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
cytopathic effect
Dulbecco's modified essential medium
generally recognized as safe
molecular dynamics
Madin‐Darby Canine Kidney cells
minimal essential medium
Markov state modeling
SARS‐CoV‐2 nucleocapsid protein
SARS‐CoV‐2 N protein C‐terminal domain
nuclear export signal
nuclear localization signal
SARS‐CoV‐2 N protein N‐terminal domain
nucleoprotein
protein database
radii of gyration
root mean square deviation
real‐time quantitative PCR
Verda reno cells
p‐cymene inhibits SARS‐CoV‐2 and impairs Influenza H1N1 viral replication, in vitro, at non‐toxic concentrations. This action is exerted by p‐cymene interaction with SARS‐CoV‐2 nucleocapsid and influenza nucleoprotein, as revealed by extensive
An unforeseen International effort led to the identification of the cause of the novel COVID‐19 disease (the SARS‐CoV‐2 virus) and the detailed analysis of its genome,
The
Among the multitude of products tested against the COVID‐19 disease, a number of natural products have been assayed (critically reviewed in Ref. [
The sequence of nucleocapsid proteins from SARS‐CoV‐2, influenza, Ebola and rabies viruses, in fasta format, was retrieved from the NCBI protein database (
Each protein (in pdb format) and ligand (in mol2) files were uploaded to the GalaxyWeb server and a fully flexible docking (involving the receptor and the ligand) was performed, followed by optimization and subsequent refinement, through the GalaxyRefine algorithm.
The sequence of human coronavirus 229E nucleocapsid protein mRNA (GenBank: J04419.1), part of the whole SARS‐CoV‐2 mRNA, was used as a template, in order to simulate the interaction of SARS‐CoV‐2 N‐protein‐viral mRNA. It was introduced to the RNAfold web server (
For protein–protein and protein–RNA interactions, we have used the HEX 8.0.0 program (
The crystal structure of the SARS‐CoV‐2 nucleocapsid protein (C‐terminal dimerization domain, residues 249–389, PDB entry: 6WJI
The Amber03
Classical MD production trajectories were run with the leap‐frog integrator in GROMACS 2020
Α detailed description of the MD/enhanced sampling implementation and data analysis are presented in
SARS‐CoV‐2 (isolate 30–287) was obtained through culture in Vero E6 cells (ATCC® CRL‐1586), from an infected patient, in Alexandroupolis, Greece. Virus stock was prepared by infecting fully confluent Vero E6 cells in DMEM, 10% fetal bovine serum, with antibiotics, at 37°C, 5% CO2. Four days after inoculation, the supernatant was frozen at −80°C until use. Infections were carried out in 96‐well plates, using SARS‐CoV‐2 (m.o.i. of 0.1) on Vero E6 cells. Cells were treated with different concentrations of p‐cymene, in a volume of 15 μl, per 150 μl of medium, for 48 h. Cell morphology was observed with phase contrast, in an inverted microscope, to record CPE. Culture supernatants were analyzed using real‐time RT‐PCR.
Madin‐Darby Canine Kidney cells (MDCK; ATCC® CCL34™) cells were grown according to standard conditions, as described in detail in.
Analysis of the effects of p‐cymene on the cell viability was performed in MDCK and Vero E6 cells using the tetrazolium MTT assay for 24, 48, and 72 h respectively.
Titration of SARS‐CoV‐2 infected cells was carried in 96‐well plates, using Vero E6 cells and TCID50 was calculated according to the method of Reed and Muench
The antiviral activity of p‐cymene on Influenza A virus was assayed in plaque‐reduction assay (expressed in the number of forming plaques), with Ribavirin as a positive control (25 μg/ml). After 3–4 days and upon plaque formation, cells were fixed with paraformaldehyde and cell monolayers were stained with crystal violet. Following staining the plaques that developed were counted, and the viral titer was evaluated by the formula:
To determine the SARS‐CoV‐2 viral load, RNA was extracted from 96‐well supernatants (100 μl) using NucleoSpin Dx Virus according to the manufacturer (Macherey Nagel). Multi‐target real‐time RT‐PCR was performed using COVID‐19 SARS‐Cov‐2 Real‐TM according to the manufacturer (Sacace Biotechnologies).
For influenza experiments, MDCK cells (2 × 105) were plated in 12‐well plates and after 24‐h infected with influenza A FM/1/47/H1N1 or HRV14 for 2 h at 37°C. Subsequently, cells were incubated in the absence (control) or the presence of p‐cymene for 12 h. Total RNA was extracted with NucleoSpin RNA kit (Macherey‐Nagel). The
Madin‐Darby Canine Kidney cells were plated on glass coverslips in 24‐well plates. After 24 h, they were infected with influenza A FM/1/47/H1N1 for 1 h at 37°C. MEM supplemented or not with p‐cymene was added, and cells were fixed 6 h after the infection with 4% PFA and permeabilized with 0.1% TritonX‐100, for 10 min. Cells were stained with a mouse monoclonal anti‐NP antibody (Santa Cruz), followed by a secondary FITC‐labeled goat anti‐mouse antibody (Santa Cruz). The nuclei were stained with DAPI. Fluorescent images were acquired with an epifluorescent Leica DMIRE2 microscope equipped with a Leica DFC300FX digital camera.
For the analysis of whole‐cell extracts, cells were collected and pelleted at 2500 g for 10 min. The M‐PER Mammalian Protein Extraction Reagent (Cat. No 78503; Thermo Scientific) along with protease inhibitors (Cat. No 78415; Thermo Scientific) was used. Samples were boiled in SDS gel‐loading buffer, separated by electrophoresis and transferred on to PVDF membrane. Membrane was blocked with TBST buffer with 5% (w/vol) dried non‐fat milk and incubated with primary antibody of NP (sc‐80481; Santa Cruz). Following incubation with the primary antibody, membrane was incubated with secondary antibody (goat Anti‐mouse IgG Antibody, Peroxidase Conjugated, H+L [AP124P] Sigma). The membrane was developed using Luminata Forte Western HRP Substrate (Cat. No WBLUF0100; Millipore) by the ChemiDoc™ MP System (Cat. No 170‐8280; Bio‐Rad).
Statistical analysis was performed in GraphPad Prisma V6.05 (GraphPad Software Inc.) and Origin 2018 (OriginLab Co.), with the tests described in Section 3. A threshold of
Key protein targets in this article are hyperlinked to corresponding entries in
Nucleocapsid SARS‐CoV‐2 is a large protein of 419 amino acids (NCBI Reference Sequence: YP_009724397.2). Until today, the crystal structure of the complete protein has not been resolved. By contrast, a number of crystals (PDB codes 6M3M, 6VYO, 6YI3) of the N‐, RNA‐binding, terminal part, and the C‐ dimerization part of the protein (PDB code 6WJI) have been reported (Figure
(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. Δ
Molecular docking simulation of the p‐cymene binding on SARS‐CoV‐2 N protein revealed a high‐affinity association of the ligand in the structured C‐terminal part of the molecule (Δ
Molecular dynamics simulations of N protein complex with Importin A, in the absence or the presence of p‐cymene (Figure
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
Previous reports suggest that SARS‐CoV‐2 protein dimerizes through an interaction of its N‐CTD η1 domain.
A number of RNA viruses contain NPs, which may shuttle between the cytoplasm to the nucleus. This transfer depends also on the binding of NPs to host importins and the subsequent nuclear internalization, through nuclear pores. Here, we confirmed the presence of a NLS for Importin α (NLSα, Figure
(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
p‐Cymene interacted with Influenza A (H1N1) NP through binding with Thr177, the first of amino acid of NLS of this NP (Figure
Treatment of SARS‐CoV‐2 infected Vero cells with variable concentrations of p‐cymene resulted in a significant decrease of plaque formation (Figure
(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
Incubation of MDCK cells with variable concentrations of p‐cymene for 72 h revealed a modest effect of plaque formation, much lower than that of ribavirin (25 μg/ml), used as a positive control (Figure
(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. *
COVID‐19 pandemics imposed a number of, not yet resolved, problems to the scientific community. In spite of the combined world‐wide scientific effort and the analysis of SARS‐CoV‐2 virus,
Here, we propose that p‐cymene can act as a potential novel agent for the treatment of RNA viruses‐induced diseases (influenza, rabies, Ebola), including SARS‐CoV‐2‐induced COVID‐19. In addition, our preliminary
Our results on SARS‐CoV‐2 were performed in the African green monkey kidney Vero E6 cell line. These cells are the standard cell line used for SARS‐CoV and SARS‐CoV‐2 antiviral screening, as they are highly permissive to virus infections and lack interferon production allowing for CPE observation. However, resent studies
In contrast to SARS‐CoV‐2 infected cells, the effect of p‐cymene did not induce a complete elimination of influenza viral infection, at non‐toxic concentrations, suggesting that, this agent should be used in combination with other anti‐viral compounds. A previous study
p‐Cymene is a naturally occurring organic compound that is classified as a hydrocarbon, related to a monoterpene. p‐Cymene is an isoprenoid lipid molecule with one aromatic ring. It exists as a solid and is considered to be practically insoluble in water, with a molecular weight of 134.222 g/mol, water solubility 23.4 mg/L and boiling point 177.1°C. It is a constituent of the essential oils of more than 100 plant species, such a
SARS‐CoV‐2 N protein is mainly cytoplasmic, with very rare reports suggesting a possible nuclear translocation. This 46‐kDa protein presents a ~90% homology to the SARS‐CoV N, and a major factor conferring the enhanced pathogenicity of SARS‐CoV‐2.
Interestingly, p‐cymene binds also in the vicinity of the NLSα sequence of NPs of other RNA viruses (influenza, rabies, and Ebola), as revealed by simulation, modifying significantly the 3‐D conformation of the NLS site, and inhibiting its interaction with importin α, a mechanism known for influenza virus and suggested for Ebola, through an alternative O‐glycosylation and O‐phosphorylation,
Is therefore p‐cymene a potential anti‐viral compound? p‐Cymene's rapid absorption, leading to a time of maximum concentration of 0.33 h,
In conclusion, data presented in this study suggest that p‐cymene, a non‐toxic natural compound, may be used alone or as a companion therapeutic for the control of RNA viral diseases, including COVID‐19 and influenza. However, the
AP, MT, CL, MK, SP, GS, and EC are inventors in a PCT patent application (priority # GR‐is 20200100068, GB‐2002141.6, PCT/EP2021/053389), related to the subject of this work. MK, GS, CL, SP, and EC are shareholders of the University of Crete spin‐off company Nature Crete Pharmaceuticals PC.
Castanas, Sourvinos, Pirintsos, and Karakasiliotis participated in research design. Panagiotopoulos, Tseliou, Karakasiliotis, Kotzambasi, Daskalakis, and Kesesidis, Kampa conducted experiments. Karakasiliotis, Daskalakis, Notas, Sourvinos, and Castanas performed data analysis. Karakasiliotis, Daskalakis, Lionis, Kampa, Pirintsos, Sourvinos, and Castanas wrote or contributed in the writing of the manuscript.
Ethics issues are not applicable to this work.
Supplementary Material
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We acknowledge PRACE for awarding us access to Joliot‐Curie at GENI@CEA (Irene), France, through the “PRACE support to mitigate impact of COVID‐19 pandemic” call and the project “Epitope vaccines based on the dynamics of mutated SARS‐CoV‐2 proteins at all atom resolution.” We also acknowledge Greece and the European Union (European Social Fund‐ESF) for funding through the Operational Program «Human Resources Development, Education and Lifelong Learning» in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS‐5000432), implemented by the State Scholarships Foundation (ΙΚΥ)» to AAP (PhD scholarship) and a Hellenic Foundation for Research and Innovation (H.F.R.I.) Grant to MK (# 3725). The financial support of Galenica SA is also acknowledged. All data are included in the main and the Supplemental part of this work.
All data are included in the text, figures, and supporting information provided.