In vitro and in silico studies of the antiviral activity of polyhydrated fullerenes against influenza A (H1N1) virus

Abstract

As of today, influenza viruses remain a relevant target for the development of antiviral compounds due to their rapid evolution and acquisition of the resistance to existing drugs. Fullerene derivatives have already shown the ability to successfully interact with viruses, and polyhydrated fullerenes (or fullerenols) are particularly attractive due to their compatibility with biological fluids and low toxicity. Therefore, the goal of this work was to study the effect of two batches of a mixture of polyhydrated fullerenes with a mass ratio of 78.1% C₆₀/C₇₀ and 21.9% C₇₆/C₇₈/C₈₄ on the influenza A (H1N1) virus. It was determined that the mixture of fullerenols, along with the low toxicity, showed high antiviral activity with a decrease in the viral infectious titer up to 4 orders of magnitude. In addition, studied fullerenols did not affect the hemagglutination process and did not show any significant prophylactic activity. With the help of molecular docking and molecular dynamics simulation, the likely target of fullerenols' action was determined-the binding site of the RNA primer of the viral RNA-dependent RNA polymerase. Therefore, we assume that the high antiviral effect of polyhydrated fullerenes on influenza A virus is related to their interaction with the viral RNA polymerase.

Figure 1

The effect of hydrated fullerenes on the reproduction process of influenza A (H1N1) virus and its infectious titer: (A) The antiviral activity of HyFn050 according to the post-exposure administration scheme; (B) The value of infectious titers de novo in TCID50 (log10) in the supernatant after determining the antiviral activity of HyFn050; (C) The antiviral activity of HyFn400 according to the post-exposure administration scheme; (D) The value of infectious titers de novo in TCID50 (log10) in the supernatant after determining the antiviral activity of HyFn400; Ref oseltamivir phosphate; IAV virus control.

Figure 2

The prophylactic effect of hydrated fullerenes on the process of influenza virus infection: (A) Indicators of the prophylactic activity for HyFn050; (B) The value of infectious titers de novo in the supernatant after determining the prophylactic effect of HyFn050; (C) Indicators of the prophylactic activity for HyFn400; (D) The value of infectious titers de novo in the supernatant after determining the prophylactic effect of HyFn400; Ref oseltamivir phosphate; IAV virus control.

Figure 3

The RdRp of H1N1 in complex with primer RNA (A) and C₆₀(OH)₆₀ (B). Amino acids that directly surround a fullerenol during its interaction with viral polymerase are highlighted in pink. It can be clearly seen that the same amino acids are directly involved in the interaction of H1N1 RdRp with vRNA.

Figure 4

The root-mean-square deviation of the ligand-receptor complex and the root-mean-square fluctuation of RdRp PA subunit. At the beginning of the simulation, the average RMSD value of C₆₀(OH)₆₀ quickly reached its maximum at ~ 3 Å, and after that stabilized. And the mentioned value remained until the end of the simulation with the exception of minor oscillations. At the same time, the amplitude of the RMSD of fullerenol is ~ 1 Å. The RMSF of RdRp PA showed a high degree of stability of most of the amino acids involved in the interaction with C₆₀(OH)₆₀. Residues Y393, S395, D396, and P398 are exceptions because they are a part of the highly labile disordered loop.

Figure 5

The number of hydrogen bonds between C₆₀(OH)₆₀ and RdRp. On average, it varies between 2–3.