key: cord-0946333-et94m6xj
authors: Feng, Jingwen; Li, Dongyang; Zhang, Jin; Yin, Xiushan; Li, Jian
title: Crystal structure of SARS-CoV 3C-like protease with baicalein
date: 2022-04-22
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2022.04.086
sha: b1e842cd9543a4f8eabf774e3fd0d3650b82cc16
doc_id: 946333
cord_uid: et94m6xj

The 3C-like protease (M(pro), 3CL(pro)) plays a key role in the replication process in coronaviruses (CoVs). The M(pro) 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 M(pro) in vitro experiments. In this study, we resolved the SARS-CoV M(pro) with baicalein by X-ray diffraction at 2.25 Å (PDB code7XAX), which provided a structural basis for the research and development of baicalein as an anti-CoVs drug.

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 J o u r n a l P r e -p r o o f 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.

M pro 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 M pro 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 M pro crystal structure in the apo state was more flexible than the ligand-binding mode [11] [12] . Various complexes of the M pro 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 acticity of M pro . Studies showed herbacetin, gallocatechin gallate and rhoifolin can block the enzymatic activity of SARS-CoV M pro due to S1, J o u r n a l P r e -p r o o f 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 M pro . Previous data showed baicalein was identified as potential noncovalent inhibitors for SARS-CoV-2 M pro of IC 50 values at 0.94μM [19] [20] . In this paper, we resolved the crystal structure of SARS-CoV M pro -baicalein at 2.25Å, analyzed and compared with the structure of SARS-CoV-2 M pro -baicalein. It provides a structural basis and theoretical basis for the drug research and development of treating CoVs in the near future.

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] .

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 hours, and the Xray diffraction data were collected at beamline02U1 (BL02U1) at the J o u r n a l P r e -p r o o f 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.

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

The binding modes of SARS-CoV M pro -baicalein were compared with structure of SARS-CoV-2 M pro -baicalein. In order to identify the key residues binding to baicalein, we obtained the crystal structure of SARS-CoV M pro with baicalein at 2.25Å (PDB code 7XAX) (Fig. 1A) . SARS-CoV-2 M pro with baicalein at 2.20Å (PDB code 6M2N) (Fig. 1B) . The structure of baicalein has been shown in (Fig. 1C) . The M pro 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 M pro of SARS-CoV and SARS-CoV-2 (Fig. 1D ).

J o u r n a l P r e -p r o o f 

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. 2A) . From the structure of SARS-CoV M pro , the S2 and S2′subsites are critical for substrate binding to the SARS-CoV M pro [26] [27] [28] . SARS-CoV M pro 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 M pro with baicalein is shown in (Fig. 2C) . Three phenolic hydroxyl groups of catalytic dyad (Fig. 3A and 3D) . Baicalein in the active site of SARS-CoV M pro and SARS-CoV-2 M pro (Fig. 3B and 3E) . The phenolic hydroxyl group of baicalein in SARS-CoV M pro forms hydrogen bonds with mains chains and side chains has been shown in (Fig. 3D) . The SARS-CoV-2 M pro 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 ). 

Rencently, 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. M pro 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. M pro 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 M pro [37] .

Correspondingly, baicalein which belongs to flavonoid compounds has been shown the specific activity for SARS-CoV M pro in vitro experiments.

SARS-CoV M pro 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 M pro 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.

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This work was supported by Jiangxi natural science Foundation for distinguished young scholar 

The authors declare no conflflicts of interest.