200 μM. The tissue concentration of copper is a thousand times lower than the serum concentration, at 1 to 12 μg/g vs. 1000 μg/ (15 μM). For this reason, the scientists chose to evaluate the effect of up to 25 μM of copper gluconate, even though the observed effect fell far short of antiviral drugs in efficacy.

The researchers call attention to the presence of copper in all eukaryotic cells. This could explain and predict a complex antiviral effect of copper in vivo rather than just reducing the infection rate.

Mechanism of copper toxicity

Copper gluconate may damage the viral membranes and denature the viral genome through a direct effect since, in this experiment, the copper concentration was kept steady throughout the experiment. Secondly, rising copper gluconate levels up to 100 μM were linked to a fall in mean fluorescent intensity, MFI. Interestingly, GFP in the recombinant SARS-CoV-2 is fused to the non-structural protein nsp7. Therefore, reduced MFI could indicate that copper disrupts the production of viral proteins.

In fact, earlier modeling studies predicted an inhibitory role for metals like cobalt or copper in ionic form on the main protease of SARS-CoV-2. More research will confirm this to be the case.

Another mechanism could be increased Cu/Zn superoxide dismutase 1 (SOD1) expression, which has been shown to be linked to reduced viral replication in vitro. Finally, coronavirus replication requires a replication complex that depends on cellular components associated with autophagy.

Copper is known to alter the rate of autophagy and could thus reduce or prevent the formation of this complex. Which of these potential mechanisms is most important in the antiviral effect of copper remains to be teased out in future research.

Conclusion

Much more study is required to understand what role copper plays in acute viral infection, beginning from copper ion concentrations in blood, nails, hair, and other tissues at all stages and at different levels of clinical severity of COVID-19.

The current study shows that copper gluconate administration can reduce infection rates with SARS-CoV-2 in vitro.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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