Screening of Natural Products Inhibitors of SARS-CoV-2 Entry

The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC50) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC50) of 91.65 µg/mL, a CC50 of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors.


Introduction
Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and is still a major global health problem [1]. SARS-CoV-2 is a beta-coronavirus that emerged in late 2019. It is related to the human pathogens severe acute respiratory syndrome coronavirus-1 (SARS-CoV) and to the Middle East respiratory syndrome coronavirus (MERS-CoV), which previously appeared in 2002 and 2012, respectively [2,3]. During the COVID-19 pandemic, SARS-CoV-2 vaccines have been developed, and millions of people have been immunized [4]. However, COVID-19 continues to be a health problem, and in addition to the use of vaccines, antivirals are necessary for the effective treatment of acute SARS-CoV-2 infections [5].
Despite there being several antivirals in different stages of development, there are no approved antivirals that inhibit the entry of the virus into the cell [6,7]. Therefore, new effective entry inhibitors are needed for the better management of COVID-19. Viral entry Molecules 2022, 27, 1743 2 of 10 into the host cell is one of the most critical steps in the viral cycle. This process is mediated by the interaction between the SARS-CoV-2 spike glycoprotein and the cellular receptor angiotensin-converting enzyme 2 (ACE-2) [8]. Thus, antivirals that inhibit the viral entry process have important therapeutic potential [9].
Components of natural origin, such as products derived from plants and microorganisms, can be a source of antiviral compounds. Some natural products have shown activity against other coronaviruses, such as SARS-CoV [10]. Additionally, over 85% of patients infected with SARS-CoV-2 in China have received herbal formulae from traditional Chinese medicine along with conventional treatment [11].
In addition, bioinformatics studies such as molecular docking have identified natural products with potential antiviral activity against SARS-CoV-2 by blocking the interaction between the spike protein and the ACE2 receptor. Some of the compounds identified by this strategy are artemisinin and gamma mangostin [12,13]. It is important to note that experimental validation of these data based on bioinformatics models is necessary.
SARS-CoV-2 is highly infective, generates severe clinical cases, and is classified as an aerosol biosafety level (BSL)-3 pathogen [1]. In this situation, the use of pseudotyped viruses represents a robust, fast, and safe alternative to the evaluation of entry inhibitor antivirals in a BSL-2 environment. Due to its versatility, this system has been used for screening entry inhibitors for other viruses [14]. Pseudotyped viral systems have been a valuable tool during the COVID-19 pandemic, especially for the determination of virusspecific neutralizing antibodies [15]. In addition, using this strategy, already-approved drugs were identified as SARS-CoV-2 entry inhibitors [16].
In this work, we screened a library of 41 natural products, including plant extracts, essential oils, and compounds from plants and microorganisms, using an HIV-1-based SARS-CoV-2 pseudotype to identify products with viral entry inhibitory activity. We found that the extract of Stachytarpheta cayennensis and β-caryophyllene were able to inhibit SARS-CoV-2 entry into cells. Moreover, our work indicates the usefulness and practicality of this pseudotyped virus in the screening of SARS-CoV-2 entry inhibitors.
In silico analyses have suggested that some essential oil components may be binders of SARS-CoV-2 spike or ACE2 [17]; for that reason, we decided to evaluate the inhibitory effect of essential oils. However, in our experimental conditions, we did not identify essential oils with antiviral activity against SARS-CoV-2. In contrast, some essential oils were found to increase the infectivity of the pseudotyped virus ( Table 2).
Since the inhibitory effect of pseudovirus infection can be caused at different stages of infection and not only on the interaction of spike with ACE2, we evaluated the entry inhibition of the best candidates by using a pseudotyped virus carrying the vesicular stomatitis virus glycoprotein G (VSV-G) instead of the spike protein. As shown in Figure 1, Stachytarpheta cayennensis extract and β-caryophyllene showed low inhibitory activity against the entry of the VSV-G-pseudotyped virus, showing a specific effect towards the SARS-CoV-2 spike-pseudotyped virus. In the case of Phoradendron liga, cytotoxicity was detected in the presence of the VSV-G-pseudotyped virus at 500 µg/mL of the extract; for this reason, the analysis was performed using 250 µg/mL of extract. A greater entry inhibition of the VSV-G-pseudotyped virus (72.46%) compared to the spike-pseudotyped virus (51.89%) was observed, indicating non-specific inhibition.    HEK-293T ACE2 cells were infected with the corresponding spike-or VSV-G-pseudotyped virus in the presence and absence of different natural products. After 24 h.p.i., the luciferase activity was measured. The % of inhibition was determined as the ratio between treated and untreated cells.
Given that some of the essential oils tested showed an increase in infectivity by the spike-pseudotyped virus, the specificity of infection was also evaluated using the VSV-G-pseudotyped virus. As seen in Figure 2, eucalyptus essential oil increased infectivity only with the spike-pseudotyped virus, indicating that this essential oil could facilitate virus entry mediated by the SARS-CoV-2 spike protein. These results should be confirmed using the whole SARS-CoV-2 virus. Recently, molecular docking analysis has suggested that compounds present in the eucalyptus essential oil could inhibit the SARS-CoV-2 MPro protease. For that reason, it has been proposed as a possible alternative for the treatment of COVID-19 [18]. Our results suggest that caution should be used with the use of eucalyptus essential oil in COVID-19 therapy. HEK-293T ACE2 cells were infected with the corresponding spike-or VSV-G-pseudotyped virus in the presence and absence of different natural products. After 24 h.p.i., the luciferase activity was measured. The % of inhibition was determined as the ratio between treated and untreated cells. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 2.
Increased infectivity of pseudotyped virus by essential oils. HEK-293T ACE2 cells were infected with spike-or VSV-G-pseudotyped virus in the presence and absence of essential oils. After 24 h.p.i., luciferase activity was measured. The % of infection was determined as the ratio between the treated and untreated cells. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
contributes to demonstrating the potential of pseudotyped viruses as a tool for the screening of a large number of natural products that inhibit the entry of SARS-CoV-2 into the cell.

Cell cultures and plasmids
HEK293T and stable HEK293T-ACE2-expressing cells [15] were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), antibiotics (Sigma-Aldrich, Saint Louis, MO, USA ), and non-essential amino acids (Gibco) at 37 °C and 5% CO2 atmosphere. HEK293-ACE2 cells also contained puromycin at a final concentration of 1 µg/mL. The plasmids used were as follows: pNL4.3-ΔEnv-FLuc, spike-G614-Δ19 [15], and pCMV-VSV-G. contributes to demonstrating the potential of pseudotyped viruses as a tool for the screening of a large number of natural products that inhibit the entry of SARS-CoV-2 into the cell.

Cell cultures and plasmids
HEK293T and stable HEK293T-ACE2-expressing cells [15] were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), antibiotics (Sigma-Aldrich, Saint Louis, MO, USA ), and non-essential amino acids (Gibco) at 37 °C and 5% CO2 atmosphere. HEK293-ACE2 cells also contained puromycin at a final concentration of 1 µg/mL. The plasmids used were as follows: pNL4.3-ΔEnv-FLuc, spike-G614-Δ19 [15], and pCMV-VSV-G. Members of the genus Stachytarpheta have been associated with different biological activities, such as analgesic, hypotensive, antacid, antihelminthic, diuretic, laxative, lactagogue, purgative, sedative, spasmogenic, vasodilator, vulnerary, and vermifuge properties [25]. Recently, the popular use of Stachytarpheta jamaicensis (L.) Vahl and Stachytarpheta cayennensis (Rich.) Vahl has been reported for the treatment of COVID-19 in Jamaica [26]. Notably, both species showed low toxicity in laboratory animals [25,27]. Furthermore, Stachytarpheta cayennensis and Stachytarpheta jamaicensis have been used in traditional medicine to treat inflammation, pain, and fever [25]. The anti-inflammatory activity of both species has been identified in rat and mouse inflammation models [28,29]. Recent therapeutic approaches for COVID-19 patients recommend the use of anti-inflammatories in combination with antivirals [30]. Since Stachytarpheta cayennensis extract has anti-inflammatory activity in addition to antiviral activity, it could also be used in the inflammatory stages of COVID-19. In conclusion, our data, together with the aforementioned results, indicate that the Stachytarpheta cayennensis extract has potential as an inhibitor of SARS-CoV-2 entry and as a therapeutic agent for COVID-19. However, clinical studies are needed to validate its therapeutic use.
Although there are not many reports of the chemical composition of Stachytarpheta cayennensis, Stachytarpheta jamaicensis has been reported to be rich in secondary metabolites, showing the presence of several major groups of secondary metabolites, such as alkaloids, flavonoids, phenols, steroids, and terpenoids [25]. Coumarins, flavonoids, tannins, and saponins have been reported among the phenolic compounds present [25]. However, β-caryophyllene, thymol, citral, 1,8-cineole, carvone, and limonene were identified in the essential oil of Stachytarpheta cayennensis. Although we identified the SARS-CoV-2 entry inhibitory activity of β-caryophyllene, it is not primarily responsible for the effect observed with the Stachytarpheta cayennensis extract, since the extract presented greater activity than β-caryophyllene. It is important to note that there may be other compounds that act synergistically with β-caryophyllene. Molecular docking studies have indicated that thymol, carvone, and limonene can interact with ACE2 and SARS-CoV-2 spike [17]. However, the inhibitory activity of these compounds needs to be confirmed by in vitro or in vivo assays. It is noteworthy that we did not observe inhibitory activity for limonene (Table 3).
Limited reports are available on the use of pseudotyped viruses for the screening of natural products that inhibit SARS-CoV-2 entry. At the beginning of the pandemic, pseudotyped viruses with the spike protein of MERS and SARS-CoV were used to search for compounds that could inhibit SARS-CoV-2 [31]. Recently, it was reported that the extract of Punella vulgaris inhibited the entry of SARS-CoV-2 when using a pseudotyped virus with SARS-CoV-2 spike [32], similar to the system used in this work. Our study also contributes to demonstrating the potential of pseudotyped viruses as a tool for the screening of a large number of natural products that inhibit the entry of SARS-CoV-2 into the cell.
The methanolic plant extracts, essential oils, and compounds (Tables 1-3) were diluted in DMSO, except for the compound guaiol, which was diluted in methanol.

Cytotoxicity Assay
The maximum non-toxic concentration was assessed using the resazurin method. Briefly, HEK293T-ACE2 cells were cultured at 1 × 10 4 cells/well in a 96-well plate in the presence of different concentrations of the natural products. Vehicle (DMSO)-treated cells were used as a control and normalizer. Resazurin was added 48 h later, and after 3 h, cell viability was determined. Absorbance was measured on the MultiskanTM GO (ThermoScientific, Waltham, MA, USA) at 570 and 630 nm. The MNTC is the maximum concentration with a cytotoxicity lower than 10%.

HIV-1-Based SARS-CoV-2 Pseudotyped Particles
Pseudotyped viral particles were generated by cotransfection of pNL4.3-∆Env-FLuc and spike-G614-∆19 plasmids (molar ratio, 3:2). HEK293T cells were transfected using the calcium phosphate method [39]. Briefly, 3.2 × 10 6 cells in a 10 cm plate were transfected with 20 µg of the plasmid mix, 2.5 M calcium chloride, and HEPES-buffered saline 2X (0.28 M NaCl, 0.05 M HEPES, 1.5 mM Na 2 HPO 4 , pH 7.05). The mixture was incubated for 20 min at room temperature and added to the cells. Sixteen hours after transfection, cells were washed, and fresh medium was added. The pseudotyped virus-containing supernatant was collected 48 h after transfection and centrifuged at 3500 rpm for 5 min at room temperature. Viral stocks were aliquoted and stored at −80 • C. Stocks were diluted, HEK293T and HEK293-ACE2 cells were transduced, and firefly luciferase activity was measured 48 hours later using the Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA) and Fluoroskan FL (Thermo Scientific). HEK293T cells transduced with the pseudotyped particles were used as a negative control.

Antiviral Activity
The assay was performed using the maximum non-toxic concentration (MNTC) of each natural product in HEK293-ACE2 cells. A total of 1 × 10 4 cells in suspension were added to each well of 96-well plates. The mix (50% natural product dilution and 50% virus dilution) was added to the cells. Firefly luciferase activity was measured 48 h later using Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA) and Fluoroskan FL (Thermo Scientific). HEK293T-ACE2 cells transduced with the pseudotyped virus without any natural products were used as control untreated cells. The following formula was used to calculate the percentage of inhibition: 100 − (RLUs of treated cells/RLUs of control untreated cells) × 100. In all assays, RLUs of untreated pseudotyped-virustransduced HEK293T-ACE2 cells were at least 10-fold over the RLUs of untransduced cells. The selectivity index (SI) was determined as the ratio of CC 50 versus IC 50 .

Statistical Analysis
Data are presented as mean ± standard deviation (SD) of at least 3 independent experiments. Differences within each group were subjected to the t-test. Statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.001) are indicated by lines between the groups being compared.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.