Zinc inhibits SARS-CoV-2’s main protease in vitro

Zinc is well established as a critical element in COVID-19 (coronavirus disease 2019) treatment. COVID-19 patients with low levels of zinc can often result in poor clinical outcomes, including an extended duration of convalescence, higher morbidity, and higher mortality in older adults

Zinc has antiviral properties. However, the detailed kinetics and the mechanism of zinc in the inhibition of viruses, particularly, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – the causative virus of COVID-19 – is unclear.

To probe the role of zinc as an anti-SARS-CoV-2 agent at the structural and molecular level, researchers in India studied the binding kinetics and the inhibition mechanism of zinc with SARS-CoV-2’s viral proteins. The team recently released their findings as a preprint on the bioRxiv* server.

Study: Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro. Image Credit: Yuliia Sihurko / Shutterstock

While the SARS-CoV Main protease (Mpro) and RNA-dependent RNA polymerase (RDRP) are known as the potential molecular target of zinc, the researchers took a closer look at the mechanism of ionic zinc targeting SARS-CoV-2 Mpro.

“The structure of SARS-CoV-2 RDRP suggests a structural role for zinc rather than an inhibitory one,” said the researchers while establishing a strongly inhibitory role for ionic zinc in this study. Supported by complex crystal structure and subsequent inhibition of viral replication in vitro, the researchers demonstrated that zinc inhibits SARS-CoV-2 Mpro enzyme activity.

Using surface plasmon resonance (SPR), the team studied the binding kinetics of zinc acetate with purified SARS-CoV-2 Mpro. They reported an association rate constant (ka) of 8,930±30 M^-1s^-1 and the dissociation rate constant (kd) of 0.01755±10 s^-1 and equilibrium dissociation constant (KD) of 1.965E-06 M. They also computed the half-life (t_1/2=ln [0.5]/kd) of the zinc-Mpro complex to be ~10s.

Zinc supplements on the market are available as zinc glycinate and inc gluconate. The researchers assessed the inhibitory effects of Zn2+ binding on the proteolytic activity of SARS-COV-2 Mpro in the presence of zinc acetate. They reported its IC50 values, including that of zinc glycinate and zinc gluconate complexes.

Importantly, the researchers checked the reversibility of Zn²⁺-mediated inhibition using EDTA and found it completely reversible. This suggests that inhibition by the metal ion is not because of the oxidation of catalytic cysteine (Cys145).

To further understand the structural basis of SARS-CoV-2 Mpro inhibition by Zn²⁺ ion, the researchers solved the crystal structure of Zn²⁺ bound complex at 1.9 Å. They found that Zn²⁺-bound complex shows a tetrahedral coordination geometry at the Mpro active site.

An unambiguous electron density for Zn²⁺ shows that the metal ion is coordinated by the catalytic dyad His41 and Cys145, which is absent in the control datasets collected for apo-enzyme crystals grown in the same condition.”

At the active site of the enzyme, the zinc ions form a stable complex with two water molecules that are absent in the apoenzyme state.

As the Zn²⁺ coordinating catalytic dyad; Cysteine and Histidine are present across all coronaviral 3C-like protease (Mpro) active sites, including the ones coded by virulent strains of SARS-CoV-2; the mode of Zn²⁺ inhibition is expected to be conserved.”

Further, the researchers compared the inhibitory potential of the zinc complexes: zinc acetate, zinc glycinate, and zinc gluconate. Except for zinc acetate, the others failed to produce any antiviral effects in the cell culture experiments, despite showing effective enzyme inhibition in vitro. “This is in agreement with recent clinical trial data that shows no significant effect of zinc gluconate in clinical outcome in COVID-19 patients,” noted the researchers.

Quercetin is a natural zinc ionophore that increases the bioavailability of zinc within cells. Significantly, the researchers showed that quercetin aids in the inhibition of SARS-CoV-2 replication by increasing the intracellular concentration of zinc. They observed a >2-fold viral inhibition in the presence of quercetin when zinc acetate with quercetin (at 1:2 molar ratio) was tested for antiviral SARS-CoV-2 activity in infected Vero E6 cells.

This study supports and recommends a combination of zinc salt, which provides ionic zinc, with ionophores, to possibly have a better clinical outcome in COVID-19 therapy.

Because of the short half-life (~10s) of the Mpro-zn²⁺ complex, fast association and dissociation rates, and water-soluble nature of zinc acetate observed in this study, the researchers suggested that constant doses of zinc-ionophore combination may be required for effective inhibition of SARS-CoV-2 Mpro.

*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.

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.