key: cord-0830815-ixun0c8g
authors: Su, Haixia; Yao, Sheng; Zhao, Wenfeng; Li, Minjun; Liu, Jia; Shang, WeiJuan; Xie, Hang; Ke, Changqiang; Gao, Meina; Yu, Kunqian; Liu, Hong; Shen, Jingshan; Tang, Wei; Zhang, Leike; Zuo, Jianping; Jiang, Hualiang; Bai, Fang; Wu, Yan; Ye, Yang; Xu, Yechun
title: Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro
date: 2020-04-14
journal: bioRxiv
DOI: 10.1101/2020.04.13.038687
sha: 834c9f01bfe24ff36355dc80462859c4fb9780fc
doc_id: 830815
cord_uid: ixun0c8g

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a “shield” in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.

Traditional Chinese medicines (TCMs) have evolved over thousands of years and are an invaluable source for drug discovery and development. As a notable example, the discovery of artemisinin (Qinghaosu), which was originally isolated from the TCM Artemisia annua L.

(Qinghao), is a milestone in the treatment of malaria. TCMs as well as purified natural products also provide a rich resource for novel antiviral drug development. Several herbal medicines and natural products have shown antiviral activities against viral pathogens (8) (9) (10) (11) . Among these, the roots of Scutellaria baicalensis Georgi (Huangqin in Chinese) are frequently used in TCM for the prophylaxis and treatment of hepatitis and respiratory disorders (12) (13) (14) . In the present study, an enzymatic assay was performed to test if the ingredients isolated from S. baicalensis are inhibitors of SARS-CoV-2 3CLpro. As a result, baicalin and baicalein, two bioactive components from S. baicalensis, are identified as novel inhibitors of SARS-CoV-2 3CLpro with an antiviral activity in the SARS-CoV-2 infected cells. A crystal structure of SARS-CoV-2 3CLpro in complex with baicalein, the first non-covalent, non-peptidomimetic small-molecule inhibitor, was also determined, revealing a unique binding mode of this natural product with the protease.

The pivotal role of the 3CL protease in processing polyproteins into individual functional proteins for viral replication and a highly conserved substrate specificity of the enzyme among various CoVs make it a promising target for screening of inhibitors. A fluorescence resonance energy transfer (FRET) protease assay was applied to measure the proteolytic activity of the recombinant SARS-CoV-2 3CLpro on a fluorogenic substrate. The detail of the assay is described in the Experimental section. This FRET-based protease assay was utilized to screen natural products as novel inhibitors of SARS-CoV-2 3CLpro. It was first used to determine the inhibitory activities of the total aqueous extract, fractionations, and purified compounds from S. baicalensis against SARS-CoV-2 3CLpro (see Supplementary Materials, Fig. S1 ). As the result, two fractions from S. baicalensis showed significant inhibition on SARS-CoV-2 3CLpro at 10.0 g/mL (Table S1) . Surprisingly, baicalin, the major component in fraction 8, shows an IC50 of 6.41 M against the protease, while baicalein, the major component in fraction 12, has an IC50 of 0.94 M (Fig. S2 ; Table 1 ). Accordingly, baicalin and baicalein are identified as novel non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro with single-digit micromolar potency.

To validate the binding of baicalin and baicalein with SARS-CoV-2 3CLpro and exclude the suspicion of being the pan-assay interference compounds (PAINS) (15) , their binding affinities with the protease were measured by isothermal titration calorimetry (ITC), widely known as an invaluable tool used to determine thermodynamic parameters of protein-ligand interactions such as Kd (Fig. 1, A and B ; Table 1 ). The resulting Kd of baicalin and baicalein binding with SARS-CoV-2 3CLpro is 11.50 and 4.03 M, respectively, which has a good correlation with the IC50s mentioned above, demonstrating that specific binding of the compounds with the enzyme is responsible for their bioactivities. Moreover, the ITC profiles in combination with their chemical structures suggest that baicalin and baicalein act as noncovalent inhibitors of SARS-CoV-2 3CLpro with a high ligand binding efficiency.

Native state electrospray ionization mass spectrometry (ESI-MS) has been used extensively to directly observe native state proteins and protein complexes, allowing direct detection of protein-ligand non-covalent complexes with Kds as weak as 1 mM (16) . The determination of m/z between [protein + ligand] m/z and [unbound protein] m/z is able to identify a ligand as a binder with the correct molecular weight, while the ratio of the intensity of the [protein + ligand] peaks relative to [unbound protein] peaks provides a qualitative indication of the ligand-binding affinity. Herein, an ESI-MS analysis using high-resolution magnetic resonance mass spectrometry (MRMS) was carried out to detect the binding of baicalin and baicalein with SARS-CoV-2 3CLpro. For the free protease performance optimization, the mass range around the change stated 18+ was isolated with a center mass of the quadrupole of m/z 3750 (Fig. S3 ).

For the ligand-binding screening studies, two charge states (18+ and 19+) have been used for calculation of the free protease and protein-ligand complex intensities. The representative spectra of samples containing SARS-CoV-2 3CLpro (1 M) and baicalin (3.13 M) or baicalein (0.31 M) acquired under both optimized and screening conditions are shown in Fig.  2C and D, demonstrating a specific binding of baicalin or baicalein with the protease. Moreover, the plot of the fraction of the bound protease versus the total concentration of baicalin or baicalein obtained Kds of 12.73 and 1.40 µM for baicalin and baicalein, respectively (Fig. 1E and F), in keeping with the results from the ITC measurements.

The mode of action of baicalein and the structural determinants associated with its binding with SARS-CoV-2 3CLpro were further explored using X-ray protein crystallography. The crystal structure of SARS-CoV-2 3CLpro in complex with baicalein was determined at a resolution of 2.2 Å ( Fig. 2 ; Table S2 ). The protease has a catalytic Cys145-His41 dyad and an extended binding site, features shared by SARS-CoV 3CLpro and MERS-CoV 3CLpro ( hydrophobic interactions. Consequently, baicalein is perfectly ensconced in the core of the substrate-binding pocket and interacts with two catalytic residues, the oxyanion loop (residues 138-145), Glu166, and the S1/S2 subsites, which are the key elements for recognition of substrates as well as peptidomimetic inhibitors (17) . Although baicalein did not move deeply into the S1 sub-pocket, its phenolic hydroxyl groups did form contacts with the crucial residue of this sub-pocket, His163, via the water molecule. By the aid of an array of direct and indirect hydrogen bonds with Leu141/Gly141/Ser144, baicalein fixed the conformation of the flexible oxyanion loop, which serves to stabilize the tetrahedral transition state of the proteolytic reaction. These results together provide the molecular details of baicalein recognition by SARS-CoV-2 3CLpro and an explanation for the observed potent activity of such a small molecule against the protease.

The amino sequence of SARS-CoV-2 3CLpro displays 96% sequence identity to SARS-CoV 3CLpro. There are 12 residues differed in two proteases and none of them participates in the direct contacts with baicalein. The high level of sequence identified between two proteases allows one to assume that baicalein will bind to the SARS-CoV 3CLpro in the same way as it does to SARS-CoV-2 3CLpro. The inhibition assay shows that baicalein can also inhibit SARS-CoV 3CLpro, with an IC50 of 1.18 ± 0.37 µM. Thus, a three-dimensional model of SARS-CoV 3CLpro in complex with baicalein was constructed by superimposing the crystal structure of SARS-CoV-2 3CLpro/baicalein with that of SARS-CoV 3CLpro/TG-0204998 (PDB code 2ZU4) (Fig. S4A ). The binding mode of baicalein with SARS-CoV 3CLpro is distinctively different from those of known inhibitors. All of the crystal structures of the inhibitor-bound SARS-CoV 3CLpro were collected for a comparison analysis (Fig. S4B ). If those peptidomimetic inhibitors are delineated like "swords" to compete with the binding of substrate, baicalein works as a "shield" in front of two catalytic dyads to prevent the approach of the substrate to the active site (Fig. S4B ). Such a unique binding mode in combination with its high ligand-binding efficiency and small molecular weight renders baicalein valuable for drug development.

We substrate-binding sites, particularly for the crucial S1/S2 subsites (7, 19, 20) . Accordingly, substrate analogs or mimetics attached with a chemical warhead targeting the catalytic cysteine were designed as peptidomimetic inhibitors of 3CLpros with a covalent mechanism of action (6). A series of diamide acetamides acting as non-covalent SARS-CoV 3CLpro inhibitors and their binding modes examined by crystal structures have been reported, but they are more or less peptidomimetic inhibitors and a continuous development of these compounds is absent (21) . Although several other small molecules have been declared as 3CLpro inhibitors, a solid validation of their binding with 3CLpros by ITC or complex structure determination is lacking.

As none of the known inhibitors has been moved to clinical trials, considerable efforts to discover novel small molecule inhibitors of 3CLpros are urgently needed.

In 

The authors sincerely thank Prof. Zihe Rao and Prof. Haitao Yang for kindly providing the protein as well as the substrate for the enzymatic assay. We also thank the staff from beamlines BL17U1 and BL18U1 at Shanghai Synchrotron Radiation Facility. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. 

The authors declare no competing interests.

The SARS-CoV-2 3CLpro-baicalein complex structure was deposited with the Protein Data Bank with accession code 6M2N. All other data are available from the corresponding author upon reasonable request.

Figures S1-S4

Tables S1 and S2

References (24-29) Chemical shifts were reported in ppm (δ) coupling constants (J) in hertz. Chemical shifts are reported in ppm units with Me4Si as a reference standard.

The cDNA of full length SARS-CoV-2 3CLpro or SARS-CoV 3CLpro was cloned into the For ligand binding screening and dissociation constant (Kd) determination, the magnitude size was set to 128 K and 1000 single scans were added. (24).

The purified SARS-CoV-2-3CLpro protein was concentrated to 7 mg/mL for crystallization.

One hour incubation of the protein with 10 mM baicalein was carried out before crystallization condition screening. Crystals of the complex were obtained at 20 °C by mixing equal volumes of protein-baicalein and a reservoir (16 % PEG6000, 100 mM MES, pH 5.8, 3% DMSO) with a handing-drop vapor diffusion method. Crystals were flash frozen in liquid nitrogen in the presence of the reservoir solution supplemented with 20% glycerol. X-ray diffraction data were collected at beamline BL18U1 at the Shanghai Synchrotron Radiation Facility (25) . The data were processed with HKL3000 software packages (26) . The complex structure was solved by molecular replacement using the program PHASER (27) with a search model of PDB code 6LU7. The model was built using Coot (28) and refined with a simulated-annealing protocol implemented in the program PHENIX (29) . The refined structure was deposited to Protein Data Bank with an accession code listed in Table S1 . The complete statistics as well as the quality of the solved structure are also shown in Table S1 .

The Vero E6 cell line was obtained from American Type Culture Collection (ATCC, Manassas, USA) and maintained in minimum Eagle's medium (MEM; Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen, UK) in a humid incubator with Baicalein is shown as green spheres and other inhibitors together with two catalytic residues are shown as sticks. 

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After that, the virus-compound mixture was removed and cells were further cultured with a fresh compound containing medium. At 48 h p.i., the cell supernatant was collected and the viral RNA in supernatant was subjected to qRT-PCR analysis as described previously (18). DMSO was used in the controls. The experiments were performed in triplicates and three

HPLC-MS profiling of the active fraction 8 and 12

HPLC-MS profiling of fration 8. (B) HPLC-MS profiling of fration 12