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Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors


A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019–2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1,2,3,4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds—including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds—as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 μM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Data availability

The coordinates and structure factors for SARS-CoV-2 Mpro in complex with the inhibitor N3 have been deposited in the PDB with accession number 6LU7, deposited on the 26 January 2020 and released on the 5 February 2020. While this work was under review, we solved the complex structure at a higher resolution (1.7 Å); the relevant coordinates and structure factors have been deposited in the PDB with accession number 7BQY. Any other relevant data are available from the corresponding authors upon reasonable request.


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We thank Y. Lei and J. Kong from High Throughput Platform and staff from Analytical Chemistry Platform at the Shanghai Institute for Advanced Immunochemical Studies for their technical support; the National Centre for Protein Science Shanghai and The Molecular and Cell Biology Core Facility of the School of Life Science and Technology, ShanghaiTech University for use of their instrumentation and technical assistance; Z. Liu and H. Su for discussion; and the staff from beamlines BL17U1, BL18U1 and BL19U1 at the Shanghai Synchrotron Radiation Facility. This work was supported by grants from National Key R&D Program of China (grant no. 2017YFC0840300 to Z.R. and no. 2020YFA0707500), Project of International Cooperation and Exchanges NSFC (grant no. 81520108019 to Z.R.), Science and Technology Commission of Shanghai Municipality (grant no. 20431900200) and Department of Science and Technology of Guangxi Zhuang Autonomous Region (grant no. 2020AB40007).

Author information




Z.R. and H.Y. conceived the project; Z.J., H.J., Z.R. and H.Y. designed the experiments; Z.J., X.D., Y. Duan, J.Y., T.Y., Xiaoce Liu and Xiuna Yang cloned, expressed, purified and crystallized proteins; Z.J., Y.Z., B.Z. and F.L. collected the diffraction data; B.Z. and Xiang Liu solved the crystal structure; Z.J., X.D., Y. Duan and J.Y. performed enzymatic activity and inhibition assays, high-throughput drug screening and IC50 measurement; L.W. and F.B. performed virtual screening and molecular docking; Y.X., L.Z. and H.L. performed enzymatic inhibition, cell-based antiviral and cytotoxicity assays for cinanserin; Y. Deng and X.L. performed qRT–PCR analysis and cytotoxicity assay of N3; M.L., R.J. and Xinglou Yang performed plaque-reduction assays; C.P. performed intact protein and MS/MS analyses; Z.J., X.D., Y.X., Y. Deng, C.P., F.B., H.L., Xiang Liu, K.Y., L.G., W.X., G.X., C.Q., Z.S., H.J., Z.R. and H.Y. analysed and discussed the data; Z.J., X.D., F.B., Xiang Liu, L.G., G.X., C.Q., Z.S., H.J., Z.R. and H.Y. wrote the manuscript.

Corresponding authors

Correspondence to Hualiang Jiang or Zihe Rao or Haitao Yang.

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The authors declare no competing interests.

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Peer review information Nature thanks Julien Lescar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 The purification of SARS-CoV-2 Mpro and the inhibitory assay of the N3 compound.

a, SDS–PAGE gel of SARS-CoV-2 Mpro. First lane, marker; second lane, Mpro before treating with rhinovirus 3C protease; third lane, Mpro after the cleavage of C-terminal His tag. For gel source data, see Supplementary Fig. 1. b, Size-exclusion chromatography profile of Mpro. c, The chemical structure of the N3 inhibitor. d, Inhibition mechanism for N3. e, Typical inhibition curves for N3. f, Cytotoxicity assay of N3 on Vero cells. Data are shown as mean ± s.e.m., n = 3 biological replicates. The data in a, b, e, f are representative of three independent experiments with similar results. Source Data

Extended Data Fig. 2 The interactions between SARS-CoV-2 Mpro and N3.

a, The Fo − Fc omit map (contour level of 3σ, shown as blue mesh). b, Detailed view of the interactions between the inhibitor N3 and SARS-CoV-2 Mpro. Mpro residues are shown in blue (protomer A) and salmon (protomer B); N3 is in green; and water is in black. The hydrogen bonds are shown as black dashed lines. The covalent bond between N3 and the C145 of protomer A is in purple.

Extended Data Fig. 3 Comparison of the binding modes between SARS-CoV-2 Mpro–N3 and SARS-CoV Mpro–N1.

a, The chemical structure of the N1 inhibitor. b, The binding mode of SARS-CoV-2 Mpro (blue sticks) with N3 (green sticks). c, The binding mode of SARS-CoV Mpro (grey sticks) with N1 (pink sticks). The hydrogen bonds formed by water (W1) are indicated by the dashed lines.

Extended Data Fig. 4 Cinanserin is an inhibitor of SARS-CoV-2 Mpro.

a, The docking result of cinanserin. The structure of SARS-CoV-2 Mpro is shown as a white cartoon, cinanserin is shown as cyan balls and sticks, and residues predicted to be interacting with cinanserin are shown as sticks. b, Inhibitory activity of cinanserin on Mpro. c, Antiviral activity of cinanserin determined by qRT–PCR. d, Cytotoxicity assay of cinanserin on Vero E6 cells. All data are shown as mean ± s.e.m., n = 3 biological replicates. Source Data

Extended Data Fig. 5 The detergent-based assay for drug leads.

a-f, The IC50 values determined in the presence or absence of 0.01% Triton X-100, which show that detergent did not affect the results. g, Different concentrations of Triton X-100 notably affected IC50 curves for TDZD-8. All data are shown as mean ± s.e.m., n = 3 biological replicates. Source Data

Extended Data Fig. 6 MS/MS analysis reveals that ebselen, PX-12 and carmofur are able to covalently bind to C145 of SARS-CoV-2 Mpro.

a, Molecular weight of apo SARS-CoV-2 Mpro and compound-treated Mpro. The mass shifts (∆m) of the proteins indicate that more than one molecular of the compounds can be covalently bonded to one molecular of Mpro. be, A higher-energy collisional dissociation MS/MS spectrum recorded on the [M + H]2+ ion, at m/z 787.3852 of the Mpro unmodified peptide TIKGSFLNGSCGSVGF (b); at m/z 998.4152 of the Mpro modified peptide FTIKGSFLNGSCGSVGF containing a modification (–C13H9NOSe) induced by ebselen on C145 (c); at m/z 831.4080 of the Mpro modified peptide TIKGSFLNGSCGSVGF containing a modification(–C4H8S) induced by PX-12 on C145 (d); and at m/z 850.9414 of the Mpro modified peptide TIKGSFLNGSCGSVGF containing a modification(–C7H13NO) induced by carmofur on C145 (e). Some of the predicted b- and y-type ions are listed above and below the peptide sequence, respectively. The experiment was performed once.

Extended Data Fig. 7 Docking poses of different SARS-CoV-2 Mpro inhibitors.

a, The crystal structure of the SARS-CoV-2 Mpro–N3 complex. bd, The docking results of three drug leads. Mpro is shown as grey background, and inhibitors are in different colours. The inhibitors identified through the high-throughput screening are likely to occupy the same pocket as N3. e, Predicted binding affinities for the drug leads to SARS-CoV-2 Mpro by using the MM-GBSA module, integrated in Schrödinger.

Extended Data Fig. 8 Images for the plaque-reduction assay using ebselen.

As the concentration of ebselen increases, there is a considerable reduction in the numbers of the plaques by comparison with negative control (NC) and DMSO. Results are shown as representative of four biological replicates. For image source data, see Supplementary Fig. 2.

Extended Data Table 1 Data collection and refinement statistics

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Jin, Z., Du, X., Xu, Y. et al. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature (2020).

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