Anticoronavirus Activity of Water-Soluble Pristine C₆₀ Fullerenes: In Vitro and In Silico Screenings

The emergence of a new member of the Coronaviridae family, which caused the 2020 pandemic, requires detailed research on the evolution of coronaviruses, their structure and properties, and interaction with cells. Modern nanobiotechnologies can address the many clinical challenges posed by the COVID-19 pandemic. In particular, they offer new therapeutic approaches using biocompatible nanostructures with "specific" antiviral activity. Therefore, the nanosized spherical-like molecule (0.72 nm in diameter) composed of 60 carbon atoms, C₆₀ fullerene, is of interest in terms of fighting coronaviruses due to its high biological activity. In here, we aim to evaluate the effectiveness of anticoronavirus action of water-soluble pristine C₆₀ fullerene in the model and in vitro systems. As a model, apathogenic for human coronavirus, we used transmissible gastroenteritis virus of swine (TGEV), which we adapted to the BHK-21 cell culture (kidney cells of a newborn Syrian hamster).

The shape and size of the particles present in C₆₀ fullerene aqueous colloidal solution (C₆₀FAS) of given concentration, as well as C₆₀FAS stability (value of zeta potential) were studied using microscopic (STM, scanning tunneling microscopy, and AFM, atomic force microscopy) and spectroscopic (DLS, dynamic light scattering) methods. The cytopathic effect of TGEV was determined with the help of a Leica DM 750 microscope and the degree of monolayer changes in cells was assessed. The microscopy of the viral suspension was performed using a high resolution transmission electron microscope (HRTEM; JEM-1230, Japan). Finally, the search for and design of optimal possible complexes between C₆₀ fullerene and target proteins in the structure of SARS-CoV-2 coronavirus, evaluation of their stability in the simulated cellular environment were performed using molecular dynamics and docking methods.

It was found that the maximum allowable cytotoxic concentration of C₆₀ fullerene is 37.5 ± 3.0 μg/ml. The investigated C₆₀FAS reduces the titer of coronavirus infectious activity by the value of 2.00 ± 0.08 TCID₅₀/ml. It was shown that C₆₀ fullerene interacts directly with SARS-CoV-2 proteins, such as RdRp (RNA-dependent RNA polymerase) and 3CLpro (3-chymotrypsin-like protease), which is critical for the life cycle of the coronavirus and, thus, inhibits its functional activity. In both cases, C₆₀ fullerene fills the binding pocket and gets stuck there through stacking and steric interactions.

Pioneer in vitro study to identify the anticoronavirus activity of water-soluble pristine C₆₀ fullerenes indicates that they are highly promising for further preclinical studies, since a significant inhibition of the infectious activity of swine coronavirus of transmissible gastroenteritis in BHK-21 cell culture was found. According to molecular modeling results, it was shown that C₆₀ fullerene can create the stable complexes with 3CLpro and RdRp proteins of SARS-CoV-2 coronavirus and, thus, suppress its functional activity.