p-cymene impairs SARS-CoV-2 and Influenza A (H1N1) viral replication: In silico predicted interaction with SARS-CoV-2 nucleocapsid protein and H1N1 nucleoprotein

Abstract

Therapeutic regimens for the COVID-19 pandemics remain unmet. In this line, repurposing of existing drugs against known or predicted SARS-CoV-2 protein actions have been advanced, while natural products have also been tested. Here, we propose that p-cymene, a natural monoterpene, can act as a potential novel agent for the treatment of SARS-CoV-2-induced COVID-19 and other RNA-virus-induced diseases (influenza, rabies, Ebola). We show by extensive molecular simulations that SARS-CoV-2 C-terminal structured domain contains a nuclear localization signal (NLS), like SARS-CoV, on which p-cymene binds with low micromolar affinity, impairing nuclear translocation of this protein and inhibiting viral replication, as verified by preliminary in vitro experiments. A similar mechanism may occur in other RNA-viruses (influenza, rabies and Ebola), also verified in vitro for influenza, by interaction of p-cymene with viral nucleoproteins, and structural modification of their NLS site, weakening its interaction with importin A. This common mechanism of action renders therefore p-cymene as a possible antiviral, alone, or in combination with other agents, in a broad spectrum of RNA viruses, from SARS-CoV-2 to influenza A infections.

Keywords: Ebola; SARS-CoV-2; importin A; influenza A; nucleocapsid protein; nucleoprotein; p-cymene; rabies.

FIGURE 1

(A) Sequence of the SARS‐CoV‐2 N protein. In purple are shown the crystalized parts of the protein, while in green is presented the nuclear localization signal (NLS) sequence. (B) In silico simulation of the binding of the IMPα‐IMPβ‐RanGDP complex and viral RNA with SARS‐CoV‐2 N protein. ΔG for the interaction of N protein with the IMPα‐IMPβ‐RanGDP complex and with viral RNA are also shown. (C) Docking of p‐cymene within the NLS sequence of N protein shown in yellow. As shown the ligand interacts preferentially with Arginine 262 and Threonine 265 at the center of the NLS sequence (aa 258–268), therefore impairing the interaction of N with importins

FIGURE 2

Free energy surfaces for the dissociation of the nucleocapsid–Importin α complex in the absence (A) and the presence of p‐cymene (B). (C) The associated structures at the M1–M2 minima of the complex

FIGURE 3

(A) Comparison of nuclear localization signal (NLS) sequences in Influenza H1N1, Ebola, Rabies, and SARS‐CoV‐2 viruses. (B–D) Simulation binding of p‐cymene (red color) on Influenza H1N1, Rabies, and Ebola nucleoproteins (NPs). The NLS sequence of each protein is presented in blue color. (E) Table summarizing the RNA and Importin binding to Influenza H1N1, Rabies, and Ebola NPs in the absence or the presence of p‐cymene

FIGURE 4

(A) Phase‐contrast photographs of cells infected with SARS‐CoV‐2 and co‐treated or pre‐treated for 2 h with the indicated concentrations of p‐cymene. (B) The inhibition of SARS‐CoV‐2 RNA in the supernatant of cell cultures is presented (mean ± SD) of cells co‐treated or pre‐treated for 2 h with the indicated concentrations of p‐cymene together with a sigmoidal fit of data. The obtained IC₅₀s are also presented

FIGURE 5

(A) Plaque reduction assay of Madin‐Darby Canine Kidney (MDCK) cells infected with Influenza H1N1 virus (0.05 PFU/cell) and incubated with variable concentrations of p‐cymene for 72 h. Figure shows mean ± SD of two experiments in triplicates. Ribavirin (red dot) at 25 μg/ml is presented as a positive control. *p < .05 as compared to non‐treated cells. (B) p‐cymene reduces the production of progeny virus, as assayed by quantitative PCR of the supernatants of MDCK cells were pretreated with p‐cymene (20 μg/ml) and infected with influenza A/H1N1 virus (0.1 PFU per cell) for 24 h. (C) Quantification by qRT‐PCR of FluA M1 genome copies in MDCK cells infected with FluA/H1N1 at MOI 0.5 PFU/cell. Data shown are means ± SD of two independent experiments. (D) Immunoblot analysis of influenza nucleoprotein (NP) in total MDCK cell lysates incubated for 10 and 24 h post‐infection with variable concentrations of p‐cymene. (E) The role of p‐cymene in the NP distribution. MDCK cells were infected with FluA/H1N1 (MOI 5) in the absence or presence of p‐cymene. Six hours after the infection, cells were fixed and subsequently stained using influenza A anti‐NP primary antibody followed by FITC secondary antibody (green). DAPI was used to visualize the nucleus area (blue)