key: cord-277253-vy0mvzeb
authors: Liu, Hongbo; Ye, Fei; Sun, Qi; Liang, Hao; Li, Chunmei; Lu, Roujian; Huang, Baoying; Tan, Wenjie; Lai, Luhua
title: Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro
date: 2020-04-11
journal: bioRxiv
DOI: 10.1101/2020.04.10.035824
sha: 
doc_id: 277253
cord_uid: vy0mvzeb

COVID-19 has become a global pandemic that threatens millions of people worldwide. There is an urgent call for developing effective drugs against the virus (SARS-CoV-2) causing this disease. The main protease of SARS-CoV-2, 3C-like protease (3CLpro), is highly conserved across coronaviruses and is essential for the maturation process of viral polyprotein. Scutellariae radix (Huangqin in Chinese), the root of Scutellaria baicalensis has been widely used in traditional Chinese medicine to treat viral infection related symptoms. The extracts of S. baicalensis have exhibited broad spectrum antiviral activities. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredient compounds. We found that the ethanol extract of S. baicalensis inhibits SARS-CoV-2 3CLpro activity in vitro and the replication of SARS-CoV-2 in Vero cells with an EC50 of 0.74 μg/ml. Among the major components of S. baicalensis, baicalein strongly inhibits SARS-CoV-2 3CLpro activity with an IC50 of 0.39 μM. We further identified four baicalein analogue compounds from other herbs that inhibit SARS-CoV-2 3CLpro activity at microM concentration. Our study demonstrates that the extract of S. baicalensis has effective anti-SARS-CoV-2 activity and baicalein and analogue compounds are strong SARS-CoV-2 3CLpro inhibitors.

Coronaviruses (CoVs) are single stranded positive-sense RNA viruses that cause severe infections in respiratory, hepatic and various organs in humans and many other animals [1, 2] . Within the 20 years of the 21st century, there are already three outbreaks of CoV-causing global epidemics, including SARS, MERS, and COVID-19. The newly emerged CoV infectious disease (COVID-19) already caused more than 1.5 million confirmed infections and 89 thousands deaths worldwide up to April 9, 2020 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports).

There is an urgent call for drug and vaccine research and development against COVID-19.

COVID-19 was confirmed to be caused by a new coronavirus (SARS-CoV-2), whose genome was sequenced in early January 2020 [3, 4] . The genomic sequence of SARS-CoV-2 is highly similar to that of SARS-CoV with 79.6% sequence identity [5] and remain stable up to now [6] . However, the sequence identities vary significantly for different viral proteins [7] . For instance, the spike proteins (S-protein) in CoVs are diverse in sequences and even in the host receptors that bind due to the rapid mutations and recombination [8] . Although it has been confirmed that both SARS-CoV and SARS-CoV-2 use ACE2 as receptor and occupy the same binding site, their binding affinities to ACE2 vary due to subtle interface sequence variations [9] . On the contrary, the 3Clike proteases (3CL pro ) in CoVs are highly conserved. The 3CL pro in SARS-CoV and SARS-CoV-2 share a sequence identity of 96.1 %, making it an ideal target for broad spectrum anti-CoV therapy.

Although many inhibitors have been reported for SARS-CoV and MERS-CoV 3CL pro [10] [11] [12] [13] , unfortunately none of them has entered into clinical trial. Inspired by the previous studies, several covalent inhibitors were experimentally identified to inhibit the 3CL pro activity and viral replication of SARS-CoV-2, and some of the complex crystal structures were solved [14, 15] . In addition, a number of clinically used HIV and HCV protease inhibitors have been proposed as possible cure for COVID-19 [16] and some of them are now processed to clinically trials [17] . Several computational studies proposed potential SARS-CoV-2 3CL pro inhibitors by virtual screening against the crystal or modeled three-dimensional structure of SARS-CoV-2 3CL pro as well as machine intelligence [18] [19] [20] [21] [22] [23] . Highly potent SARS-CoV-2 3CL pro inhibitors with diverse chemical structures need further exploration.

Traditional Chinese medicine (TCM) herbs and formulae have long been used in treating viral diseases. Some of them have been clinically tested to treat COVID-19 [24] . anti-microbial and anti-inflammatory activities have been reported [27] . Remarkably, the extracts of S. baicalensis have exhibited broad spectrum anti-viral activities, including ZIKA [28] , H1N1 [29] , HIV [30] and DENV [31] . In addition, a multicenter, retrospective analysis demonstrated that S. baicaleinsis exhibits more potent antiviral effects and higher clinical efficacy than ribavirin for the treatment of hand, foot and mouth disease [32] . Several S. baicalensis derived mixtures or pure compounds have been approved as antiviral drugs, such as Baicalein capsule (to treat hepatitis) and

Huangqin tablet (to treat upper respiratory infection) in China. Most of the S. baicaleinsis ingredients are flavonoids [33] . Flavonoids from other plants were also reported to mildly inhibit SARS and MERS-CoV 3CL pro [34, 35] . Here we studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis inhibits SARS-CoV-2 3CL pro activity and the most active ingredient baicalein exhibits an IC50 of 0.39 M. In addition, the ethanol extract of S. baicalensis effectively inhibits the replication of SARS-CoV-2 in cell assay. We also identified four baicalein analogue compounds from other herbs that inhibit SARS-CoV-2 3CL pro activity at microM concentration.

We prepared the 70% ethanol extract of S. baicalensis and tested its inhibitory activity against SARS-CoV-2 3CL pro . We expressed SARS-CoV-2 3CL pro and performed activity assay using a peptide substrate (Thr-Ser-Ala-Val-Leu-Gln-pNA) according to the published procedure of SARS-CoV 3CL pro assay [11, 36] . The inhibitory ratio of S. baicalensis extract at different concentrations on SARS-CoV-2 3CL pro activity were 6 shown in Figure 1A . The crude extract exhibits significant inhibitory effect with an IC50 of 8.5 g/ml, suggesting that S. baicalensis contains candidate inhibitory ingredients against SARS-CoV-2 3CL pro . 

We tested the inhibitory activity of four major ingredients from S. baicalensis: baicalein, baicalin, wogonin and wogonoside in vitro. Baicalein showed the most potent anti-SARS-CoV-2 3CL pro activity with an IC50 of 0.39 M ( Figure 1B and Table 1 ). Baicalin inhibited SARS-CoV-2 3CL pro activity for about 41% at 50 μM, while wogonin and wogonoside were not active at this concentration.

A B We performed molecular docking to understand the inhibitory activity of S. baicalensis ingredients. In the docking model, baicalein binds well in the substrate binding site of SARS-CoV-2 3CL pro with its 6-OH and 7-OH forming hydrogen bond interactions with the carbonyl group of L141 and the backbone amide group of G143, respectively ( Figure 2A ). In addition, the carbonyl group of baicalein is hydrogen bonded with the backbone amide group of E166. The catalytic residues H41 and C145 are well covered by baicalein, accounting for its inhibitory effect. As the 7-OH in baicalin is in close contact with the protein, there may not be enough space for glycosyl modification, explaining the low activity of baicalin. As for wogonin, the absence of 6-OH together with its additional 8-methoxyl group alters the binding orientation and weakens the binding strength ( Figure 2B ). Hydrogen bond is observed between its 5-OH and the backbone carbonyl group of L141, while the interaction with E166 by its 8-methoxy group is weaker than that formed by the carbonyl group in baicalein. 

We searched for baicalein analogues from available flavonoid suppliers and selected 8 flavonoids and 2 glycosides for experimental testing. Four flavonoid compounds were found to be potent SARS-CoV-2 3CL pro inhibitors. Among them, scutellarein is mainly distributed in genus Scutellaria and Erigerontis herba (Dengzhanxixin or Dengzhanhua in Chinese) in its glucuronide form, scutellarin. Scutellarin has long been used in cardiovascular disease treatment for its ability to improve cerebral blood supply [37] . For all the active flavonoid compounds that we found, the introduction of glycosyl group, as in the case of baicalein and baicalin, decreased the inhibition activity, probably due to the steric hindrance of the glycosyl group, which is also true for scutellarein/scutellarin, and myricetin/myricetrin. As glycosides and their A B

C D corresponding aglycones are often interchangeable in vivo, for instance, baicalin was reported to be metabolized to baicalein in intestine [39] , while baicalein can be transformed to baicalin by hepatic metabolism [40] , we expect that both the flavonoid form of the active compounds and their glycoside form will function in vivo. We suggest that these compounds can be further optimized or used to search for other TCM herbs containing these compounds or substructures for the treatment of COVID-19. Targetmol.

The DNA of SARS-CoV-2 3CL pro (referred to GenBank, accession number MN908947) was synthesized (Hienzyme Biotech) and amplified by PCR using primers n3CLP-Nhe (5'-CATGGCTAGCGGTTTTAGAAAAATGGCATTCCC-3') and n3CLP-Xho (5'-CACTCTCGAGTTGGAAAGTAACACCTGAGC-3'). The PCR product was digested with Nhe I/Xho I and cloned into the pET 21a DNA as reported previously [41] . The resulting SARS-CoV-2 pET 3CL-21x plasmid encodes a 35 064 Da SARS-CoV-2 3CL pro with a C-terminal 6xHis-tag. The SARS-CoV-2 pET 3CL-21x plasmid was further transformed to E. coli BL21<DE3> for protein expression as reported [41] . The recombinant protein was purified through a nickelnitrilotriacetic acid column (GE Healthcare) and subsequently loaded on a gel filtration column Sephacryl S-200 HR (GE Healthcare) for further purification as previously described [42] .

A colorimetric substrate Thr-Ser-Ala-Val-Leu-Gln-pNA (GL Biochemistry Ltd) and assay buffer ( 

The structure of SARS-CoV-2 3CL pro (PDB ID 6LU7) [14] and S. baicalensis concentrations for 48h. The supernatant was collected and the RNA was extracted and analyzed by relative quantification using RT-PCR as in the previous study [3, 43] .

Viral RNA was extracted from 100 μL supernatant of infected cells using the automated nucleic acid extraction system (TIANLONG, China), following the manufacturer's recommendations. SARS-COV-2 virus detection was performed using the One Step system (Roche, Rotkreuz, Switzerland). ORF 1ab was amplified from cDNA and cloned into MS2-nCoV-ORF1ab and used as the plasmid standard after its identity was confirmed by sequencing. A standard curve was generated by determination of copy numbers from serially dilutions (10 3 -10 9 copies) of plasmid. The following primers used for quantitative PCR were 1ab-F: 5ʹ-AGAAGATTGGTTAGATGATGATAGT-3ʹ;

1ab-R: 5ʹ-TTCCATCTCTAATTGAGGTTGAACC-3ʹ; and probe 5ʹ-FAM-TCCTCACTGCCGTCTTGTTG ACCA-BHQ1-3ʹ. The individual EC50 values were calculated by the Origin 2018 software.

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