Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity

The SARS-CoV-2 protease (3CLpro) is one of the key targets for the development of efficacious drugs for COVID-19 treatment due to its essential role in the life cycle of the virus and exhibits high conservation among coronaviruses. Recent studies have shown that flavonoids, which are small natural molecules, have antiviral activity against coronaviruses (CoVs), including SARS-CoV-2. In this study, we identified the docking sites and binding affinity of several natural compounds, similar to flavonoids, and investigated their inhibitory activity towards 3CLpro enzymatic activity. The selected compounds were then tested in vitro for their cytotoxicity, for antiviral activity against SARS-CoV-2, and the replication of other coronaviruses in different cell lines. Our results showed that Baicalein (100 μg/mL) exerted strong 3CLpro activity inhibition (>90%), whereas Hispidulin and Morin displayed partial inhibition. Moreover, Baicalein, up to 25 μg/mL, hindered >50% of SARS-CoV-2 replication in Vero E6 cultures. Lastly, Baicalein displayed antiviral activity against alphacoronavirus (Feline-CoV) and betacoronavirus (Bovine-CoV and HCoV-OC43) in the cell lines. Our study confirmed the antiviral activity of Baicalein against SARS-CoV-2 and demonstrated clear evidence of its pan-coronaviral activity.


Introduction
The pandemic threat, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still affecting people worldwide and viral variants contribute to the periodic rise in COVID-19 cases. Despite the development of multiple SARS-CoV-2 vaccines and the good degree of immunological protection achieved through vaccination, the mutation of newer variants and the uncertainty about how these detected mutations affect the efficacy of existing anti-spike vaccines have focused research on the development of efficacious drugs for COVID-19 therapeutic intervention.

Chemistry
All the tested compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) (Table 1) are known structures belonging to the inhouse library of natural products available from the Organic Chemistry Laboratory of the Department of Chemistry and Technology of Drugs of Sapienza University of Rome, Italy [21]. The chemical identity of the compounds was assessed by re-running NMR experiments and proved to be in agreement with the literature data reported below for each compound. The purity of all the compounds, checked by reversed-phase High-Performance Liquid Chromatography (HPLC), was always higher than 95%.

Chemistry
All the tested compounds (1-12) ( Table 1) are known structures belonging to the inhouse library of natural products available from the Organic Chemistry Laboratory of the Department of Chemistry and Technology of Drugs of Sapienza University of Rome, Italy [21]. The chemical identity of the compounds was assessed by re-running NMR experiments and proved to be in agreement with the literature data reported below for each compound. The purity of all the compounds, checked by reversed-phase High-Performance Liquid Chromatography (HPLC), was always higher than 95%.  [25] 302. 24 C 15 H 10 O 7 Moriaceae family [22] 2 Quercetin

Chemistry
All the tested compounds (1-12) ( Table 1) are known structures belonging to the inhouse library of natural products available from the Organic Chemistry Laboratory of the Department of Chemistry and Technology of Drugs of Sapienza University of Rome, Italy [21]. The chemical identity of the compounds was assessed by re-running NMR experiments and proved to be in agreement with the literature data reported below for each compound. The purity of all the compounds, checked by reversed-phase High-Performance Liquid Chromatography (HPLC), was always higher than 95%.

In Silico Screening of Natural Compounds
The crystallographic structure of SARS-CoV-2 3CLpro in complex with a small molecular fragment at 1.95 Å resolution and coded by PDB-ID: 5R81 was selected [50]. In order to relax possible structural constraints, 500 ns of MD simulations were run with AMBER18 using the ff14SB force field for the protein and GAFF for the small molecule. An already validated protocol for classical MD simulations was used [51][52][53]. A representative structure was extracted from the MD trajectory using a cluster analysis, and it was used as a rigid receptor in the subsequent structure-based virtual screening of a proprietary library of natural products [21]. Virtual screening was carried out with the FRED docking program, version 3.3.0.3 (OpenEye Scientific Software) [54,55], in a binding site of 390 Å 3 that was centered on the binding pose of the crystallographic ligand. For virtual screening purposes, 3D conformers of natural products included in the proprietary library were generated from SMILES using the OMEGA software version 3.1.0.3 (OpenEye Scientific Software) [56], whereas the protonation state at a pH of 7.4 was assigned using QUACPAC version 2.0.0.3 (OpenEye Scientific Software) [57]. Ligand energy minimization was carried out using SZYBKI, version 1.10.0.3 (OpenEye Scientific Software) [58], using the MMFF94S force field.

In Silico Screening of Natural Compounds
The crystallographic structure of SARS-CoV-2 3CLpro in complex with a small molecular fragment at 1.95 Å resolution and coded by PDB-ID: 5R81 was selected [50]. In order to relax possible structural constraints, 500 ns of MD simulations were run with AMBER18 using the ff14SB force field for the protein and GAFF for the small molecule. An already validated protocol for classical MD simulations was used [51][52][53]. A representative structure was extracted from the MD trajectory using a cluster analysis, and it was used as a rigid receptor in the subsequent structure-based virtual screening of a proprietary library of natural products [21]. Virtual screening was carried out with the FRED docking program, version 3.3.0.3 (OpenEye Scientific Software) [54,55], in a binding site of 390 Å 3 that was centered on the binding pose of the crystallographic ligand. For virtual screening purposes, 3D conformers of natural products included in the proprietary library were generated from SMILES using the OMEGA software version 3.1.0.3 (OpenEye Scientific Software) [56], whereas the protonation state at a pH of 7.4 was assigned using QUACPAC version 2.0.0.3 (OpenEye Scientific Software) [57]. Ligand energy minimization was carried out using SZYBKI, version 1.10.0.3 (OpenEye Scientific Software) [58], using the MMFF94S force field.

SARS-CoV-2 3CLpro Assay
In order to examine the compounds with structural analogies to active flavonoids 7,8-Hydroxyflavone and Luteolin for their inhibitory activity against SARS-CoV-2, a preliminary screening using an enzymatic assay [3CLpro, Untagged SARS-CoV-2 Kit, AMSBio (Abingdon, UK) was performed. We tested the compounds at different concentrations and the control inhibitor compounds were incubated with 3CLpro for 30 min, following the manufacturer's instructions. Subsequently, the substrate of protease was added, and after 4 h of incubation, the fluorescence intensity following the cleavage of the enzyme substrate was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The "blank" value (fluorescence intensity of the substrate in an assay buffer) was subtracted from each reading and the percentage of inhibition shown by each compound was calculated with respect to untreated 3CLpro activity (0%) and to the control inhibitor compound (100%). All the conditions were performed in triplicate.

Cell Viability Assay to Determine Compounds' Toxicity
Cell viability was assessed using the Cell Proliferation kit II (XTT) (Roche Diagnostics, Merck, Darmstadt, Germany) as previously described [59]. In this assay, the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) was cleaved by viable cells in order to form an orange formazan dye that is then quantified photometrically at 450 nm. Briefly, the cells (4 × 10 5 cells/mL) were cultured in 96-well plates for 24 h. The culture medium was then replaced by medium containing serial dilutions of the 3CLpro inhibitors, and the cells were incubated for 24, 48, 72, and 96 h. Then, XTT was added to each well and the plates were incubated for 2 h. Optical density was measured at 450 nm (reference wavelength-650 nm) using a Multiskan GO plate reader (Thermo Scientific Instruments, Waltham, MA, USA). For the quantifications, the background levels of media without cultured cells were subtracted.

SARS-CoV-2 Titration
Virus stocks were titrated using the Endpoint Dilutions Assay (EDA, TCID 50 /mL), as described [59]. Briefly, Vero E6 cells (4 × 10 5 cells/mL) were seeded into 96 wells plates and infected with base 10 dilutions of the virus stock. After 1 h of adsorption at 37 • C, the cell-free virus was removed, and complete medium was added to the cells after a PBS wash. After 72 h, the cells were observed to evaluate CPE. The Reed and Muench method was used to calculate the 50% endpoint using serial dilutions.

Feline-CoV, Bovine-CoV, and HCoV-OC43 In Vitro Assays and Titration
HRT-18 (CCL-244; ATCC) and CRFK (CCL-94; ATCC) cells were cultured in DMEM supplemented with NEAA, P/S, Hepes buffer, and 10% FBS. A clinical isolate of F-CoV (kind gift from Prof. Buonavoglia, University of Bari) was obtained and propagated in CRFK cells, whereas B-CoV (kind gift from Prof. Buonavoglia, University of Bari) was isolated and propagated in HRT-18 cells. HCoV-OC43 was a kind gift from Prof. Baldanti (S. Matteo Hospital, Pavia). A titer of each CoV was measured using RT/Real-Time PCR assays, as previously described [60]. Briefly, the following primers and probes were used [60]

Coronaviruses In Vitro Assays
The cells (4 × 10 5 cells/mL) were seeded into 96-well plates 24 h prior to the experiment. The cells were pretreated with different concentrations of selected 3CLpro inhibitors (1:2 serial dilutions) for 1 h at 37 • C, and were then infected for 1 h with each CoV (0.1 multiplicity of infection, MOI) in the presence of each compound. After a PBS wash to remove cell-free virus particles, 3CLpro-inhibitor-containing medium with 2% FBS was added to the cells and was maintained until the end of the experiment [48 h post-infection (hpi) for F-CoV (CRFK), 72 hpi for SARS-CoV-2 (Vero E6), and 96 hpi for B-CoV (HRT-18) and HCoV-OC43 (HRT- 18)]. Then, the SARS-CoV-2 cytopathic effect (CPE) was assessed using a scoring system (0 = uninfected; 0.5 to 2.5 = increasing number/area of plaques; 3 = all the cells are infected), as described [59]. The infection control (score 3) was set as 100% infection and the uninfected cells (score 0) were set as 0% infection. The whole surface of the wells was considered for the analysis (5× magnification). F-CoV, B-CoV, and HCoV-OC43 titers were evaluated through RT/Real-Time PCR and absolute quantifications were calculated using a standard curve [60]. All the conditions were tested in triplicate.

Statistical Analysis
A two-way ANOVA and Dunnett's multiple comparisons test were performed for cell viability assessment. The CPE observed was normalized to corresponding SARS-CoV-2 infection control. Then, a two-way ANOVA and Tukey's multiple comparisons test was performed for the evaluation of CPE scoring results (GraphPad Prism 8). The antiviral activity of the compounds tested against F-CoV, B-CoV, and HCoV-OC43 were calculated as the percentage of the reduction of viral RNA copies between untreated infected cells and treated infected cells. A p-value below 0.05 was considered significant.

Virtual Screening of the in-House Natural Products Library
The natural products included in the in-house library (available at Sapienza University of Rome in the research group of Prof. Bruno Botta [21]) were prepared for virtual screening through (i) the generation of 3D conformers, (ii) ionization at a pH of 7.4, and (iii) energy minimization in water solvent with the MMFF94S force field. The crystallographic structure of SARS-CoV-2 3CLpro in complex with the non-covalent inhibitor Z1367324110 (PDB ID: 5R81) was selected as a receptor in structure-based virtual screening [49]. To remove any possible structural bias from protein crystallization, molecular dynamics (MD) simulations were carried out on the 3CL/Z1367324110 crystallographic complex using a previously validated protocol. After 500 ns of unrestrained production of the MD trajectories, the MD frames were clustered using a hierarchical agglomerative algorithm, and the centroid frame of the most populated cluster was selected as representative structure for subsequent structure-based virtual screening.

SARS-CoV-2 3CLpro Assay
We evaluated the inhibitory activity of compounds 1-12 against the 3CLpro of SARS-CoV-2 in on-plate enzymatic assays at the following concentrations: 100 µg/mL and serial 1:2 dilutions reaching a final concentration of 0.78 µg/mL. We observed substantial inhibitory activity of Baicalein (94.72%) at the concentration of 100 µg/mL (Table 2), whereas for Hispidulin and Morin, partial inhibition of the protease activity was observed at 100 µg/mL (55.62% and 53.64%, respectively) ( Table 2). Isokaempferide exhibited a detectable effect on 3CLpro when used at concentrations ranging from 100 µg/mL to 8 of 18 25 µg/mL, leading to a percentage of reduction of its activity of 49.98%, 40.06%, and 19.69%, respectively. We also examined the inhibitor effects of Luteolin and 7,8-dihydroxyflavone, for which antiviral activity has been previously reported [20,61], finding low activity against 3CLpro at 100 µg/mL (38.12% and 39.80%, respectively), and undetectable activity at 25 µg/mL (Table 2). By contrast, no activity against 3CLpro was observed for Taxifolin, Steppogenin, Quercetin, Chrysin, Sakuranetin, Alnusin, Galangin, and Isosakuranetin when used at concentrations ranging from 100 µg/mL to 6.125 µg/mL (data not shown). Given the Morin, Baicalein, Hispidulin, and Isokaempferide had detectable activity against SARS-CoV-2 3CLpro, we investigated the efficacy of these compounds in the impairment of SARS-CoV-2 replication on Vero E6 cells with an in vitro assay.

Effect of the 3CLpro Inhibitors on VERO E6 Viability
Vero E6 cells were tested with serial dilutions of Morin, Baicalein, Hispidulin, Isokaempferide, Luteolin, and 7,8-dihydroxyflavone in order to assess their cytotoxicity. The results showed no difference between the treated and untreated cells when Baicalein and Morin were used, even after testing higher concentrations for 72 h (Figure 1A,B). Instead, the other compounds induced some degree of cytotoxicity. In detail, Hispidulin ( Figure 1C) was found to be toxic when used at 50 µg/mL after 24 and 48 h (p < 0.0001 and p < 0.01, respectively) and at 25 µg/mL after 72 h of treatment (p < 0.01). Luteolin ( Figure 1D) impaired cell viability starting from 12.5 µg/mL at each considered time point (p < 0.001 after 48 and 72 h, p < 0.01 after 24 h), as well as Isokaempferide ( Figure 1E) starting from 50 µg/mL (p < 0.001 at all the time points), and 7,8-dihydroxy-flavone starting from 6.25 µg/mL (p < 0.0001) at all the time points. stead, the other compounds induced some degree of cytotoxicity. In detail, Hispidulin ( Figure 1C) was found to be toxic when used at 50 µg/mL after 24 and 48 h (p < 0.0001 and p < 0.01, respectively) and at 25 µg/mL after 72 h of treatment (p < 0.01). Luteolin ( Figure  1D) impaired cell viability starting from 12.5 µg/mL at each considered time point (p < 0.001 after 48 and 72 h, p < 0.01 after 24 h), as well as Isokaempferide ( Figure 1E) starting from 50 µg/mL (p < 0.001 at all the time points), and 7,8-dihydroxy-flavone starting from 6.25 µg/mL (p < 0.0001) at all the time points. Figure 1. Cell viability analysis. Different concentrations (100-0.4 µg/mL) of the selected 3CLpro inhibitors (A-F) were tested in order to assess their toxicity on Vero E6. Absorbance was reported as mean values ± SD, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Antiviral Activity of 3CLpro Inhibitors against SARS-CoV-2
Vero E6 cells were treated with serial dilutions of the selected compounds, relying on cell viability data to determine the highest concentrations to test: 100 µg/mL for Baicalein and Morin (Figure 2A,B), 25 µg/mL for Hispidulin ( Figure 2C), 10 µg/mL for Luteolin and 7,8-dihydroxy-flavone ( Figure 2D,F), and 50 µg/mL for Isokaempferide ( Figure 2E). The inhibitors were added one hour before inoculation with the SARS-CoV-2 Omicron BA.5 variant (0.1 Multiplicity of infection, MOI), and were monitored for cytopathic effects at 72 hpi. The results showed that no compound, at any concentration, could fully protect the cells from infection with the virus. Only the treatment with Baicalein hindered virus replication above 50%, starting from a concentration of 25 µg/mL. Luteolin and 7,8-dihydroxy-flavone ( Figure 2D,F), and 50 µg/mL for Isokaempferide ( Figure 2E). The inhibitors were added one hour before inoculation with the SARS-CoV-2 Omicron BA.5 variant (0.1 Multiplicity of infection, MOI), and were monitored for cytopathic effects at 72 hpi. The results showed that no compound, at any concentration, could fully protect the cells from infection with the virus. Only the treatment with Baicalein hindered virus replication above 50%, starting from a concentration of 25 µg/mL.

Effect of the 3CLpro Inhibitors on CRFK and HRT-18 Viability and Pan-Coronaviral Activity
In order to investigate possible anti-pan-coronaviral activity, we tested the efficacy of Baicalein and Luteolin against Feline-CoV (F-CoV), Bovine-CoV (B-CoV) and human-CoV (HCoV) OC43 replication.
CRFK and HRT-18 were tested with serial dilutions of Baicalein and Luteolin in order to assess their cytotoxicity. The highest concentrations with no toxic activity were 50 µg/mL for Baicalein and 3.125 µg/mL for Luteolin in both the cell lines. In order to test pan-coronavirus antiviral activity, Baicalein and Luteolin were added 1 h before inoculation with F-CoV on CRFK cells, B-CoV, and HCoV-OC43 on HRT-18 cells (0.1, Multiplicity of infection, MOI) and were monitored 48 hpi (F-CoV) and 96 hpi (B-CoV and HCoV-OC43). Only Baicalein showed antiviral activity (Table 3); it inhibited F-CoV and B-CoV at 50 µg/mL and at lower concentrations (up to 12.5 µg/mL) inhibited HCoV-OC43 replication (Table 3).

Predicted Binding Mode of Most Effective SARS-CoV-2 3CLpro Inhibitors
The possible binding mode of the bioactive compounds discussed above within the catalytic site of SARS-CoV-2 3CLpro was investigated by molecular docking simulations. Since all the docked compounds are members of the flavone family, although they bear different substitution patterns, it is not surprising that they bind within the catalytic site of SARS-CoV-2 3CLpro in a highly consistent manner to each other, as well as to the docking-based binding mode of other flavonoids from the literature [62]. Specifically, Morin, Luteolin, and the 7,8-dihydroxyflavone bind with opposite orientations with respect to Baicalein, Hispidulin, and Isokaempferide. The latter of these have the polyhydroxylated ring A of the flavone scaffold projected to the inner core of 3CLpro that is in the proximity of Asn142 (Figure 3). Besides stacking to the side chain of His41, the natural compounds establish H-bond interactions with the backbone of Asn142 (Baicalein, Isokaempferide, and Morin), with the phenolic side chain of Try54 (Luteolin), with the backbone of Gln189 (Morin and Luteolin), with the backbone of Thr190 (Morin, Hispidulin, and Luteolin), and with the side chain of Cys44. Only 7,8-dihydroxyflavone establishes an H-bond interaction with the side chain of Glu166 ( Figure 3). Notably, Baicalein and Hispidulin are able to H-bond a water molecule, which was identified by the MD simulations and is found in X-ray structures ( Figure 3A,C).
The predicted binding mode supports the inhibition of SARS-CoV-2 3CLpro by these natural compounds, as was confirmed by the experimental studies. The predicted binding mode supports the inhibition of SARS-CoV-2 3CLpro by these natural compounds, as was confirmed by the experimental studies.

Discussion
The COVID-19 pandemic has revealed the urgent need for novel antiviral drugs. Flavonoids have been largely studied as possible inhibitors of 3CLpro and several studies

Discussion
The COVID-19 pandemic has revealed the urgent need for novel antiviral drugs. Flavonoids have been largely studied as possible inhibitors of 3CLpro and several studies found them to be active against SARS-CoV and MERS-CoV proteases [19]. It has previously been shown that several of these compounds can impair HCoV-229E replication in vitro [63]. The antiviral activity of Quercetin was previously observed in vitro against HSV-1 [64] and during in vitro and in vivo Rhinovirus infection [65], and its derivates also show antiviral activity against Respiratory Syncytial Virus (RSV) [66] and Influenza A H1N1 virus replication in cell cultures [67]. During the COVID-19 pandemic, many flavonoids were examined against SARS-CoV-2 3CLpro. In particular, it has been observed that myricetin covalently binds to the Cys300 and Cys41 of 3CLpro [68].
Then, we tested these flavonoids in a 3CLpro enzymatic assay, finding four of them, Morin, Baicalein, Hispidulin, and Isokaempferide, to be active against 3CLpro. Baicalein, the most promising compound, inhibited 3CLpro activity above 90%, and Morin, Hispidulin, and Isokaempferide were partially active against SARS-CoV-2 3CLpro. The antiviral activity of Morin was observed against the Herpesviridae family [69], whereas Hispidulin has been associated with Influenza A H1N1 neuraminidase inhibition [70].
In order to assess their anti-coronaviral activity, we tested these compounds against SARS-CoV-2 replication with an in vitro assay, but only Baicalein had more than 50% activity against virus replication at concentrations that were non-toxic for Vero E6 cells. In agreement, Seri et al. [4] found Baicalin, but also Herbacetin and Pectolinarin, to be effective against SARS-CoV-2. Other studies have reported in vitro Baicalein to have antiviral activity against SARS-CoV-2 [71]. The anti-SARS-COV-2 properties of Baicalein were, in part, explained through the observation of in silico interactions between this compound and 3CLpro [15,72]. Furthermore, a crystallographic picture of this molecular interaction was obtained with SARS-CoV-1 3CLpro [73], which has 96% similarity with SARS-CoV-2 [74].
Since the emergence of SARS-CoV-1 in 2002 [75], MERS-CoV in 2012 [76], and SARS-CoV-2 in 2019 [77], research has uncovered many details of the life cycle of coronavirus and its pathogenesis; however, there are currently no approved drugs with anti-pancoronaviral activity.
Thus, we extended our observations on the anti-SARS-CoV-2 activity of Baicalein, testing its inhibitory potency against additional members of the Coronaviridae family. To our knowledge, for the first time we found that this compound is highly active against F-CoV, B-CoV, and HCoV-OC43 replication. F-CoV [78,79] and HCoV-OC43 [80] have been previously used as SARS-CoV-2 surrogates in order to test the virucidal and antiviral activity of several compounds.
Considering that F-CoV belongs to the alpha coronavirus genus, Baicalein could be active against the human alpha Cov species, i.e., HCoV-229E which usually infects the upper respiratory tract, but can cause more severe diseases in newborns and in immunocompromised individuals [81], and HCoV-NL63 which causes bronchiolitis in infants [82] and pneumonia.
Furthermore, from a translation point of view, the activity of Baicalin against HCoV-OC43 is relevant. Indeed, this HCoV causes very mild disease in immunocompetent adults [81], but it is a threat for newborns (0-1 year) causing bronchiolitis [83].
The broad-spectrum of the anti-pancoronaviral action of Baicalein against F-CoV, B-CoV, and HCoV-OC43 could be explained by the fact that 3CLpro is highly conserved within the Coronaviridae family [74]. Of note, the anti-inflammatory activity of flavonoids has also been shown, modulating the expression of inflammatory markers, such as IL1-B and Tumor Necrosis Factor Alpha (TNF-a) [84], and it can modulate the NLRP3 inflammasome [85,86]. Interestingly, IL1-B and TNF-a blood levels were associated with post-COVID-19 illness [87] and the use of flavonoid-derived compounds could mitigate post-COVID-19 symptoms.
In this context, Baicalein has been shown to interact with the NLRP3 inflammasome [86] and with Nf-kB through Nrf2 activation [88].
The limitations of this study include a lack of other HCoV models, including low (HCoV-NL63 and HKU1) and high (MERS-CoV) human pathogenetic coronavirus, but also additional animal coronaviruses (bat coronaviruses) in order to confirm the anti-pancoronaviral activity of Baicalein. The availability of a compound with proven activity against emerging coronaviruses would represent great value as coronaviruses infecting animals still represent a threat. Indeed, a novel CoV infecting pigs, named swine acute diarrhea syndrome (SADS)-CoV that recently caused an outbreak in China [89], is capable of infectibg human derived cells [90], suggesting that it might also be capable of jumping to humans. Moreover, several compounds were found to be toxic for the cells and were tested against SARS-CoV-2 in low concentrations. The latter could have underestimated the evaluation of the antiviral potency of these molecules and indicates the need to improve the synthesis processes and test new Baicalein-derived compounds against coronaviruses.
In conclusion, our study confirmed the in vitro antiviral activity of Baicalein against SARS-CoV-2 replication, but also demonstrated clear evidence of the anti-pan-coronaviral activity exerted by this compound. Given that Baicalein and the other bioactive compounds highlighted in this work belong to a very large family of natural compounds, this work paves the way for the exploration of naturally occurring flavonoids in the identification of additional and effective 3CLpro inhibitors of SARS-CoV-2 replication. Starting from our results, future investigations are warranted in order to elucidate the possible activity of Baicalein and its derivatives against other steps of the coronavirus life cycle. Furthermore, we underline the importance of using an experimental approach combining in silico and in vitro analyses in order to uncover novel antiviral drugs that are suitable for treating SARS-CoV-2 in this current pandemic or for the next emerging viral pathogen. Funding: This work was supported by a grant from Sapienza University (ATENEO H2020, PH120172-B4BA8CAF) to A.G. Funding sources were not involved in the study design; in the collection, analysis and interpretation of data; in the writing of this manuscript; and in the decision to submit the article for publication.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The datasets analysed during this study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.