Therapeutic potential of Cordyceps militaris mushroom against SARS-CoV-2 : Virtual screening against Mpro and in vitro validation

doi:10.21203/rs.3.rs-3598125/v1

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Therapeutic potential of Cordyceps militaris mushroom against SARS-CoV-2 : Virtual screening against Mpro and in vitro validation

https://doi.org/10.21203/rs.3.rs-3598125/v1

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Pathogenic coronavirus, including COVID-19, threatens human health, and there are strong demands for efficient therapeutics. Cordyceps militaris is a medicinal mushroom that has long been used for immune enhancement, anticancer, and antiviral effects. Therefore, the inhibitory potentials of constituents of C. militaris against COVID-19 were analyzed using various virtual screening analyses. Among ten constituents of C. militaris, cordycepin, the major component, and 3´-deoxyuridine and 2´-O-methyl-adenosine showed strong binding affinity to Mpro, a potential target for COVID-19 therapeutics. Considering the structure activity relationship, nucleosides having deoxyribose and methoxyribose moiety are important for the affinity to Mpro. Cordycepin is also bound to Mpro mutants, and the binding mechanisms between cordycepin and Mpro were further demonstrated by MD simulation, RMSA, and MM/PBSA analysis. Principal component analysis also confirmed the conformational change of Mpro by cordycepin, which inhibits the function of Mpro. In vitro, the efficacy of cordycepin was measured using Vero cells infected with SARS-CoV-2, which showed excellent inhibition with an IC50 value of 29 μM. Conclusively, the constituents of C. militaris are expected to inhibit SARS-CoV-2 replication through binding to Mpro. Therefore, C. militaris can be an important therapeutic for coronavirus through the synergistic effect of its constituents.

Since the first outbreak of Coronavirus disease 2019 (COVID-19), this pandemic has caused major global health issues and threatened worldwide medical settings ^1-2. The risk of COVID-19 appears to be somewhat decreased compared to the beginning, and it is still threatening public health along with the emergence of new variants ³.

Pathogenic coronavirus, which includes COVID-19, also contains Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus which caused SARS and MERS, respectively. COVID-19 is caused by SARS-CoV-2, the positive-sense single-stranded RNA virus ^4-5. The viral entry of SARS-CoV-2 within the host cell is mediated by binding its surface spike glycoprotein (S protein) to the host angiotensin-converting enzyme 2 (ACE2)^6-7. Comprising of 14 open reading frames (ORFs), the SARS-CoV-2 genome encodes polyprotein precursors (pp1a and pp1ab), four structural (spike, envelope, nucleocapsid, and membrane protein) and accessory proteins ^8-10. The two polyproteins (pp1a/pp1ab) cleave into individual nonstructural proteins to replicate the viral genome. This cleavage of pp1a/pp1ab is mediated by two main proteases, main protease (M^pro) and papain-like protease (PL^pro) ⁸. Therefore, for the replication and infectious capacity of SARS-CoV-2, proteolytic enzymes, SARS-CoV-2 M^pro and SARS-CoV-2 PL^pro are essential and thus have become important antiviral targets ^11-13. Notably, SARS-CoV-2 M^pro (M^pro) is considered a potential target for COVID-19 therapeutics due to the difference with the human protease and the consequent side effects ^13-14.

Cordycepin is the major constituent of Cordyceps militaris, a parasitic fungus that develops on the bodies of insects in the wild. It has diverse pharmacological effects such as anti-cancer and immunostimulatory activity ^15-17. It also exerted anti-viral activity against EBV, HIV and influenza virus ^18-19.

Cordycepin is 3'-deoxyadenosine, a nucleoside analog, and it has drawn special attention due to its similarity with nucleosides. Derivatives of nucleic replace normal nucleic acids due to their structural similarity and inhibit viral replication ^21-22. Accordingly, remdesivir or GS-441524, synthetic substances similar to nucleic acids, inhibit the replication of SARS-CoV-2 and have been used as therapeutic agents for COVID-19 ^23-24. Based on this mechanism, the therapeutic potential of cordycepin against COVID-19 has been investigated by many researchers.

As mentioned above, cordycepin is an adenosine derivative, which does not have a 3′ hydroxy group of ribose ²⁵. Due to this similarity, cordycepin suppresses the transcription by binding to adenosine receptors and eventually inhibits its proliferation ²⁶. Various computational analyses predicted the inhibitory efficacy of cordycepin against SARS-CoV-2. Cordycepin exhibited strong interaction with RdRp, which might induce premature protein termination and inhibit polyadenylation ²⁷. The affinity of cordycepin against other protein targets such as S protein and M^pro was also demonstrated by molecular docking analysis ^28-29.

Cordyceps mushroom contains various compounds in addition to cordycepin ^30-31. Various compounds including cordyrrole, a novel compound were purified from Cordyceps in our previous study ³². In this study, we attempted to analyze other constituents of Cordyceps against SARS-CoV-2 M^pro with the expectation that they would also have therapeutic potential against COVID-19. The mutation is a big problem in developing antiviral therapeutic agents, therefore, the effect on mutants was further analyzed for compound showing efficacy. In vitro antiviral evaluation of active compound was also conducted using Vero cells infected with SARS-CoV-2.

Interaction of compounds of Cordyceps and M^pro

In order to investigate the inhibitory effect of compounds of Cordyceps against SARS-CoV-2, the interactions of ten compounds with a different skeleton (Fig. 1) with M^pro were first analyzed.

Among the compounds, cordycepin (2) showed the strongest PLP score of 52.02 followed by 2´-deoxyuridine (6) (48.81) and 2´-O-methyl-adenosine (3) (48.54). Adenosine (1), xanthosine (4), and uridine (5) also showed affinities with M^pro with PLP scores of 45.69, 45.05, and 45.86, respectively. However, cordyrrole A (9), uracil (7), and nicotinamide (8) showed relatively weak affinities (< 40.0). The study showed several active site residues, M49, P52, and R188, which facilitate the ligand binding within the active site of M^pro, prominently involved in making hydrogen bonds with cordycepin (Fig. 2 and Table 1). The Cys-His dyad within the active site of M^pro has been well studied to show protease activity. Cordycepin was found to be making Pi-Alkyl interaction with H61 of this catalytic dyad. M49, a member of the substrate union triad ^33–34 was also found to make a hydrogen bond with cordycepin. Along with these interactions, several other residues contribute to accommodating the molecules by making hydrophobic or pi-cation interactions (Fig. 2).

Considering the chemical structures of compounds, cordycepin (2) and 2ʹ-O-methyl-adenosine (3) share the same structures as adenosine (1) except for the removal of a hydroxy group (OH) at 3ʹ of ribose or the addition of methoxy group (OCH₃) to 2ʹ of ribose, respectively. Xanthosine (4) also has a ribose moiety as adenosine (1) but differs in nucleotide moiety. All these compounds showed strong affinities to M^pro which suggested the importance of ribosyl moiety, as also observed in uridine (5) and 2´-deoxyuridine (6). However, cordycepin (2) and 2´-O-methyl-adenosine (3) showed a stronger affinity to M^pro compared to that of adenosine (1). Similarly, the affinity of 2´-deoxyuridine (6) to M^pro was stronger than that of uridine (5). Therefore, ribose moiety is important for the affinity to M^pro and the removal of hydroxy or the addition of methoxy moiety to ribose increased the affinity. Consistently, the compounds 7–10 without ribose moiety showed relatively weak affinities to M^pro. These results suggested that not only cordycepin but also other compounds of Cordyceps also have an inhibitory effect on M^pro and can be useful as therapeutics against SARS-CoV-2.

Table 1. Detailed interaction of all the compounds within the active site of M^pro.

Compound	ClemPLP Score	Bond	Distance	Type
Adenosine (1)	45.69	SER144:HG - C05:O2	2.22	Hydrogen Bond
		C05:H12 - GLU166:OE2	2.02	Hydrogen Bond
		C05:H13 - PHE140:O	2.98	Hydrogen Bond
		C05:H13 - GLU166:OE2	1.95	Hydrogen Bond
		HIS172:HD2 - C05:O4	2.95	Hydrogen Bond
		C05:H1 - GLU166:O	2.54	Hydrogen Bond
		C05:H10 - ASN142:OD1	2.65	Hydrogen Bond
		C05:N3 - HIS41	4.4	Pi-Cation
		C05 - MET165	5.15	Pi-Alkyl
Cordycepin (2)	52.02	C06:H13 - MET49:O	1.51	Hydrogen Bond
		PRO52:HD2 - C06:O3	2.5	Hydrogen Bond
		C06:H6 - ARG188:O	1.9	Hydrogen Bond
		C06 - MET165	5.4	Alkyl
		HIS41 - C06	4.70	Pi-Alkyl
		MET165 - C06	4.14	Pi-Alkyl
		C06 - MET165	5.13	Pi-Alkyl
2ʹ-O-Methyl-adenosine (3)	48.54	SER144:HG - C07:O2	2.11	Hydrogen Bond
		C07:H10 - ASN142:OD1	1.97	Hydrogen Bond
		MET165:HA - C07:O1	2.57	Hydrogen Bond
Xanthosine (4)	45.05	C08:H10 - HIS164:O	2.09	Hydrogen Bond
		C08:H11 - HIS164:O	2.23	Hydrogen Bond
		C08:H3 - GLN189:OE1	2.43	Hydrogen Bond
		C08 - CYS145	5.03	Pi-Alkyl
		C08 - MET165	5.49	Pi-Alkyl
Uridine (5)	45.86	SER144:HG - C09:O6	2.96	Hydrogen Bond
		CYS145:SG - C09:O6	3.43	Hydrogen Bond
		GLU166:HN - C09:O2	1.91	Hydrogen Bond
		C09:H10 - PHE140:O	2.95	Hydrogen Bond
		C09:H10 - GLU166:OE2	1.93	Hydrogen Bond
		C09:H11 - GLU166:OE2	1.94	Hydrogen Bond
		HIS172:HD2 - C09:O5	2.94	Hydrogen Bond
2′-deoxyuridine (6)	48.81	C10:H11 - THR190:O	1.91	Hydrogen Bond
		C10:H12 - THR190:O	1.72	Hydrogen Bond
		C10:H2 - ARG188:O	2.25	Hydrogen Bond
		C10:H4 - GLU166:O	2.97	Hydrogen Bond
		C10:H5 - GLU166:O	2.29	Hydrogen Bond
		C10:H9 - THR190:O	2.91	Hydrogen Bond
		C10 - MET165	4.68	Alkyl
		C10 - MET165	4.41	Pi-Alkyl
Uracil (7)	29.54	C12:H3 - GLN189:OE1	2.22	Hydrogen Bond
		C12:H4 - HIS164:O	1.96	Hydrogen Bond
		ASP187:HA - C12:O1	2.89	Hydrogen Bond
		HIS41:NE2 - C12	4.59	Pi-Cation
		HIS41 - C12	4.31	Pi-Pi Stacked
		C12 - MET49	4.93	Pi-Alkyl
Nicotinamide (8)	36.39	C03:N1 - ASP187:OD2	5.08	Attractive Charge
		C03:H6 - HIS164:O	1.64	Hydrogen Bond
		C03:H3 - ASP187:O	2.42	Hydrogen Bond
		C03:H4 - HIS164:O	2.57	Hydrogen Bond
		HIS41:NE2 - C03	4.69	Pi-Cation
		C03:N1 - HIS41	4.00	Pi-Cation
		HIS41 - C03	4.18	Pi-Pi Stacked
		C03 - MET49	4.62	Pi-Alkyl
		C12 - MET49	4.93	Pi-Alkyl
Cordyrrole A (9)	38.35	C01:H4 - ASN142:OD1	2.20	Hydrogen Bond
		GLU166:HN - C01:O3	2.15	Hydrogen Bond
		C01 - CYS145	4.90	Pi-Alkyl
		HIS163 - C01	5.05	Pi-Alkyl
2-Hydroxy-1-[1-(2-hydroxyethyl)-1H-pyrrol-2-yl]-ethanone (10)	42.68	ARG188:HN - C02:O2	2.65	Hydrogen Bond
		C02:H8 - ASP187:O	2.57	Hydrogen Bond
		C02:H11 - ARG188:O	1.80	Hydrogen Bond
		ARG188:HA - C02:O1	2.47	Hydrogen Bond
		HIS41:NE2 - C02	4.43	Pi-Cation
		C02 - MET49	4.86	Pi-Alkyl

Table 2: The MM/PBSA binding free energy components of M^pro in complex with cordycepin

	VDWAALS	EEL	EGB	ESURF	GGAS	GSOLV	TOTAL
Energy Average (kcal/mol)	-20.02	39.25	-21.54	-3.08	19.23	-24.62	-5.39

Binding affinity of cordycepin against SARS-CoV-2 M ^pro mutants

As cordycepin showed the strongest affinity, further studies about the action against SARS-CoV-2 M^pro were conducted. The occurrence of mutation is a common phenomenon in the viral systems, which further challenges vaccine/drug candidate identification ³⁵. The emergence of mutations in SARS-CoV2 M^pro is susceptible to potential drug resistance ³⁶. Therefore, we further studied active site mutations within M^Pro to confirm the binding affinity of cordycepin against these mutants. As shown in Fig. 3, 10 out of the 14 high-frequency point mutations near the binding pocket of M^pro protease were found in the wild and mutant sequence alignment. Mutations M49I, Y54F, N142S, G143S, S144F/A, C145S, Q189K, Q192T, A193S/T and E166R/N/Q were found within 5Å distance of the ligand pocket.

We further investigate the binding potential of cordycepin against 14 different mutant structures by the clustering of docking results. The top representative binding orientations were broadly clustered into 7 distinct pose clusters (Fig. 4). The largest pose cluster encompassing E166R, C145S, G142S, A193S, and N142S mutations was found with RMSD less than 1Å. The effect of these mutations on the ligand binding is minimal, with no significant changes in the interacting partners (Fig. 5).

MD simulation analysis of cordycepin against SARS-CoV-2 M ^pro

MD simulation study was also conducted to assess the extent of the interaction of cordycepin with the M^pro. The structures of apo- and cordycepin-bound Mpro were subjected to 100 ns MD simulations and evaluated using various investigational parameters. Then, the thermodynamically stable simulated M^pro-cordycepin complex was compared with the initial docked complex to understand the dynamics of ligand and the secondary structure elements of protein. As shown in Fig. 6, a translational shift in the ligand pose within the binding pocket was observed. In addition, the hyper-flexible linker region surrounding the pocket which lies between D176-T198 and I43-S62 displayed a varying orientation throughout the simulation period. The two hydroxy groups on the tetrahydrofuran on the ligand are more conductively oriented toward the antiparallel β-barrel structure of the catalytic pocket.

RMSD measurement of cordycepin against SARS-CoV-2 M ^pro

RMSD measures the protein backbone's deviation from its starting structural conformation to its final conformation. These backbone deviations during the simulation period can be used to determine the protein stability concerning its structural conformation ^37–38. Therefore, the fluctuation in the Cα backbone of M^pro in the presence and absence of cordycepin was assessed by the backbone RMSD measurement. The backbone of apo and cordycepin bound Mpro reached an equilibrium confirmation after 60 and 70 ns, respectively. The RMSD of both the apo and cordycepin bound structures of M^pro were constant throughout the rest of the period (Fig. 7a). Next, the structural compactness level within the cordycepin unbound and bound structures of M^pro were determined by the radius of gyration (Rg) and SASA analysis ³⁹. The Rg of Cα atoms of the apo and cordycepin bound M^pro during the 100 ns simulation time is shown in Fig. 7b. The Rg of cordycepin bound M^pro was found to be slightly higher than the apo form, which suggests that the apo form of M^pro was somewhat more stable than the cordycepin bound form. Consistent with the results of RMSD and Rg, the SASA plot also showed a slightly higher value for the cordycepin bound structure of M^pro (Fig. 7c). Furthermore, the analysis of RMSD showed a clear difference in the fluctuation of the residues between the cordycepin bound and apo M^pro conformations (Fig. 7d).

Molecular-mechanics Poisson-Boltzmann Surface Area (MM/PBSA) analysis

The molecular-mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method is applied to estimate the difference in the binding free energy of the complex ⁴⁰. The binding free energy is calculated by considering the vacuum potential energy, solvation-free energy (polar), and solvation-free energy (nonpolar). Polar and nonpolar solvation energy terms were estimated using the Poisson–Boltzmann equation and solvent-accessible surface area (SASA) methods ^41–42. The Poisson–Boltzmann equation approximates the electrostatic component of biological macromolecules. It assesses the ligand-binding affinity of the protein, while the SASA method aids in identifying the surface of the protein with van der Waals contact probed by the solvent sphere ⁴³.

The MM/PBSA result suggested the predominance of hydrophobic forces between the ligand and the target protein throughout the simulation time (Table 2). The binding free energy for cordycepin calculated by the Molecular mechanic force field was found to be quite high as -5.39 kcal/mol, probably due to the minor presence of the polar fields in the binding pocket.

Principal component analysis of cordycepin against M ^pro

Principal component analysis (PCA), a method that accounts for the essential dynamics ⁴⁴, was employed to investigate the higher atomic motions patterns among all the motions within the cordycepin unbound and bound state of M^pro. Figure 8a shows the conformational sampling of tertiary structure for the apo and cordycepin bound structure in the essential subspace along eigenvectors 1 and 2. It was noticed from PC1 and PC2 projections that the cordycepin bound structure shows a less compact cluster of stable states. The analysis depicts that the cordycepin bound M^pro covers a wide range of phase spaces (higher level of internal motions). This study indicates the apo form of M^pro has comparatively fewer internal motions, indicating higher stiffness and stability of this structure. Additionally, we plotted the free energy landscapes to understand better the apo and cordycepin bound structures of M^pro (Figs. 8b and 8c). The knowledge of the free energy landscape of a protein offers the possibility of characterizing essential structural aspects, including its stability, folding pattern, and molecular recognition ⁴⁵.

Figure 8. (a) The PCA of the cordycepin unbound and bound complex of M^pro The graphical representation of the free energy landscape of the (b) apo and (c) cordycepin bound complex of M^pro.

Here we analyzed for any differences in the protein-folding patterns in both structures (apo and cordycepin bound). A slight deviation in the projection of free energy was noticed with relatively stable conformation and energetically favored for the apo form of M^pro compared to the cordycepin bound complex. All the investigation clearly indicates that the binding of cordycepin with M^pro slightly perturbs its conformation, subsequently inhibiting its activity.

In vitro evaluation of cordycepin using SARS-CoV-2 infected Vero cells

We analyzed the inhibitory mechanism of cordycepin against M^pro through various analyses and finally tried to prove its efficacy through in vitro evaluation on the Vero cells after SARS-CoV-2 infection and then treated at different concentrations

Cordycepin showed a CC₅₀ of more than 50 µM whereas IC₅₀ value of 29 µM, nearly halfway to its CC₅₀, which is comparable to the referenced drugs, chloroquine, remdesivir, and lopinavir (Fig. 9). The immunofluorescent staining of Vero cells treated with cordycepin clearly shows complete inhibition at 50 µM and partial inhibition a 25 µM; as no green staining was observed for Alexa-four-488 tagged viral nucleocapsid protein (Fig. 9). Our findings could be further authenticated using appropriate in vivo animal models and other subsequent methods to provide additional therapeutic routes.

To evaluate Cordyceps militaris as a potential candidate for coronaviruses, an analysis of constituents of C. militaris against SARS-CoV-2 M^pro was conducted by virtual screening analysis. Among ten compounds of C. militaris, cordycepin showed the strongest affinity to SARS-CoV-2 M^pro, an important target for COVID-19 therapeutics. Further analysis of the structure activity relationship showed the importance of the ribosyl group of nucleoside derivatives and cordycepin with 3ʹ-deoxyribose and 2ʹ-methoxyribose exerted stronger affinity than adenosine with ribose. Cordycepin, which showed the strongest affinity to M^pro also bound to M^pro mutants. In addition, the binding mechanisms between cordycepin and M^pro were further demonstrated by MD simulation, RMSA, and MM/PBSA analysis. PCA analysis also confirmed the conformational change of M^pro by cordycepin, which inhibits the function of M^pro. Finally, the efficacy of cordycepin was evaluated in vitro using Vero cells infected with SARS-CoV-2, which showed excellent inhibition with an IC₅₀ value of 29 µM. Taken together, the constituents of C. militaris are expected to inhibit SARS-CoV-2 replication through binding to M^pro. Therefore, C. militaris can be an important therapeutics for coronavirus through the synergistic effect of its constituents.

Structure analysis of SARS-CoV-2 M ^pro

The structure of SARS-CoV-2 Main Protease (M^pro) was retrieved from the RCSB protein data bank (pdb id: 6lu7) ⁴⁶. The structure consists of 309 amino acids. Mutant structures with single point mutaions (IDs 8EHM, 8EHK, 8E26, 8E1Y, 8DZA, 8DZ9, 8DZ6, 8DZ1, 8DGB, 8DDM, 8DDI, 8D4N, 8D4L, 7ZB8, 7ZB7, 7U29, 7U28 and 7N6N) were analyzed.

Virtual screening

The structure of SARS-CoV-2 Main Protease (M^pro) was retrieved from the RCSB protein data bank (PDB id: 6lu7) ⁴⁷. The selected compound structures were retrieved from the PubChem compound database. All the compounds were screened against the M^pro, and the top-scoring compound was selected based on the PLP score.

Molecular dynamics simulation

The structure of cordycepin, the top-scoring compound in complex with M^pro was subjected to Molecular dynamics simulation using Gromacs (2020.04 package) ⁴⁸. The cubic box model was done in the TIP3P water molecules for solvation with a 10 Å radius as its margin with the CHARMM27 force field. Ligand topology was generated with SwissParam. The system was made neutral using sodium ions and chloride ions. Protein-ligand complex, along with water and ions were energy minimized for 50,000 steps using the steepest descent method. Isothermal and isochoric equilibration was done using Particle Mesh Ewald for long-range electrostatics for 50,000 steps. Isothermal and isobaric equilibration also used Particle Mesh Ewald for long-range electrostatics for 50,000 steps. MD run was done for 50 ns after two equilibrations.

Free energy calculation

The MMPBSA.py module was used to perform MM-PBSA calculations using the AMBER software. This approach calculates the binding free energy (ΔG binding) based on the following equation:

Isolation of cordycepin

Cordycepin was purified from the dried C. militaris, as previously reported.⁴⁹. Briefly, the dried C. militaris was extracted with 80% methanol for one night. The extract was evaporated under reduced pressure to give a methanolic extract. After the extract was suspended in water, column chromatography using Diaion HP-20 was conducted with the elution using a mixture of methanol and water. Cordycepin was purified from the fraction eluted with H₂O: MeOH (80:20) by semipreparative HPLC.

In vitro evaluation of Cordycepin against SARS-CoV-2

In vitro evaluation of cordycepin against SARS-CoV-2 was performed at the Institute Pasteur Korea, South Korea using the standard protocol ^50–51.

Vero cells (ATCC CCL-81) were maintained at 37°C with 5% CO2 in Dulbecco’s modified eagle medium (DMEM; Welgene), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1× antibiotic-antimycotic solution (Gibco). SARS-CoV-2 (βCoV/KOR/KCDC03/2020) was provided by the Korea Centers for Disease Control and Prevention (KCDC) and was propagated in Vero cells. All experiments involving live SARS-CoV-2 followed the guidelines of the Korea National Institute of Health (KNIH) using enhanced biosafety level 3 (BSL3) containment procedures at Institute Pasteur Korea approved for use by the KCDC.

To evaluate compounds, Vero cells were seeded at 1.2 × 10⁴ cells per well in DMEM, supplemented with 2% FBS and 1× antibiotic-antimycotic solution (Gibco), in 384-well plates 24 h before the experiment. Ten-point DRCs were generated, with compound concentrations ranging from 0.1 to 50 µM. Approximately 1 hour after compound treatment, SARS-CoV-2 (0.0125 MOI) was infected with cells in a BSL3 facility and incubated at 37°C for 24 hours. After that, the cells were fixed with 4% paraformaldehyde (PFA), and then permeabilized. The cells were then stained by treating the anti-SARS-CoV-2 nucleocapsid (N) primary antibody and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody and Hoechst 33342. Images were acquired by a Perklin Elmer Operetta (20×; Waltham, MA, USA) and analyzed by in-house developed Image Mining 3.0 (IM 3.0) plug-in software.

Lopaminivir, chloroquine, and remdesivir were obtained from SelleckChem, Sigma-Aldrich, and MedChemExpress, respectively. An anti-SARS-CoV-2 N protein antibody was purchased from Sino Biological Inc. (Beijing, China). Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody and Hoechst 33342 were purchased from Molecular Probes.

Data availability

Data of analyses in the current study are included in this article and data for evaluation of biological activity are available from corresponding authors upon reasonable request.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT, 2022R1A2C1008081) and by the Medical Research Center program (2017R1A5A2015541).

Contributions

M.K.L. and J.-Y. C. designed experiments. M.H.B. and A.T. isolated compounds and evaluated biological activity. M.H.B., A.T., P.V., J.-J.D., D.H.L. and Y.G.K. run virtual screening and analyzed the data. M.K.L. J-J.D. and J.-Y. C. wrote the manuscript. All authors reviewed the manuscript.

Conflict of interest

The authors declare no conflicts of interest.

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No competing interests reported.

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Therapeutic potential of Cordyceps militaris mushroom against SARS-CoV-2 : Virtual screening against Mpro and in vitro validation

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