Artemisia argyi potentially prevents the infections with SARS-CoV-2 variants

Abstract


Introduction
The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a serious public issue threating people's health in most of countries [1].The genomic evolution of SARS-CoV-2 creates the diverse mutant strains which become more transmittable and resistance to immune attack and anti-virus treatments [2].In order to control the outbreak of COVID-19, multiple variants of SARS-CoV-2 were classi ed into variant of interests (VOIs) and variant of concerns (VOCs) by World Health Organization (WHO) according to their transmission rate, disease severity, therapeutic response, and healthy problem of mutant viruses [3].For example, Omicron variant, identi ed as the lineage B.1.1.529in South Africa in November 2021, has been viewed as one of circulating and high lethal VOIs associated with its rapid human-to-human transmission, high risk of reinfection, and resistance to vaccines [4][5][6].SARS-CoV-2 belongs to the family of highly diverse coronaviruses (CoVs) constituted by positive-sense and single-strand RNA [7].For cellular infection, SARS-CoV-2 utilizes diverse factors for the entry of host cells, translation of RNA genome, and replication cycle, ultimately resulting in the release of viral progeny.
The cellular entry of SARS-CoV-2 relies on its receptor engagement of spike (S) protein with the speci c host receptor angiotensin-converting enzyme2 (ACE2) [8].After S protein binding to ACE2, the cleavage and priming of S protein by different proteases, such as transmembrane serine protease 2 (TMPRSS2) and cathepsin L, are typically necessary to present fusion peptide (FP) for membrane fusion with host cells [9,10].Following cellular entry, viruses release, replicate, and transcribe their genomic RNA to form essential structural proteins that compose new and complete viral particles [11,12].Since SARS-CoV-2 evolves to diverse variants, the therapeutic effectiveness of antivirus treatments is limited and scarce.It is imperative to explore the potential protective treatments or integrated approaches against COVID-19.
Artemisia argyi (A.argyi) belonging to the family of Asteraceae is a renowned traditional Chinese medicine (TCM) and food ingredients in the Far East.In the broad-spectrum pro le, A. argyi possesses several bioacitve conpounds, including avonoids, terpenoids, and caffeoylquinic acids, and contributes to anti-microbial, anti-allergic, anti-diabetic, and anti-in ammatory acitivties by modulating the immune system or defending oxidative stress [13,14].In addition, this TCM was also shown to suppress the proliferation of tumor cells with the low cytotoxicity to normal cells [15,16].Recently, A. argyi has been implied as a potential TCM against severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), and COVID-19 [17,18].However, its activity against the infection with SARS-CoV2 has not been demonstrated yet and the active phytochemicals and underlying mechanisms also remain unclear.

Artemisia argyi i s a potential TCM against COVID-19
To identify the bioactive compounds of A. argyi with the potential against the infection of SARS-CoV-2, FRET-based enzymatic activity assay as illustrated in Fig. 1A was performed to examine the effects of 14 known ingredients of A. argyi on the activity of cellular proteins invovled in the priming of S protein and viral replication.As shown in Table 1, the enzymatic activity of TMPRSS2 but not 3CLpro was dramatically attenuated by eriodictyol (100.0%±0.3%;Fig. 1B), umbelliferone (74.1%±3.5%),and 13-Oxo-9E,11E-octadecadienoic acid (13-Oxo-ODE) (76%±4.7%).13-Oxo-ODE is a metabolite from dehydrogenation of 13-hydroxyoctadecadienoic acid (13-HODE), which is a oxidized product of linoleic acid by 12/15-lipoxygenase (LOX) [19,20], and has been reported as an endogenous ligand for nuclear hormone receptor peroxisome proliferation-activated receptor gamma (PPARγ), resulting in amelioration of in ammatory bowel disease [21,22].Since activation of PPARγ has also been associated with cancer progression [23][24][25], these unfavor effects of 13-Oxo-ODE lead us to focus on only eriodictyol and umbelliferone for their anti-corona virus activity.In addition, eriodictyol not only repressed the enzymatic activity of TMPRSS2 (Fig. 1B), but also abolished furin activity (Fig. 1C) in a dose-dependent manner.
Following the priming of S protein by TMPRSS2 or furin, the cleaved S protein interacts with human cellular receptor ACE2 [26].FRET assays as illustrated in Fig. 1D further showed the inhibitory effect of eriodictyol on the interaction between S protein and ACE2 in a dose-dependent manner (Fig. 1E).Taken together, eriodictyol and umbelliferone in A. argyi potentially suppress the activity of TMPRSS2 and furin to reduce the S1/S2 priming of S protein and interferes the interaction between S protein and ACE2, thereby decreasing the binding actiivty of SARS-CoV2 to host cells.

Molecular docking of eriodictyol and umbelliferone with SARS-CoV-2 proteins
Since eriodictyol and umbelliferone suppressed the enzymatic activity of TMPRSS2 and S protein/ACE2 interaction, the binding mode of these two compounds in the catalytic pockets of TMPRSS2 and ACE2 were performed with Discovery Studio.The results indicated that eriodictyol (Fig. 2A and 2B) and umbelliferone (Fig. 3A and 3B) showed interactions with the catalytic pockets of TMPRSS2 and ACE2, and the changes in energy value were − 13.05 kcal/mol (eriodictyol/TMPRSS2), -28.991 kcal/mol (eriodictyol/ACE2), -14.82 kcal/mol (umbelliferone/TMPRSS2), and − 33.837 kcal/mol (umbelliferone/ACE2), respectively (Table 2).Futhermore, the interface between S protein and human receptor ACE2 was also interferred by these two compounds (Figs.2C and 3C) with free energy descreases by -33.478 and − 36.539kcal/mol for eriodictyol/S protein-ACE2 and umbelliferone/S protein-ACE2 complexes, respectively (Table 2).Base on these results, eriodictyol and umbelliferone are phytochemicals of A. argyi directly targeting multiple protiens associated with the cellular entry of SARS-CoV-2. A. argyi and its active compounds downregulate ACE2 and TMPRSS2 expressions in lung epithelial cells The cytotoxic activities of A. argyi, eriodictyol, and umbelliferone were next examined in Beas 2B lung epithelial cell by using MTT assay.Cytotoxicity concentration 50% (CC 50 ) values of A. argyi, eriodictyol, and umbelliferone were 4471 µg/ml (Fig. 4A), 212.6 µM (Fig. 4B), and 302.4 µM (Fig. 4C), respectively.Base on the level of CC 50 , we further addressed whether the expressions of ACE2 and TMPRSS2 are also affected by these potential anti-SARS-CoV agents in preventing the cellular entry of SARS-CoV-2.As shown in Fig. 4, they selectively suppressed the protein expressions of ACE2 and TMPRSS2 (Fig. 4D-4F), and signi cantly decreased the RNA levels of ACE2 and TMPRSS2 in a dose-depentment manner (Fig. 4G-4J), suggesting that A. argyi is able to inhibit SARS-CoV-2 infection via repressing the expressions of cellular ACE2 and TMPRSS2.
A. argyi and its phytochemicals broadly suppress the cell entry of Vpp of SARS-CoV-2 variants The above data showed that A. argyi could potentially prevent the entry of SARS-CoV-2 to cells with multiple activities including inhibition of cellular ACE2 and TMPRSS2 activity and expressions and blockade of S protein/ACE2 interaction.Therefore, we further assessed the potential e cacy of A. argyi extracts and its phytochemicals in blocking SARS-CoV-2 infection by using SARS-CoV-2 S proteinpseudotyped lentiviral particles (SARS-CoV-2 S-Vpp).HEK-293T cells stably expressing ACE2 protein were employed to test the cellular entry of SARS-CoV-2 Vpp with S protein from different variants followed by the pretreatments with A. argyi extracts.The results displayed that the infection with most SARS-CoV-2 Vpp variants including wild type (WT), B.1.351(Beta), Lineage P1 (Gamma), and B1.617.2 (Delta) was dramatically repressed by the treatments with A. argyi (Fig. 5A), eriodictyol (Fig. 5B), and umbelliferone (Fig. 5C) in a dose-dependent manner.However, the infections with B1.To validate the inhibition of Omicrone entry, HEK-293T cell expressing ACE2 was infected with the Vpp of Omicrone variants and the results showed that umbelliferone signi cantly and dose-dependently suppressed the cellular entry of Omicrone variants BA.1 (Fig. 6A), BA.1.1 (Fig. 6B), and BA.2 (Fig. 6C).Unexpectedly, eriodictyol did not interfere the infection of Omicron variants (Fig. 6A-6C).Our ndings suggeted that A. argyi and umbelliferone can be potential agents against the variants of SARS-CoV-2 Omicrone.
Eriodictyol decreases the replication of SARS-CoV-2 by enzymatic inhibition of RdRp After the entry and uncoating of SARS-CoV-2, 3-chymotrypsin-like protease (3CL pro ) and papain-like protease (PL pro ) are the virus-encoded cysteine proteases for cleaving the viral polyprotein and then generating nonstructural proteins (nsps) during viral replication [27,28].RNA-dependent RNA polymerase (RdRp; also known as nsp12) is a viral replicase for the syntehsis of the complementary RNA during the transcriptional cycle of SARS-CoV-2 [29].Therefore, we further examined the inhibitory effects of tested compounds on the enzymatic activity of 3CL pro , PL pro , and, RdRp by performing FRET-base enzymatic activity assays (Fig. 1A) and found that eriodictyol dramatically reduced the activities of these three viral enzymes (Fig. 7A-7C).However, the 50% inhibition concentrations of eriodictyol for 3CL pro and PL pro were over 240 µM (Fig. 7B and 7C).These results displayed that A. argyi and its phytochemicals may interfer the cellular entry and RNA replication of SARS-CoV-2 at relative lower doses and showed inhibitory effects on the viral protease activities at higher doses.

Discussion
As of 6th December 2022, COVID-19 pandemic has caused more than 60 million con rmed cases and 6 million deaths worldwide, threatening people's health and economy globally.The emergency of SARS-CoV-2 variants that facilitate viral replication, transmission, immune escape, and weaken the protective ability of recently developed vaccines [2,30], creates great concerns on the prevention of SARS-CoV-2 infection.Traditional chinese medicines have been explored as potential therapeutics against COVID-19 and can improve the e cacy of routine treatments while diminishing disease deterioration [31,32].For example, Taiwan Chingguan Yihau (NRICM101) has been shown to disrupt virus invasion and host in ammation in patients with SARS-CoV2 infections [33,34].Our data showed that A. argyi, a well-known herbal used in the Far East, can be a new strategy to prevent the infections with multiple variants of SARS-CoV2 by suppressing their cellular entry as well as viral replication via targeting cellular proteins TMPRSS2 and ACE2 and viral protein RdRp, respectively (Fig. 7D).
Although the binding a nity of jaceosidin and eupatilin to SARS-Cov-2 main protease (3CL pro ) has been predicted in molecular docking simulation [35].In the present study, however, several avonoids from A. argyi including 5,7,3'-Trihydroxy-6,4',5'-trimethoxy avone, beta-Rhamnocitrin, eriodictyol, eupatilin, hispidulin, and jaceosidin containing the basic skeleton of bezno--pyrene [36] did not showed the inhibitory effects on the activity of SARS-CoV-2 main protease as well as other related enzymes in the biochemical reaction assays (Table 1), suggesting that the avonoids in A. argyi provide an essential ability to decrease the COVID-19 deterioration by mainly targeting the activity of TMPRSS2.
SARS-CoV-2 lineage B.1.1.529,named Omicron strain, has been documented as a VOC on 26th November 2021 by WHO and becomes the major viral strain in COVID-19 pandemic.Unlike other SARS-CoV-2 variants, Omicron is marked by the large number of mutations across entire genome including its spike glycoprotein gene [37], contributing to immune escape, a attenuating ability of vaccines, and a higher frequency of reinfections [5,6].Additionally, these high mutations of S protein are associated with its increased binding e cacy to host receptor ACE2 [38].While other SARS-Cov-2 variants require the S protein priming by the host transmembrane protein TMPRSS2 for cellular entry, the infection of Omicron variants can be accomplished by an endocytic route in a TMPRSS2-indenpendednt manner [39,40].Our results found that both A. argyi and umbelliferone diminished the protein level of ACE2 (Fig. 4), accounting for their activity in blocking the cellular entry of Omicron variants (Fig. 6).Therefore, umbelliferone would be the critical phytochemical existed in A. argyi to reduce the infection with Omicron variants.

Conclusions
In general, this study explored the potent antiviral activity of A. argyi and its phytochemicals (eriodictyol and umbelliferone) against multiple SARS-CoV-2 variants with the IC 50 values ranging from 87 to 275 µg/ml and 45 to 52 µM, respectively.Mechanistically, all A. argyi, eriodictyol, and umbelliferone repress the enzymatic activity of TMPRSS2 and furin for priming S protein, impede the interaction between S protein and ACE2, and inhibit the RdRp-mediated viral replication (Fig. 7D).In addition, A. argyi and umbelliferone also showed speci cal activity against Omicron variants due to their inhibitory effect on ACE2 protein expression.Our ndings suggest that A. argyi and its phytochemicals possess the potential to be developed as the inhibitors used for preventing or treating the infections with most SARS-CoV-2 variants.

Cell lines culture
Beas 2B epithelial cell line of normal human bronchus was grown in Dulbecco's Modi ed Eagle Medium (DMEM) with low glucose (1 g/L) and sodium pyruvate (Gibco), and ACE2-expressing HEK-293T cell line was cultured in DMEM/Nutrient Mixture (F-12) medium (Gibco).All cell lines were incubated at 37°C in a humidi ed 5% CO 2 /95% incubator.

Preparation of A. argyi extraction and pure compounds
The powder of A. argyi was obtained from dry plants, and was dissolved and heated in sterilized H 2 O at 37°C for 30 minutes.Eriodictyol (Cayman), umbelliferone (Sigma-Aldrich), and other ingredients of A. argyi listed in Table 1 were dissolved in DMSO.

Infection with SARS-CoV-2 S protein-pseudotyped lentiviral particles
The virus particle pseudotyped (Vpp) of SARS-CoV-2 S protein mutants and the control vesicular stomatitis-G (VSV-G) bearing luciferase were obtained from RNA Technology Platform and Gene Manipulation Core, Academia Sinica in Taiwan.ACE2-expressing HEK-293T cells were pre-treated with A. argyi solution, eriodictyol, and umbelliferone at the indicated concentrations for 2 days and then was infected with the variants of SARS-CoV-2 pseudovirus.After 1 day, the infected cells were lysed with One-Glo ™ Luciferase assay buffer (Promega), and the luciferase intensity was measured by Luminescence Plate Reader.

Measurement of cell viability
The cell viability was measured according to the manufacturer's protocols of MTT and cell counting kit-8 (CCK-8) assays.Beas 2B cell (5000 cells/well) in 96-well plates were treated with A. argyi solution, eriodictyol, or umbelliferone in a dose-dependent manner for 2 days and then subjected to MTT assay (Sigma-Aldrich).The viability of ACE2-expressing HEK-293T cells infected with the variants of SARS-CoV-2 pseudovirus was detected in CCK-8 assays (Sigma-Aldrich).

Molecular docking simulation
The structures of proteins and compounds in this study were retrieved from Protein Data Bank (PDB, https://www.rcsb.org/)and were applied to molecular docking calculation (BIOVIA Discovery Studio) to simulate the binding e cacy of tested compounds to S protein, TMPRSS, and ACE proteins.

FRET-based enzymatic activity assay
To examine the inhibitory effects of tested compounds on the protease activity in the cleavage of S protein and 3CLpro of SARS-CoV-2, the assay buffer (25 mM Tris 8.0, 150 mM NaCl) containing 15 µg/ml of recombinant proteins with/without eriodictyol was pre-incubated at room temperature for 30 mins.After adding 20 µM of uorescent protein substrate, the reaction of substrate cleavage was monitored continuously for 6 hours by detecting mNeonGreen uorescence (excitation: 506 nm/emission: 536 nm) using Synergy™ H1 hybrid multi-mode microplate reader (BioTek Instruments, Inc.).The rst 1 hour of the reaction was used to calculate initial velocity (V 0 ).The initial velocity with each compound was calculated and normalized to DMSO control (illustrated in Fig. 1A).Additionally, the effects of tested compounds on the interaction between SARS-CoV-2 Spike S1 and human ACE2 proteins were measured by using TR-FRET assays according to manufacturer's protocol (BPS Bioscience) [41,42].Brie y, the recombinant proteins of ACE2 and SARS-CoV-2 Spike S1 were incubated with or without the tested compounds at the indicated concentrations at room temperature for 1 hour.TR-FRET signals were recorded by detecting the emission at a wavelength 620 or 665 nm with the excitation at a wavelength 340 nm (Fig. 1D).

Western blotting
Beas 2B lung cells were treated with A. argyi solution, eriodictyol, and umbelliferone in a dose-dependent manner for 2 days, and were subsequently lysed in RIPA buffer with protease and phosphatase inhibitors.The protein lysates were separated by SDS-PAGE and then transferred to PVDF membranes.The membranes were blocked in 5% milk in TBST buffer (TBS with 0.1% Tween 20), and were incubated with primary antibodies against TMPRSS2 (Santa Cruz), ACE2 (Genetex), or β-actin (Sigma-Aldrich), at 4 ºC for overnight followed by the incubation with HRP-conjugated second antibody at room temperature for 1 hour.After wash with TBST buffer, the immunoreactive signals were visualized by using enhanced chemiluminescence with ECL reagent.

Statistical analysis
Data are shown as the mean ± standard error of the mean (SEM

Figures Figure 1
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Figure 2 The
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Figure 3 The
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Figure 5 The
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Table 2
The binding energy and interaction sites of SARS-CoV-2-related proteins with eriodictyol and umbelliferone.
* Compound interacts to S protein after receptor engagement.** Compound interacts to ACE2 after receptor engagement.* Compound interacts to S protein after receptor engagement.** Compound interacts to ACE2 after receptor engagement.

Table 1 is
available in the Supplementary Files section.