Crystal structure of SARS-CoV 3C-like protease with baicalein.

The 3C-like protease (Mpro, 3CLpro) plays a key role in the replication process in coronaviruses (CoVs). The Mpro is an essential enzyme mediates CoVs replication and is a promising target for development of antiviral drugs. Until now, baicalein has been shown the specific activity for SARS-CoV Mpro in vitro experiments. In this study, we resolved the SARS-CoV Mpro with baicalein by X-ray diffraction at 2.25 Å (PDB code 7XAX), which provided a structural basis for the research and development of baicalein as an anti-CoVs drug.

Introduction

CoVs are the largest RNA viruses, which have a positive-sense, single-stranded RNA genome [1]. The severe acute respiratory syndrome (SARS) is caused by a novel species of CoVs (SARS-CoV) [2]. The symptoms of SARS-CoV infection are the lower respiratory tract disease including fever, lymphopenia, malaise and mildly elevated serum hepatic enzymes etc. [3,4]. At present, no anti-viral drugs have been found to be beneficial for SARS. The world is facing with a pandemic caused by SARS-CoV-2 now, which is a strain of CoVs spreading rapidly across the globe. But SARS-CoV-2 resulted in less widespread morbidity and mortality compared to SARS-CoV [5]. Although vaccination campaigns are underway globally, the efficacy is reduced because of the variants of concern (VOCs) [6]. Potential risk exists for SARS-CoV-2 VOCs to develop and gain some mutations similar to life threaten SARS happened in 2003. There is a need to fully understand the SARS-CoV and even the whole sub-type of coronavirus.

Mpro is an attractive drug target among CoVs due to its essential role in processing the polyproteins which were translated from the viral RNA [7]. Studies show Mpro is an essential target for inhibition by interaction with Cys145 of its catalytic site [[8], [9], [10]]. The substrate-binding site and active site of the SARS-CoV-2 Mpro crystal structure in the apo state was more flexible than the ligand-binding mode [11,12]. Various complexes of the Mpro structure of SARS-CoV-2 with natural products and novel inhibitors have emerged. The elucidation of the mechanism of shikonin against CoVs laid the foundation for more natural products and traditional Chinese medicines as a source for antivirus drug candidates [[13], [14], [15]].

Flavonoids, found in various plants, are a class of polyphenolic compounds which have a structural unit of 2-phenylchromone [16]. Some flavonoid compounds have antiviral activity against CoVs by inhibiting the activity of Mpro. Studies showed herbacetin, gallocatechin gallate and rhoifolin can block the enzymatic activity of SARS-CoV Mpro due to S1, S2 and S3 sites [17,18]. Baicalein is an ingredient of Shuanghuanglian, mainly derived from the root of Scutellaria baicalensis. Baicalein shown superior binding effect to Mpro. Previous data showed baicalein was identified as potential noncovalent inhibitors for SARS-CoV-2 Mpro of IC50 values at 0.94 μM [19,20]. In this paper, we resolved the crystal structure of SARS-CoV Mpro-baicalein at 2.25 Å, analyzed and compared with the structure of SARS-CoV-2 Mpro-baicalein. It provides a structural basis and theoretical basis for the drug research and development of treating CoVs in the near future.

Materials and methods

Expression and purification of human SARS-CoV

The codon-optimized cDNAs for the SARS-CoV was synthesized fused with 6_His at the N terminus. Synthesized gene was subcloned into the pET-28a vector. The expression and purification of protease was performed by a standard method previously described [21].

X-ray crystallography

Details of the crystallization, data collection, structure solution, and refinement are provided in Table 1 . Briefly, all crystallization trials were conducted using a sitting-drop vapor diffusion method at 20 °C. Baicalein was soaked with the crystal of SARS-CoV-apo within 12 h, and the X-ray diffraction data were collected at beamline02U1 (BL02U1) at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). The structure solution was conducted by molecular replacement using SARS-CoV-apo (PDB code 7DQZ) as an initial model. Refinement and model building were carried out using Phenix [22] and Coot [23], respectively.

Table 1

Statistics for data processing and model refinement of SARS-CoV Mpro with baicalein.

PDB code	7XAX
Synchrotron	SSRF
Beam line	BL02U1
Wavelength (Å)	0.97919
Space group	P1
a, b, c (Å)	55.51, 60.55, 68.28
α, β, γ (°)	90.95, 120.71, 108.65
Total reflections	129,291
Unique reflections	37,931
Resolution (Å)	2.25 (2.31–2.25)
R-merge (%)	7.1 (66.2)
Mean I/σ (I)	11.0/1.8
Completeness (%)	97.7 (96.2)
Redundancy	3.4 (3.5)
Resolution (Å)	66.31–2.25
Rwork/Rfree (%)	22.92/27.74
Atoms	4442
Mean temperature factor (Å2)	46.1
Bond lengths (Å)	0.008
Bond angles (°)	0.973
Ramachandran plot (%)
Preferred	97.11
Allowed	2.89
outliers	0

Data availability

Coordinates for SARS-CoV-Mpro-baicalein complexe has been deposited in the Protein Data Bank (PDB) under accession numbers 7XAX.

Results

Structures of SARS-CoV Mpro-baicalein

The binding modes of SARS-CoV Mpro-baicalein were compared with structure of SARS-CoV-2 Mpro-baicalein. In order to identify the key residues binding to baicalein, we obtained the crystal structure of SARS-CoV Mpro with baicalein at 2.25 Å (PDB code 7XAX) (Fig. 1 A). SARS-CoV-2 Mpro with baicalein at 2.20 Å (PDB code 6M2N) (Fig. 1B). The structure of baicalein has been shown in (Fig. 1C). The Mpro of SARS-CoV and SARS-CoV-2 had 96% similarity and 95% amino acid homology [24,25]. A comparison of the sequences shows that twelve residues are different between the Mpro of SARS-CoV and SARS-CoV-2 (Fig. 1D).

Fig. 1

Comparison of the binding pocket of Mfrom different CoVs. (A) Surface of SARS-CoV Mpro. Baicalein is shown in purple. Oxygen atoms are shown in red. (B) Surface of SARS-CoV-2 Mpro. Baicalein is shown in green. Oxygen atoms are shown in red. (C) The structure of baicalein. (D) Alignment of the Mpro of SARS-CoV and SARS-CoV-2. The conserved precent has been shown orange (100),blue (≥50). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Crystal structure of SARS-CoV Mpro with baicalein

The protomer is composed of three domains. Domain I and domain II have an antiparallelβ-barrel structure. Domain III contains five α-helices arranged into a largely antiparallel globular cluster, and it is connected to domain II by a long loop region [13](Fig. 2 A). From the structure of SARS-CoV Mpro, the S2 and S2′subsites are critical for substrate binding to the SARS-CoV Mpro [[26], [27], [28]]. SARS-CoV Mpro with baicalein had a Cys145-His41 catalytic dyad in the S2 subsite, which located in a cleft between domain I and domain II [19,[29], [30], [31]](Fig. 2B). SARS-CoV Mpro with baicalein is shown in (Fig. 2C). Three phenolic hydroxyl groups of baicalein form hydrogen bonds with the main chains of Cys145/Ser144/Gly143 as well as the side chains of Asn142/Asn141. The free benzene ring inserted into S2 subsite by making hydrophobic interactions with multiple residues Gln189/Arg188/Glu166/Met165/His164/Asp48/His41. With the aid of an array of direct hydrogen bonds with Cys145/Ser144/Gly143, baicalein served to stabilize the tetrahedral transition state of the proteolytic reaction (Fig. 2D).

Fig. 2

Crystal structure of SARS-CoV Mwith baicalein (PDB code7XAX). (A) Overview of homodimers shown as cartoon. (B) The binding pocket of SARS-CoV Mpro with baicalein. Baicalein is shown in purple. (C) The structure of SARS-CoV Mpro with baicalein. SARS-CoV is shown in yellow and baicalein is shown in purple. Oxygen atoms are shown in red. (D) Interactions of SARS-CoV with baicalein (purple). Residues as well as the baicalein are shown as sticks and hydrogen bonds are represented by dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Comparison of structure of SARS-CoV Mpro-baicalein and SARS-CoV-2 Mpro-baicalein

SARS-CoV Mpro and SARS-CoV-2 Mpro have the same binding mode with baicalein, both have the binding sites include the Cys145-His41 catalytic dyad (Fig. 3 A and D). Baicalein in the active site of SARS-CoV Mpro and SARS-CoV-2 Mpro (Fig. 3B and E). The phenolic hydroxyl group of baicalein in SARS-CoV Mpro forms hydrogen bonds with mains chains and side chains has been shown in (Fig. 3D). The SARS-CoV-2 Mpro with baicalein complexes are hydrogen-bonded to the Ser144/Gly143/Leu141 and side chains via the water molecule, where the only carbonyl group established a hydrogen bond with the main chain of Glu166 [[32], [33], [34]]. The free benzene ring also inserted into the S2 subsite by hydrophobic interactions with His41 residue (Fig. 3F). The electrostatic potential surface surrounding the active pocket in SARS-CoVs with baicalein are also shown in Fig. 3. It was revealed that SARS-CoV-apo (Fig. 3G) is different from that of SARS-CoV Mpro-baicalein(Fig. 3H). SARS-CoV-2-apo (Fig. 3I) is different from SARS-CoV-2 Mpro-baicalein (Fig. 3J).

Fig. 3

Crystal structures of SARS-CoVs with baicalein. (A) Electron density maps (2Fo-Fc) of baicalein at 1.0 σ (7XAX). (B) Baicalein (green) in the active site of SARS-CoV Mpro. (C) Hydrogen bonding (dashed lines) interactions between SARS-CoV Mpro and baicalein. (D) Electron density maps (2Fo-Fc) of baicalein at 1.0 σ (6M2N). (E) Baicalein (yellow) in the active site of SARS-CoV-2 Mpro. (F) Hydrogen bonding (dashed lines) interactions between SARS-CoV-2 Mpro and baicalein. (G) Electrostatic potential surface distribution of SARS-CoV-apo. (H) Electrostatic potential surface distribution of SARS-CoV Mpro-baicalein. (I) Electrostatic potential surface distribution of SARS-CoV-2-apo. (J) Electrostatic potential surface distribution of SARS-CoV-2 Mpro-baicalein. The color of the surface denotes the electrostatic potential, while red signififies negative charge and blue signififies positive charge. Baicalein is shown in sticks (green). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Discussion

Recently, as the cases of SASR-CoV-2 infections, the effective drugs and vaccines has already been found [35]. But there are currently no antivirus drugs approved for the prevention or treatment of highly virulent SARS-CoV infection. Mpro plays a key role involved in the replication and transcription of CoVs among the few available targets for anti-CoVs drugs, which has become an essential and relatively mature drug target in anti-CoVs drug research. Mpro inhibitors mainly exhibit reversible binding with the amino acid residues in S1, S2, and S4 pockets. The inhibitors contain unsymmetrical aromatic disulphides showing inhibitory activity including the flavonoids compounds [36].

Flavonoid compounds displayed good inhibition toward Mpro [37]. Correspondingly, baicalein which belongs to flavonoid compounds has been shown the specific activity for SARS-CoV Mpro in vitro experiments. SARS-CoV Mpro has a Cys145-His41 catalytic dyad in the cleft between domains I and II, can recognize the eleven cleavage sites of nsp4-16 specifically and exhibit self-hydrolytic cleavage activity [38]. Here, we resolved the crystal structure of SARS-CoV Mpro with baicalein that can bind to the substrate pocket between domain I and domain II. Three phenolic hydroxyl groups of baicalein make hydrogen bonds with the main chains of Cys145/Ser144/Gly143 as well as the side chains of Asn142/Asn141, providing a structural basis and theoretical basis for baicalein to inhibit the replication of SARS-CoV.

Declaration of competing interest

The authors have no conflicts of interest to declare.