henhaobio
Sign in

bioRxivMolecular BiologyPreprint

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro

Haixia SuiD, Sheng YaoiD, Wenfeng ZhaoiD, Minjun LiiD, Jia LiuiD, Weijuan Shang, Hang XieiD, Chang‐Qiang Ke, Mei-na Gao, Kunqian YuiD, Hong Shu LiuiD, Jingshan SheniD, Wei TangiD, Leike ZhangiD, Jianping ZuoiD, Hualiang JiangiD, Fang BaiiD, Yan WuiD, Ye YangiD, Yechun XuiD

PDFDOI
In-depth full-text digest available · Jump to digest ↓

Abstract

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.

In-depth summary

Claude Sonnet 4.6 · max effort · 8 Nov 2026 · from the full text of version 1 (14 Apr 2020)

Baicalin and baicalein from Scutellaria baicalensis inhibit SARS-CoV-2 3CL protease non-covalently; a 2.2 Å baicalein complex structure shows an unusual shield-like binding mode.

Known 3CLpro inhibitors are mostly covalent peptidomimetics, none has reached clinical trials, and other small-molecule claims lack ITC or complex-structure validation. This work screens a traditional Chinese medicine herb and validates hits by enzymology, ITC, native mass spectrometry, crystallography and a cell-based antiviral assay.

Key results

  1. ShownBoth natural products inhibit recombinant SARS-CoV-2 3CLpro in a FRET assay, with baicalein showing the lower IC50; baicalein also inhibits SARS-CoV 3CLpro.

    SARS-CoV-2 3CLpro IC50 6.41 μM (baicalin) vs 0.94 μM (baicalein); baicalein vs SARS-CoV 3CLpro IC50 1.18 ± 0.37 μM; at least three independent experiments run in triplicate. · Inhibition of SARS-CoV-2 3CLpro by baicalin and…; Unique binding mode of baicalein with SARS-CoV-2…; The inhibition assay of SARS-CoV-2-3CLpro

  2. ShownITC and native ESI-MS both detect direct binding and rank the two compounds as the enzymatic assay does, arguing against assay-interference artifacts; ITC profiles suggest non-covalent binding.

    ITC Kd 11.50 μM (baicalin) and 4.03 μM (baicalein); native ESI-MS Kd 12.73 μM and 1.40 μM, respectively; ITC repeated three times, MS in three independent experiments. · Protein-ligand binding supported by ITC and…; Isothermal titration calorimetry (ITC)…; Native state mass spectrometry studies

  3. ShownThe baicalein complex structure shows the ligand in the core of the substrate-binding pocket, contacting both catalytic residues, the oxyanion loop and the S1/S2 subsites, and acting as a shield in front of the catalytic dyad.

    Crystal structure at 2.2 Å (PDB 6M2N); H-bonds to Leu141, Gly143, Ser144, His163 and Glu166; S-π contact to Cys145 at 3.3 Å; NH2-π contact from Asn142 at 3.6 Å. · Crystal structure of SARS-CoV-2 3CLpro in…; Unique binding mode of baicalein with SARS-CoV-2…; Data availability statements

  4. ShownBoth compounds suppress SARS-CoV-2 replication in Vero E6 cells at low cytotoxicity; baicalein approaches the reported potency of chloroquine and remdesivir.

    EC50 10.27 μM (baicalin) and 1.69 μM (baicalein); CC50 over 200 μM; SI >19 and >118; reference values chloroquine EC50 1.13 μM (SI > 88), remdesivir 0.77 μM (SI > 129). · Antiviral activities of baicalin and baicalein…; The cell-based antiviral activity assay

  5. SuggestedBaicalein is proposed to bind SARS-CoV 3CLpro in the same shield-like way, based on sequence conservation and a superposition model, not a new SARS-CoV crystal structure.

    96% sequence identity; 12 differing residues, none in direct contact with baicalein; model built by superimposing 6M2N on SARS-CoV 3CLpro/TG-0204998 (PDB 2ZU4). · Unique binding mode of baicalein with SARS-CoV-2…; Figure legends

Methods, data and reproducibility

Fractions and purified compounds from a S. baicalensis aqueous extract were screened in a FRET 3CLpro assay, then characterized by ITC, native ESI-MS and X-ray crystallography, and tested for cytotoxicity and antiviral EC50 by qRT-PCR in Vero E6 cells.

  • S. baicalensis aqueous extract fractionated by HPLC; baicalin and baicalein isolated from active fractions (purity over 95%) · n = 14 fractions from 75 g decoction pieces
  • Recombinant SARS-CoV-2 3CLpro (enzymatic, ITC, MS, crystallization) and SARS-CoV 3CLpro (enzymatic assay) expressed in BL21(DE3) cells · SARS-CoV-2; SARS-CoV
  • Crystal structure of SARS-CoV-2 3CLpro with baicalein, solved by molecular replacement with search model 6LU7 · SARS-CoV-2 · n = 2.2 Å resolution · PDB 6M2N
  • Antiviral and cytotoxicity assays with a clinical SARS-CoV-2 isolate in Vero E6 cells, MOI 0.05, 48 h · SARS-CoV-2 · n = three independent experiments per compound, triplicate wells
  • Comparison set of inhibitor-bound SARS-CoV 3CLpro crystal structures used to survey binding modes · SARS-CoV · PDB 2ZU4 and others listed in S20
Methods & tools
FRET protease assay (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 substrate), GraphPad Prism 8.0, iTC200 isothermal titration calorimetry, scimaX MRMS native ESI-MS, DataAnalysis 5.2 (SNAP2 deconvolution), HKL3000, PHASER, Coot, PHENIX, CCK8 cytotoxicity assay, qRT-PCR viral quantification
Reagents & environment
GraphPad Prism 8.0 for IC50 fitting; DataAnalysis 5.2 (Bruker) with SNAP2 for mass spectra deconvolution; HKL3000, PHASER, Coot and PHENIX for structure determination.
Reproducibility gaps
Structure deposited as PDB 6M2N; all other data only available from the corresponding author on reasonable request. Supplementary tables and figures are not included in the text.
Limitations (2)
  • All antiviral evidence comes from one cell line (Vero E6) in vitro, with no animal or clinical efficacy data for COVID-19; the cited safety data come from separate healthy-subject baicalein trials. · noted in this summary
  • The SARS-CoV 3CLpro/baicalein binding mode is a superposition model from sequence identity, not an experimental structure, and only baicalein (not baicalin) was crystallized. · noted in this summary

Read this if you want enzymatic, ITC, native MS, crystallographic and cell-based evidence for natural-product, non-covalent SARS-CoV-2 3CLpro inhibitors and their binding mode.

Citations
143

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISLiu '20Hou '22Unoh '25Unoh '22Boras '20Hirose '22
86% semantic matchPharmacology & Toxicology
H. Liu, F. Ye, Q. Sun+6 more·bioRxiv·Apr 10, 2020